Characterization of casein-based nanoparticles formed upon freezing by in situ SAXS measurement

The formation of casein-based nanoparticles from sodium caseinate and sodium caseinate–pectin solutions was investigated in a frozen system by protein self-aggregation and protein–polysaccharide complexation, respectively. Casein-based nanoparticles were prepared by controlling the pH levels of the solutions followed by freezing. The formation of precipitates was confirmed in the casein solutions at pH < 5.5. However, an obvious effect of the freezing on the formation of aggregates could not be confirmed, although the freezing did have an effect on accelerating the formation of precipitates. The mean particle sizes analyzed from the produced nanoparticles suggested that freezing did not have any significant effects on altering the particle sizes. Similar trends were observed in the casein–pectin solution in terms of phase separation and particle sizes. A difference was confirmed in the solution at pH 4.6; that is, a clear phase separation was observed due to freezing. Nevertheless, it was found, both in the casein and casein–pectin systems, that the degradation rates of the freeze–thawed nanoparticles were considerably slower than that of the original nanoparticles. This suggested that the casein-based nanoparticles formed through freezing had structural features different from the ones in the unfrozen solution. It could be concluded from the SAXS analysis that the formation of the protein-based particulate systems certainly occurred in the cryoconcentrated phase associated with freezing. The present technique is advantageous for the encapsulation of heat-sensitive and/or acid-sensitive ingredients in protein nanoparticles.

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► The formation of protein-based nanoparticles in a frozen system was investigated.
► The protein self-aggregation from sodium caseinate solutions and the protein–polysaccharide complexation from sodium caseinate–pectin solutions were employed for nanoparticle productions.
► A freezing process was applied to establish a low pH environment in the cryconcentrated phase and to induce the nanoparticle formations.
► The in situ SAXS analysis of the frozen systems suggested that the formation of the protein-based particulate system certainly occurred in the cryoconcentrated phase.
► The enzymatic degradation rates of the freeze–thawed nanoparticles were slower than those of the original ones.