Exploitation And Identification Of Small Peptide From Cotton Seed Meal

A procedure for producing small peptides by enzymolysis of cottonseed meel was developed,in the purpose of exploiting low-quality cottonseed meal with high performance. Qualitative analysis and quatitative analysis was done to the water-soluble peptides, those molecular weight was low than 1kDa. The paper consists of three chapters.The first chapter deployed gel electrophoresis as a fast analysis platform for protein and peptides of cottonseed meal, in the lack of standardization of peptides analysis. The total percentage concentration (% T) of both monomers (acrylamide and the crosslinker bisacrylamide) and the percentage concentration of the crosslinker (% C) were studied and optimized, and a Tricine-SDS-PAGE method was setted up for peptides analysis. It's a fast method and its accuracy was evaluated.In the second chapter, influencing factors such as quality of enzyme(g/g) and cottonseed meal(g/v), pH, temperature and time of enzymolysis were studied in order to establish a best condition to enzymolysis cottonseed meal by Alcalase and Flavourzyme, through analysis of DH and molecular weight change which measured by Tricine-SDS-PAGE system. The results showed that the best condition for Alcalase was 3% of Alcalase (g/g), 5% of cottonseed meal, pH8.0, 60℃, 6 hours; the best condition of Flavourzyme was 3% of Flavourzyme (g/g), 5% of cottonseed meal, pH7.0, 50℃, 4 hours. In the third chaper, the water-soluble peptides of cottonseed meal were measured by qualitative analysis and quatitative analysis. As qualitative analysis respect, size exclusion chromatography and LTQ mass were deployed to analysis molecular weight and amino acid sequence of water-soluble peptides of cottonseed meal, fourteen peptides'amino acid sequence were identified, among these peptides, ten of them whose molecular weight below 1.5kDa, three of them whose molecular weight below 2kDa, only one of them whose molecular weight larger than 2kDa, the average molecular weight of the water-soluble extract was 1.5kDa. As quatitative analysis respect, peptides that molecular weight below 1.5kDa were measured as a whole, as a result, the percentage of peptides in Alcalase enzyme solution was 51.8635%; the percentage of peptides in Flavourzyme enzyme solution was 29.6607%; the percentage of peptides in double-enzyme solution was 53.2849%. It's also reminded that peptides whose molecular weight below 1.5kDa can be prepared through the procedure following:①Samples was centrifugated at 3500r×15min, the clear supernatant should be collected;②Filtrated the clear supernatant by 0.45um millipore film, the collection should be cryodesiccated;③Dissolved the cryodesiccated samples into distilled water, centrifugated the solution at 7500r×15min, the clear supernatant should be collected;④Mesured the total quatity of peptides by GB/T 6432-1994: A Method to Determine the Quatity of Crude Protein in Feedstuff.