Preparation Of Collagen From Chinese Soft-Shelled Turtle Powder And Study Of Antioxidant Properties Of Peptides

The Chinese soft-shelled turtle（Pelodiscus sinensis）is an amphibious reptile that is an important part of China’s freshwater aquaculture products and has a high nutritional value.The Chinese soft-shelled turtle has high nutritional value and medicinal efficacy components.In the last decade,domestic and international research results have shown that the collagen and other bioactive components in the skirt and dorsal carapace of the Chinese soft-shelled turtle have unique functional properties that work in food,health products and pharmaceutical products.With the rising annual aquaculture population,individual deaths caused by a series of problems such as individual tearing and wound infections are common in high density feeding environments,and the waste of turtle resources is particularly serious.In this study,the main raw material is the turtle powder obtained by grinding the turtle powder at low temperature after the internal organs of the turtle have been removed after death.1.The Chinese soft-shelled turtle powder was in the form of dark minced meat,with some viscosity and poor homogeneity at room temperature,so all experiments were carried out using the lyophilised powder of the Chinese soft-shelled turtle,except for the analysis of the composition of the powder itself.Protein accounted for18.22±0.02%of the overall fraction of the Chinese soft-shelled turtle powder,second only to water.In the protein evaluation analysis,essential amino acids accounted for28.13%of the total amino acids and 52.24%of the presenting amino acids,while the crude fat and ash contents of the turtle powder were 5.5±0.06%and 7.79%,respectively.The crude fat and ash contents of the turtle powder were measured to be5.5±0.06%and 7.79±0.02%respectively,indicating that the turtle powder is a low-fat,high-protein raw material with some potential for development.2.The physical and chemical structures of collagen extracted by different enzymes were experimentally analysed in terms of collagen extraction rate,protein microstructure,functional group composition,thermal denaturation temperature and odour composition.The pepsin extraction had the highest extraction rate and the flavoured protease had the lowest extraction rate;the citric acid and pepsin methods could see obvious filamentous cross-linked structures under scanning electron microscopy and had similar resonance bands in the infrared spectra;the thermal denaturation temperatures of the collagen obtained by the four enzymatic methods were not significantly different,all of them were between 37.5-38℃,with the thermal denaturation temperature of pepsin slightly higher than the other In the analysis of the effect of different extraction methods on the odour of collagen,the collagen extracted by pepsin contained the least number of volatile odour components and no irritating odour components,while papain collagen contained the most volatile odour components,amounting to 14.The highest proline and hydroxyproline contents were found in the pepsin method with 8.81±0.15%and 2.34±0.25%,while the lowest hydroxyproline content was 0.59±0.30%in the flavour protease method.The pepsin-derived turtle powder collagen was found to be of good commercial value.3.The extraction process of collagen from turtle powder was optimized by a two-step method of"citric acid+complex enzyme"（the complex enzyme was a mixture of pepsin and acidic protease in a certain ratio）.The optimal extraction conditions for the acid method and the complex enzyme method were firstly determined by single-factor experiments,with the optimal extraction time being 8 h,the optimal feed-to-liquid ratio being 1:35 and the optimal extraction concentration being 10%for the acid method;for the complex enzyme method,the optimal extraction p H was 2,the optimal extraction time was 12 h,the optimal enzyme combination ratio was 1:2（acid protease:pepsin）and the optimal enzyme addition was 3%.Based on the results of the single-factor experiment,a three-factor,three-level Box-Behnken experiment was set up to optimize the response surface of the acid method and the enzyme complex method.After software analysis,when the extraction time was 8.7 h,the material-liquid ratio was 1:31.632 and the acid concentration was9.05%,the maximum extraction rate of the acid method was 8.502%,and the yield of the acid method was 8.47±0.02%through the validation experiment;when the p H value was 2.876,the amount of enzyme added was 2.696%and the enzyme ratio was1:2,the maximum extraction rate of the complex enzyme was 31.833%,and after the validation experiment,the The yield of the complex enzyme method was 30.6±0.06%,and 37.1±0.03%of collagen extraction was obtained by the two-step acid-complex enzyme method.4.The solubility,foaming,foam stability,emulsification,emulsion stability,oil-holding and other functional properties of the acid-complex enzyme two-step collagen were determined.The solubility,foaming and emulsification of collagen under acidic conditions were better than alkaline;foam and emulsification stability could be influenced by p H,and oil-holding properties could reach 45 g/g at low temperature,which was influenced by temperature.5.Collagen peptides were extracted by alkaline protease enzymatic digestion,and the scavenging ability,reducing power and metal Fe²⁺chelation rate of DPPH⁺·,·OH and ABTS⁺·free radicals were measured to evaluate the antioxidant properties of collagen peptides.The results showed that the turtle powder collagen peptides had good antioxidant properties,and the scavenging rate of·OH free radicals and the chelation rate of Fe²⁺were better than that of reduced glutathione,but the overall antioxidant properties were weaker than that of reduced glutathione.