CAN PULSED LIGHT MODULATE BOVINE MICELLAR CASEIN DIGESTION TOWARD HUMAN MILK-MIMICKING INFANT FORMULAS?

Breastfeeding is the primary recommendation for infant nutrition because it provides nutrients and bioactive compounds essential for immune and gastrointestinal development. When breastfeeding is not possible, infant formula is the most appropriate alternative; however, it does not fully reproduce the functional properties of human milk. Among its components, caseins are particularly relevant because they are high-biological-value proteins whose concentration, composition, and structural organization differ among species. Bovine caseins tend to form denser gastric curds in the immature stomach than human caseins, reducing protein accessibility to digestive enzymes and impairing digestion. This may result in suboptimal protein utilization and gastrointestinal disturbances in formula-fed infants, potentially affecting growth and development. In this context, emerging technologies have been investigated to modify casein structure and modulate its digestive behavior. Pulsed light (PL) stands out for inducing structural modifications in proteins through photochemical, photothermal, and photophysical effects. This study evaluated the effects of different PL intensities on the structural characteristics and digestive behavior of micellar casein (MC). MC was prepared in simulated milk ultrafiltrate at 0.4% (w/v), a concentration similar to that found in human milk, and subjected to 5, 10, or 20 PL pulses (5P, 10P, and 20P), alongside an untreated control (CT). Samples were evaluated in three independent experiments and subjected to in vitro digestion using the INFOGEST protocol adapted to infant physiological conditions. Proteolysis was monitored by Tricine-SDS-PAGE electrophoresis and the trichloroacetic acid (TCA) solubility index after protein precipitation with 10% TCA and Lowry quantification of the soluble fraction. Structural changes were evaluated by particle size distribution, and confocal microscopy. Experiments were performed in triplicate, and data were analyzed by ANOVA and Tukey’s test (p < 0.05). Electrophoresis revealed similar protein profiles among treatments and progressive proteolysis during digestion in all treatments. The TCA-soluble nitrogen index increased during digestion in all treatments, reflecting the progressive release of soluble low-molecular-weight nitrogenous compounds, with the highest values for 20P at all digestion stages. This result was accompanied by changes in particle size distribution: 10P and 20P exhibited more defined peaks at smaller size ranges, whereas CT and 5P showed broader profiles and a greater contribution of larger particles. Consistently, confocal microscopy indicated the formation of smaller gastric flocs in 20P. Overall, the highest PL intensity (20P) modified micellar casein organization, favoring a more dispersed structure and higher release of TCA-soluble nitrogenous compounds during digestion. Therefore, PL shows potential to modulate the structure and proteolytic behavior of casein, reducing the apparent formation of gastric flocs. These findings support PL as a promising strategy for developing human milk-mimicking infant formulas with improved digestive behavior.