DEVELOPMENT OF HEAT-TREATED WHEY PROTEIN EMULSIONS LOADED WITH OLIVE LEAF CAROTENOIDS: EFFECT OF CALCIUM-INDUCED STRUCTURING ON TEXTURE PROPERTIES

Developing structured emulsions provides a promising strategy to protect lipophilic bioactive compounds, such as carotenoids. Moreover, ionic crosslinking can control protein–protein interactions and gelation behavior, which is relevant for extruding new foods using 3D printing. Therefore, this study aimed to use heat-treated whey protein to develop emulsions containing carotenoids recovered from olive-leaf waste and to investigate the effect of ionic crosslinking on texture profile analysis (TPA). For this reason, olive leaves from olive oil production were lyophilized, ground, and subjected to ultrasound-assisted extraction using 95% ethanol at a solid-to-solvent ratio of approximately 1:15. Extraction was performed at 50 °C for 30 min using 600 W ultrasound with pulsed operation. The extract was centrifuged, and the supernatant was concentrated by rotary evaporation, then vacuum-dried. The resulting carotenoid-rich extract was incorporated into corn oil. For emulsion preparation, whey protein (5 g) was hydrated overnight in 35 g distilled water, adjusted to pH 7.0, thermally treated at 85 °C for 30 min, and cooled to 25 °C. The carotenoid-enriched oil phase (10 mL) was then incorporated and homogenized at 15,000 rpm for 5 min. Cold-set gelation was induced by adding CaCl₂ to obtain final concentrations of 0, 5, 10, 15, and 20 mM, followed by refrigerated maturation. After 24 h, the structured emulsions were characterized by TPA to evaluate the influence of Ca²⁺ on emulsion gelation and potential suitability for extrusion-based 3D printing. The dry extract showed a total carotenoid content of 831.1 µg g⁻¹ extract, corresponding to an extraction yield of 148.2 µg g⁻¹ biomass. Regarding the TPA results, emulsions containing 0, 5, 10, 15, and 20 mM CaCl₂ exhibited compression work values of 32.73, 28.62, 31.81, 40.64, and 56.73 g s, respectively. The increase at 15–20 mM CaCl₂ suggests enhanced Ca²⁺-induced structuring of the whey protein network, which could be associated with electrostatic screening and ionic interactions involving negatively charged carboxylate groups. The higher resistance to deformation at these concentrations could favor shape retention after deposition during extrusion-based 3D printing. Springiness indices were 0.690, 0.801, 0.739, 0.748, and 0.844, respectively. Indeed, all CaCl₂-containing emulsions showed higher values than the control, with the maximum at 20 mM. This behavior can indicate greater structural recovery after extrusion. Adhesiveness values were 0, 0, 0, −6.93, and −22.38 g s, respectively, suggesting increased adhesive character at the highest CaCl₂ concentrations. In fact, greater adhesiveness could favor interactions between deposited layers. Therefore, Ca²⁺-induced structuring improved the gelation properties of the carotenoid-loaded whey protein emulsions, supporting their potential suitability for extrusion-based 3D printing.

Acknowledgments: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul.