The generality of pH-induced liquid-liquid phase separation in plant proteins extends to commercial legume flours

Summary

This study explores the phenomenon of liquid-liquid phase separation in protein-containing dispersions from commercial soybean, yellow pea, and fava bean flours. Phase separation in these cases is also referred to as simple coacervation. The phenomenon is shown to occur for any of these legume sources. Flours were dispersed at alkaline conditions at concentrations ranging from 15 %w/w – 21 %w/w, insoluble fractions were removed, and the soluble fractions were slowly acidified. For all flours, we observe three characteristic pH ranges using microscopic analysis. A high pH range of soluble proteins, an intermediate pH range in which microscopic (roughly spherical) protein particles are formed (so-called coacervation range), and a low pH range in which the spherical protein particles cluster and create larger scale structures. Particle size distribution measurements confirmed these observations, allowing the boundaries between the different pH regions to be delineated more precisely. We find some, but contrary to other studies, no extensive fractionation of proteins of distinct types over the spherical protein particles and the continuous phase in the pH range of coacervation. Our work points to the generality of liquid-liquid phase separation in flours of legumes. We demonstrate that simple acidification can induce phase separation across different legume protein mixtures, offering a generalisable method not restricted by the conditions of specific protein fractions. Unlike conventional salt-induced methods, our approach requires no additional salt or purification, maintaining the product's suitability for food applications.