EFFECT OF ULTRASOUND-ASSISTED ENZYMATIC MODIFICATION ON THE MACROSTRUCTURAL PROPERTIES OF GOAT'S MILK

Goat milk kefir, despite its nutritional benefits, has textural and stability limitations. This study investigated the impact of US-assisted enzymatic modification on the macrostructural (mean particle size (MPS), polydispersity index (PDI), zeta potential) and structural (FTIR and intrinsic fluorescence) properties of LC for kefir production. US-assisted enzymatic modification of LC was performed in an ultrasonic bath (25 kHz, 450 W) for periods of 15, 25, or 35 minutes, complementing the total enzyme activation time to 35 minutes in a conventional bath. As a control, a sample enzymatically modified with TG was maintained exclusively in a conventional bath. After the enzymatic process, the milk was fermented at 25°C to pH 4.6. Zeta potential, MPS, and PDI were measured at 25°C with a Zetasizer Nano ZS (Malvern). FTIR spectra were obtained in the range of 4000–500 cm⁻¹ using a Cary 630 (Agilent). Intrinsic fluorescence was analyzed in a SpectraMax M5 spectrophotometer (Molecular Devices) with excitation at 290 nm and emission between 300– 420 nm. Sonication promoted a significant reduction in MPS and PDI (up to 10.2% and 40.4%, respectively, (p < 0.05)), while the zeta potential showed an increase in negative charge (33.8%), suggesting greater electrostatic stability due to the exposure of ionizable groups. Intrinsic fluorescence spectra showed that sonication for 25 and 35 minutes caused a shift in the emission peak (from 293 to 296 nm). Furthermore, the fluorescence intensity increased significantly (up to 75.8% after 35 minutes), suggesting increased exposure of aromatic amino acids and conformational changes in the protein structure. FTIR spectroscopy revealed that US treatment promoted significant changes in the secondary structure of the modified milk. The amide I bands progressively shifted, indicating the involvement of amino and carboxyl groups in the formation of amide bonds catalyzed by TG, which favored intermolecular interactions and conformational rearrangement. With increasing sonication time, there was a reduction in β-sheet content, associated with loosening of the protein structure and greater exposure of peptide bonds, facilitating enzymatic action. At the same time, an increase in α-helix content was observed, reflecting greater structural stabilization and protein rigidity, factors that contribute to the overall stability of the system. These results demonstrate that the combination of US and TG was effective in modifying the macrostructural and structural properties of LC, being essential for improving the texture of LC-based fermented products.