Multi-scale Characterization On Multilevel Structural Nature And Aggregation Behavior Of Casein

Casein is the most abundant protein in milk, so it is the core of research in dairy science. To obtain the multilevel structural information and aggregation behavior of casein, a variety of modern analytic techniques, including HPLC-ESI-MS/MS, circular dichroism, second-derivative UV absorption spectroscopy and light scattering have been used to systemically investigate the multilevel structures ofα_s-casein,β-casein, casein micelles and integrated analyze the enzymatic hydrolysis process of casein-pepsin system. The main conclusions were as following:Separation and identification of casein monomers:The individual caseins were effectively separated by RP-HPLC, electrophoresis and membrane. The individual caseins were identified by ESI-MS.Study on the secondary structure of casein:Changes in secondary structure ofα_s- andβ-casein at different temperature and pH were investigated by far UV circular dichroism. The result indicated that theα-helix of both individual caseins increased gradually upon heating and the process was irreversible at experimental conditions except heating ofβ-casein at pH8.0. The result also showed that both caseins were more thermal stabile at pH8.0. The influence conditions including temperature, ethanol concentration and esterification on casein micelles also indicated that they possessed more stabile secondary structure.Tertiary structure analysis of casein:The tertiary structure of individual caseins at various pH and temperature were studied by near UV circular dichroism and second derivative UV absorption spectroscopy. The result indicated that the aromatic amino acid side chains of caseins were inclined to incorporate into a more buried, less polar environment as elevating the temperature at different pH, which showed irreversibility after cooling. Tertiary structures of both caseins were also more stabile at pH8.0. The near-UV CD investigation on casein micelles indicated that the tertiary structure of micelles was stabile after heating. The aromatic side chains were incorporated into a more buried, less polar environment as increasing the ethanol proportion, but were exposed more polar environment after esterification.The quaternary structure and aggregation behavior of casein:Dynamic light scattering (DLS) analysis on the aggregation behavior ofα_s- andβ-casein indicated that the aggregation increased as elevating the temperature. The process was dependent on pH, which showed less stabile at pH8.0. The conformation parameters of casein micelles were obtained by static light scattering (SLS). Size-exclusion chromatography coupled with a multi-angle laser light scattering (SEC-MALLS) result showed that there might be a dynamic system between individual caseins and casein micelles. The effect of temperature, protein concentration, pH, ionic strength, ethanol concentration, cation strength and enzymatic hydrolysis on the average hydrodynamic radius (Rh) and light scattering intensity (Is) of casein micelles were also studied using DLS. The result described the aggregation behavior of casein.Product characterization of pepsin induced hydrolysis of casein:The product of enzymatic hydrolysis were separated and identified by HPLC-ESI-MS/MS. The sequence and the release characteristics of each peptide could be obtained. Changes in average hydrodynamic radius, molecular weight and its distribution were investigated by DLS and SEC-MALLS. Moreover, the kinetic appearance of five molecular weight fractions was also analyzed during peptic hydrolysis of casein.