FIELD-FLOW FRACTIONATION TECHNIQUES FOR MILK PROTEIN CHARACTERIZATION

Milk contains two types of proteins: Caseins and whey proteins. There are four caseins: α/β/γ/κ caseins that formed micelles in milk. Whey proteins are globular proteins between 12 and 200 kDa: α-lactalbumin; β-lactoglobulin; Serum albumin; Immunoglobulin and Lactoferrin. Industrials fractionate milk to produce whey protein and casein isolate to make ingredients for the formulation of dairy products. Whey protein isolate can be heated to functionalize or pasteurize them. The objective of these three studies are to characterize the isolates of whey proteins and the impact of heating treatment on whey protein fractions and formulations using different FFF techniques: Asymmetrical-FFF; 2D AsFlFFF-RPLC and Centrifugal-FFFWhey protein isolate heated at high temperature were studied by Centrifugal-FFF coupled with MALS and RI. Aggregates with the lowest density and size elute in the void peak and represent 51% of the mass fraction. For the bigger one, there are two populations: one smaller than 1 micrometer and one higher, up to 4.5 micrometers. For size determination, the biggest population fractionated has been collected and analyzed by DLS in batch. Infant milk formulation without fat was investigated by Asymmetrical Flow FFF coupled with MALS; QELS and RI. The formulation contains bovine milk and isolate whey protein fractions. The technique separates the non-aggregated whey protein to the casein micelles. The addition of lactoferrin partially disintegrated the casein micelles. The formulations are heated at 67.5°C or 80°C. Native whey protein content decreases with the heat treatment, leading to their aggregation on with the casein micelles. The phenomena depend on the temperature and protein composition. At 80°C, shape ratio Rg/Rh of casein micelles increase with the temperature when the formulation is enriched in lactoferrin. The Rg and Rh decrease when the formulation is enriched in α-lactalbumin and lactoferrin. Transmission Electronic Microscopy are correlated with these results. At 80°C, whey protein aggregates are fixed outside the micelle for the first formulation and they are fixed inside the micelles for the second formulation.Whey protein isolate contains few quantities of caseins. The second objective of this study is to identify the presence of trace of caseins in whey protein isolate using 2D AsFlFFF-RP-HPLC. This technique allows to separate proteins by their size and by their polarity. A whey protein standard mix is studied: α-lactalbumin, β-lactoglobulin A and B, BSA, and 3 caseins. A 2D map is obtained with all separated proteins.