Two-phase microfluidic approaches to study food protein functionality

Food proteins are essential for the functional performance of many emulsion-based products because they adsorb at the oil-water interface and prevent droplet destabilization. The interfacial performance of these proteins depends on the protein structures and solution conditions, making outcomes difficult to predict with traditional emulsification methods that couple droplet break up with coalescence and also require large sample volumes. The challenge is intensified for recombinant proteins (lab-grown) which are typically available only in milligram-scale quantities during early development. In this thesis, two phase microfluidics is used to: (i) study microfluidic emulsification in flow-focusing devices (FFDs) and identify droplet breakup criteria in the presence of emulsifiers including food proteins, (ii) quantify time-dependent interfacial protein loading over minutes to hours using ex situ calorimetric assays; and (iii) evaluate the functional performance of recombinant proteins relative to their bovine counterparts under food-processing relevant conditions. Droplet breakup behavior in FFDs was mapped across 1,800 flow conditions spanning two device geometries, two wettability conditions, 20 fluid combinations, 4 emulsifier types, and viscosity ratios (0.03–30). Four patterns were observed, squeezing, dripping, jetting, and plugging. Across this space, regime transitions can be described by two dimensionless groups: the continuous-phase Weber number (W_out) and the dispersed-phase Reynolds number (R_in). Jetting was observed at W_out≈1, while plugging was observed when αR_in≈1, where α is a viscosity dependent pre-factor. Comparison with literature spanning multiple FFD geometries, surfactant systems, and similar viscosity ratios showed that these criteria broadly reproduce reported thresholds and provide practical operating windows for monodisperse generation. Building on the operating map above, microfluidic emulsions were paired with low-volume ex situ colorimetry to quantify cumulative interfacial protein load over minutes to 48 h. Three dairy proteins were tested across three pH values, two ionic strengths, and seven concentrations (≈120 conditions). Loading curves were summarized by Γ_0(initial), Γ_∞(saturation), and an apparent rate parameter B. Γ_0 and Γ_∞ increased systematically with bulk concentration, with maxima near pI and 0.1 M NaCl. α-casein showed the highest loads, while β-casein and Whey Protein Isolate were comparable. Finally, scaling analysis indicated that interfacial load is primarily concentration-driven, with pH and ionic strength acting as secondary modulators. Extending the workflow, the same pipeline screened six lab-grown (precision-fermented) proteins against bovine α-casein and WPI under food-relevant pH and ionic strength, by systematically evaluating droplet formation, coalescence stability, interfacial loading, and interfacial tension. rCasein2 approached α-casein across the composite criteria, while rBLG90 was the strongest globular analogue. These measurements show that lab-grown proteins can be benchmarked quantitatively at milligram scale under controlled hydrodynamics and formulation conditions. Together, the studies establish practical operating windows for FFD emulsification and a low-material framework for quantitative assessment of protein adsorption and function under food-relevant conditions.