A methodology for monitoring globular milk protein changes induced by ultrafiltration: A dual structural and functional approach

Analysis by reverse phase chromatography. A) Shapes of G1 and G2 elution gradients. B) Improvement of the detection for BSA subpopulations (monomeric and dimeric) with G2 compared with G1. Detailed operating conditions are given in Materials and Methods. AU = arbitrary units.

Structural modifications of BSA induced by crossflow UF with restricted protein transmission. A) Comparison of the chromatographic profiles of initial solution and final retentate generated by crossflow UF of a BSA solution through a 150kg/mol zirconium oxide membrane. B) Effect of crossflow UF onto the profile of intrinsic fluorescence in the same operating conditions. C) Progressive evolution of the wavelength of maximum fluorescence intensity during BSA circulation inside the filtration pilot. AU = arbitrary units.

Modification of β-LG induced by dead-end UF. Effect of the dead-end UF of β-LG through a 30kg/mol polyethersulfone membrane. A) Surface hydrophobicity of β-LG for the initial solution and the final permeate as deduced from extrinsic fluorescence measurements. B) Intrinsic fluorescence spectra for the initial solution and the final permeate and retentate. C) Far-UV circular dichroism spectra for the initial solution and the final permeate. AU = arbitrary units.

Functional modifications of α-LA induced by dead-end UF. Effect of the dead-end permeation of an apo-α-LA enriched fraction through a 30kg/mol polyethersulfone membrane. A) Solubility profile of α-LA for the initial solution and the permeate. B) Surface hydrophobicity of α-LA for the initial solution and the permeate as deduced from extrinsic fluorescence measurements. AU = arbitrary units.

Detection of physically induced modifications of α-LA by reverse phase chromatography. A) Decrease in α-LA retention time after permeation through a 30kg/mol polyethersulfone membrane. B) Score plot of the multivariate analysis: detection of significant differences in retention times according to projections on principal component (PC) 1. AU = arbitrary units.

Detection of protein changes by near-UV circular dichroism. A and B) Spectra of α-LA tertiary structure for, respectively, the initial apo-α-LA enriched solution (molar Ca²⁺/α-LA ratio of 0.36±0.07) and the fraction resulting from dead-end permeation through a 30kg/mol polyethersulfone membrane. C) Effect of α-LA permeation on its thermal stability according to a multivariate analysis. PC = principal component; AU = arbitrary units.

Sensitivity of fluorescence techniques to minor variations of pH and ionic strength. A) Sensitivity of intrinsic fluorescence to pH and ionic force for the apo-α-LA enriched fraction (molar Ca²⁺/α-LA ratio of 0.36±0.07) as deduced from the variation of the wavelength of maximum intensity. B) Sensitivity of extrinsic fluorescence to pH and ionic force; S₀ represents the slope of maximum fluorescence intensity as a function of apo-α-LA concentration and values are compared with the value obtained in reference conditions (pH=7.0 and no added salt). C) Effect of the Ca²⁺/α-LA molar ratio onto protein surface hydrophobicity as estimated by the maximum extrinsic fluorescence intensity. AU = arbitrary units.

Immunochemistry, a pertinent tool for the detection of filtration-induced modifications. Time-resolved antigenic reaction obtained with the initial apo-α-LA enriched solution and the fraction resulting from the dead-end permeation through a 40kg/mol polyacrylonitrile membrane. A) Interaction between α-LA and the Mab273 monoclonal antibody. B) Interaction between α-LA and the Mab291 monoclonal antibody. RU = resonance units.