Effects Of Amino Acid Composition On The Stability Of Fish Collagen

In recent years,the natural collagen has been more and more widely applied in the fields of biological materials,medical hairdressing,functional food and so on.Although the considerable progress has been made in the collagen,there are still many unanswered scientific problems should be further studied and discussed.The structure-activity relationship is always the research focus of area of collagen.On the one hand,the relationship between the biological properties and molecular structure of mammalian collagen had been explored in-depth,although,the related research of fish source collagen is just beginning.On the other hand,the performance of collagen material could be improved via restructuring(self-assembly)behavior of natural collagen in vitro.However,the correlation between collagen molecular structure and its fiber assembly performance is rarely reported.In addition,the quantitative determination method of collagen,which is the key technology of collagen research and application,also needs optimization and improvement.Therefore,this paper focus on the following aspects: first,the collagen content in fish and amphibian are determinated by the commercial kits based on specificity staining principle of Sirius red,and the effect and related factors of this method is evaluated.Second,aimed for the fish collagen,the structure-activity relationship between amino acid composition and thermal stability of collagen is analysised systematically.At the same time,further research on the relationship of thermal stability of the self-assembly fiber products and the composition of the fish collagen amino acid is further revealed.These results improve the structure-activity relationship theory of collagen,and provide theoretical guidance for the development and utilization of fish source collagen.In this study,the pepsin-soluble collagen isolated from bullfrog is used as the sample.The collagen content of these samples is determined by Sirius red staining method,the precision and recovery of which are also evaluated.Based on these results,the impact of amino acid composition on the determination results is revealed by the analysis of the different sources collagen by collagen-staining method.Our results reveal the good accuracy,precision,and antijamming capability of this method to measure fish and amphibian source collagen.From the analysis of collagen dye absorbance values and collagen protein secondary structure,the dye absorbance values could be influenced by the structure integrity of collagen molecules triple helix.If the triple helix structure is damaged by the collagen thermal denaturation,the estimated value decreased significantly.Therefore,the collagen denaturation degree could be evaluated by this quantitative determination method.In addition,the determining value is also influenced by the composition of collagen amino acid.The experimental results reveal that the dye absorbance shows significant positive correlation with non-polar hydrophobic amino acids,and negative correlation with polarity of neutral amino acids,acidic amino acids and basic amino acids,which proves that the collagen amino acid composition and distribution are the "internal cause" for the Sirius red dye method.Different fish collagens are used as samples,the inner relation of thermal stability and amino acid composition is discussed by the analysis of the amino acid composition and fish collagen denaturation temperature.The experiment results mean that the collagen denaturation temperature is positive related with the proline hydroxylation rate,and the contents of basic amino acid,charged polar amino acid and total polar amino acid,while is negative related with the content of imino amino acid and nonpolar amino acid(p < 0.01).Stepwise multiple regression analysis method was used to establish the mathematical correlation model of collagen protein denaturation temperature and the proline hydroxylation ratio,the electric charge polarity amino acid is established.Experiment results proved that this model can predict the denaturation temperature of the source of fish collagen correctly.In this paper,13 different sources of collagen are used as samples,the influence of collagen composition on collagen fiber assembly are explored by measuring of the composition of collagen amino acid and the activation energy of collagen.The experimental results show that the dissociation rates of collagen fiber increasing,along with the increasing of temperature.The difference of the dissociation energy of differenct species are found in our case,which is listed as following: mammals > amphibian > fish.This result means that the compact(or stable)degree of the collagen fiber could be impacted by the molecular structure of different species.The activation energy of collagen fiber assembly Ea reveals a positive correlation to alanine,while negative correlations to phenylalanine,glutamic acid,hydroxyproline,threonine,leucine,tyrosine,aspartic acid,isoleucine,and valine.The positive correlation between dissociation activation energy of collagen fiber assembly Ea and non-polar hydrophobic amino acid indicates that the stability of triple-helix structure could be increased by the the enhancement of hydrophobic effect among collagen polypeptide chains.