Multi-Scale Characterization On Multilevel Structural Nature And Isolation Of Whry Protein In Buffalo Milk

Whey protein is rich in functional characteristics, so it is the core of research in food science, biochemistry and biomedicine. To obtain the multilevel structural information of whey protein, β-lactoglobulin and α-lactalbumin and their interactions with other biological molecules in buffalo milk,a variety of modern analytic techniques, including MS/MS, circular dichroism, fluorescence spectrum and particle size analysis have been used to investigate the primary structure and multilevel structures of whey protein, β-lactoglobulin and α-lactalbumin and research on the whey protein-biological molecules system systemically.The main conclusions were as following:Isolation of active whey protein, β-lactoglobulin and α-lactalbumin:With methods of isoelectric precipitation, NaCl precipitation, trichloroacetic acid precipitation, calcium precipitation, iron salt precipitation, chromatography separation, the whey protein, β-lactoglobulin and α-lactalbumin in buffalo milk were isolated, and its activity and recovery were analyzed, while keeping the purity.Multi-scale characterization on multilevel structural information of whey protein, β-lactoglobulin and a-lactalbumin:The β-lactoglobulin and α-lactalbumin were identified by MS/MS and the result indicated that amino acid sequence of β-lactoglobulin and a-lactalbumin in GuangXi buffalo milk were different from these in holstein milk.Circular dichroism, fluorescence spectroscopy and particle size analysis results showed that multistage structure and spatial distribution behavior of whey protein, β-lactoglobulin and α-lactalbumin were influenced significantlyby protein concentration, pH, NaCl ionic strength. Whey protein, β-lactoglobulin and a-lactalbumin intramolecular forces changed in the process of increased protein concentration and whey protein maintained the stability of tertiary structure better than β-lactoglobulin and α-lactalbumin.pH7.5-pH5.0and the range of0.00M～0.30M NaCl, secondary structure of whey protein, β-lactoglobulin and α-lactalbumin werestable,buttertiary structurechanged. Hydrophobic groups exposed of whey protein, β-lactoglobulin and α-lactalbumin wereremarkable at pH5.0and whey protein, β-lactoglobulin and α-lactalbumin gathered evidently under0.30M NaCl.Research on WP-CN system:Circular dichroism showed that secondary structure of WP-CN system was significantly influenced by interaction between WP and CN at pH6.0. The aggregation degree of WP-CN system was weaker than monomer WP/CN at pH7.0～pH6.0, to the contrary,that was stronger at pH5.0.WP-CN system was more stable than single WP/CN in present of0.00M～0.30MNaCl.Research on WP-EPS system:Electrostatic repulsion between WP and EPS resulted in the β-sheet of secondary structure of WP improved withthe EPSincreasing from0.00%to0.10%at pH7.0. Electrostatic attraction was present between WP and EPS at pH6.0and soluble complexes was formed at pH5.0.Low ionic strength ofNaCl (0.00M～0.15M) weakened the electrostatic repulsion between WP and EPS, while high ionic strength of NaCl (0.30M) induced to form the complex of WP-EPS.Research on WP-oleic acid system:Oleic acid influenced the secondary of WP, showing the increased α-helix,β-sheet content of WP at pH7.0as well as pH5.0.Theeffectof secondary structure of WP wasnot remarkable with oleic acid increasing,and the effect oftertiary structurewas obviously.Nevertheless, the forces was weaker between WP andoleic acid at pH6.0. Oleic acid slight effect on the secondary structure of WP under higher NaCl ionic strength.Oleic acid contributed to WP molecules to unfold under0.15M NaCl and to gather evidently under0.00M/0.30M NaCl.