Study On Structural Modification And Stability Of Collagen Polypeptide From Rana Residues

The research content of this dissertation is subordinated to Study on Structural Modification and Stability of Collagen Polypeptide from Rana Residues?20160204022N Y?.Northeastern Rana is a famous dual-use animal of animal and food in China.Rana is used mainly in Rana oil.The by-products of Rana to oil are often treated in very simple ways that processed into animal feed or directly discarded.It will not only causes the waste of resources,but also result in environmental problems.Therefore,how to increase additional value of Rana and use by-products efficiently has become a research hotspot for domestic Rana processing researchers.The research team of Qiu Fangping from Changchun University of Technology has prepared collagen peptide with high biological activity by enzymatic hydrolysis,which provided a new idea for realizing zero waste of Rana resources.However,some environmental factors such as pH value and temperature can cause structural changed and functional properties of peptide destroyed,which have become the main bottleneck for limiting their functional effect and commercial applications.In view of the above problems,the enzymatic high-activity peptide from Rana?molecular weight<3500 Da?was used as raw material in this study.The influencing mechanism of glycosylation on the stability in structural and functional properties of the collagen peptide was analyzed,which provided a theoretical basis for extending the application and development of Rana collagen peptide.The specific research content of this paper is as follows:1.The amino acid sequence,antioxidant stability?pH,temperature,gastrointestinal simulation?and ACE inhibitory activity of enzymatic Rana high-activity peptide?molecular weight<3500 Da?were measured in this study.The results has shown that there are 14 kinds of peptide sequences in Rana peptide,including seven essential amino acids except methionine,and it contained a variety of amino acids related to antioxidant activity and ACE inhibition.The Rana peptide had better antioxidant activity under strong acid conditions and worse thermal stability.The ACE inhibition rate of Rana peptide was 66.87%and the semi-inhibitory concentration was 753.300?mol/L.In the test of simulated gastrointestinal digestion,the results has shown that the DPPH scavenging rate and superoxide anion scavenging rate were gastric juice>blank group>intestinal fluid,and the reducing ability was intestinal fluid>blank group>gastric juice,while the hydroxyl scavenging rate of Rana peptide was lower blank group when passing through the gastric juice and intestinal juice.2.The Rana peptide was used as raw materials,and xylose/glucose was used as reducing sugar.Single-factor and orthogonal optimization experiments were performed to prepare glycosylation products by controlling reducing sugar species?xylose/glucose?and substrate mass ratio?1:0,1:1,1:2,1:4,1:6,1:8?.According to the optimal parameters,glycosylated products were prepared.The degree of glycosylation?intermediate product,degree of browning,pH,degree of grafting?,and the antioxidant activity?DPPH scavenging rate,hydroxyl scavenging rate,superoxide anion scavenging rate,reducing ability?were measured.The results has shown that glycosylation was best when the parameters were substrate concentration10 mg/ml,reactive temperature 60°C,reactive time 24 h,and pH 7,peptide:xylose=1:4,the glycosylation effect was best.According to the measurement of the degree of glycosylation,it is known that the Rana peptide and xylose/glucose do undergo glycosylation reaction,and glycosylation effect of xylose is better than that of glucose.There was a significant correlation between the degree of reaction and the antioxidant activity of Rana peptide and xylose.3.The Rana peptide-xylose glycosylated complex was prepared.Its stability?thermal stability,light stability,stability of simulated gastrointestinal digestion,effect of metal ions?and biological activity?ACE inhibitory activity?were measured.The results of thermal stability test showed that the ability of complex to scavenge DPPH free radicals was not affected by temperature,indicating that glycosylation can improve the thermal stability of Rana peptide.In the photo-stability test,the DPPH scavenging rate of complex which was placed in the sun and darkness after 30days has increased by 2.91%and 1.96%,respectively.It has indicated that light can increase the antioxidant activity of complex,and complex was less affected by light than peptide.The results of the simulated gastrointestinal digestion has shown that the hydroxyl scavenging rate in the gastric juice and intestinal fluid of peptide was significantly improved after glycosylation,and other indicators did not change significantly.The effect of metal ions on the antioxidant activity of complex has shown that the antioxidant activity of the complex decreased when Na⁺and Fe³⁺are present,and we should avoid contact with iron or sodium salts during storage and processing.The ACE inhibitory rates of peptide and complex were 66.87%and83.83%,and the semi-inhibitory concentrations were 753.300?mol/L and 414.475?mol/L,respectively,indicating that glycosylation can enhance the ACE inhibitory activity of Rana peptide.4.The effects of glycosylated modification on the structure of Rana peptide were mensurated,including DSC analysis,UV spectroscopy,fluorescence spectroscopy,infrared spectroscopy,and circular dichroism spectroscopy.The results of DSC analysis has shown that the denaturation temperature and enthalpy values of the Rana peptide decreased after glycosylation.The blue shift of the UV spectrum indicated that the structure of the Rana peptide has been extended to expose aromatic amino acids.The decrease of fluorescence intensity and the blue shift of the maximum emission wavelength indicated that the polarity around the tryptophan residues in the Rana peptide decreased and the hydrophobicity increased.By infrared spectroscopy and circular dichroism analysis,it was known that xylose was linked to Rana peptide through C-H,-C=O,C=C and C-O groups,and the glycosylation resulted in a decrease in the content of random curly structures of Rana peptide.5.Microcapsules were prepared by using complex as the core material and sodium alginate as the wall material.The embedding rate was used as an index to determine single factor and response surface tests,including concentration of calcium chloride?1⁵%?,core/wall ratio?3:1,2:1,1:1,1:2,1:3?,concentration of sodium alginate?1³%?and reactive temperature?40⁶0??.The storage stability?temperature,light,oxygen,storage time?and sustained-release test in vivo of optimal microcapsules were studied.The optimum parameters of microcapsules were sodium alginate concentration of 2.52%,core wall ratio of 0.88,calcium chloride concentration of 2.48%,reactive temperature of 50?,and embedding rate of 69.88%.The results of scanning electron microscope has shown that the prepared microcapsules had a good morphology.The storage stability test of microcapsules showed that the microcapsules had better storage stability.In the sustained-release test,the cumulative released quantity of microcapsules in gastric juice reached37.69%after 8 hours and 68.65%in intestinal juice,which can achieve better sustained-release effect.