Study On The Preparation And Application In Yeast Peptide Purificating Of Oligochitosan

Oligochitosan(OCS), the degradation product of chitosan(CTS), is the only alkaline,positively-charged, water-solubled and animal oligosaccharides found in the nature.Oligochitosan is easy absorbed by body, and showed a large number of superior biological activity. In this paper, oligosaccharides was obtained by degrading chitosin,and applied to the purification of yeast peptides, providing theoretical basis for the purification of yeast peptides from enzymatic system. The main research contents and results are as follows:1. Preparation and characterization of oligochitosanOligochitosan was prepared from chitosan degreded by double enzymes and H2O2.H2O2 was added to the system in the process of enzymatic Hydrolysis. The most suitable time to add H2O2 was the last 1 h before enzymatic Hydrolysis ended, H2O2 concentration is 10%, the amount is V(H2O2): V(solution)=1:8. Results showed the deacetylation degree was basically unchanged of obtained oligochitosan, whoes molecular weight was 1318.71. Compared with oligochitosan degreded only by double enzymes, obtained oligochitosan has smaller molecular weight and more light-colored.Result of FTIR showed the molecular structure of chitosan before and after degredition was basically unchanged, Result of TG/DSC analysis showed the obtained oligochitosan had low thermal stability than chitosan.2. Preparation of brewer’s yeast enzymatic hydrolysis solutionThis experiment using yeast extract enzyme to hydrolyze beer yeast protein. The degree of hydrolysis as response value to optimize hydrolysis condition of brewer’s yeast via response surface methodology. The optimal process is 59.9 ℃, pH 7.5, the enzyme dosage 6.0% and substrate concentration 1:10, 12 h, the degree of hydrolysis is 33.23%.Ethanol precipitation method has been used on preliminary impurity of enzymatic liquid, the removal rate of polysaccharide and nucleic acid were 52.37% and 79.09%respectively, and the loss rate of peptides was only 16.33%. Results of molecular weight distribution measured by HPSEC indicated that, the content of peptides lower than 3k Da was 47.91%, with a large proportion of MW about 5kDa and 12 kDa.3. Application of magnetid OCS microspheres on yeast peptides purificationMagnetic OSC microspheres were prepared via suspension cross-linking method,which decorate OSC no the surface of Fe3O4 magnetic particles. Also, the properties of magnetic OSC microspheres were tested by Transmission Electron Microscope(TEM)and FTIR. The results showed that the average diameter of microspheres with typical globular shell-core structure was 20 nm, Fe3O4 magnetic particles were successfully wrapped up in the composite microspheres and the infrared spectroscopy of microspheres showed Fe-O stretching vibration peak at 590cm-1.The adsorption properties of Alu-Gln and AngiotensinⅠ on magnetid OCSmicrospheres, and the adsorption model of magnetid OCS microspheres for Alu-Gln were also investigated. The results showed that: the optimal conditions of adsorption for Alu-Gln and AngiotensinⅠ were some diferent: the initial concentration of Alu-Gln was2.5 mg/mL, temperature of adsorption for Alu-Gln was 30℃, pH of buffer was 7.5, time of adsorption for Alu-Gln was 30 min, while for AngiotensinⅠwere 2.0 mg/mL, 30℃,pH 7.0, 40 min. Under the optimal conditions, the saturated adsorption of Alu-Gln was52.31mg/g and of AngiotensinⅠ was 54.94 mg/g. The adsorption of Alu-Gln on magnetid OCS microspheres accorded with Langmuir model description; The adsorption kinetics consistent with second-order kinetic adsorption kinetics. Finally, the adsorption of magnetid OCS microspheres for peptides in mix enzymatic hydrolysis system were researched. The results showed that the adsorption of magnetid OCS microspheres for peptides reached to 45%, and were few influenced by polysaccharide and nucleic acid.The molecular weight distribution of peptides showed that the proportion peptides molecular weight less than 1KDa and between 1KDa and 3KDa decreased from 23.78%and 24.13% to 17.22% and 10.39% respectively, while the proportion peptides more than3 KDa had improved. It’s feasible for magnetid OCS microspheres separate different molecular weight peptides existde in mixed enzymatic hydrolysis system.