Multilayer co-encapsulation of probiotics and γ-amino butyric acid (GABA) using ultrasound for functional food applications

Double emulsion (W1/O/W2) microcapsules containing probiotic lactic acid bacteria (LAB) and bioactive γ-aminobutyric acid (GABA) were prepared by two-step ultrasonication using dextran and whey protein as encapsulating materials. The formulation and ultrasound parameters were refined to optimise the size of the W1/O droplets and outer microcapsules (5–15 μm) for encapsulation of both LAB and GABA. Optical, fluorescence and SEM microscopy revealed the morphology of the microcapsules and confirmed the presence of encapsulated LAB. Ultrasound treatment for up to 200 s had no significant effect on bacteria viability (>10⁹ CFU/mL), while GABA helped stabilise the primary (W1/O) emulsion and was used as an encapsulation marker. All microcapsules had strongly negative ζ-potentials (−30 mV) and were quite stable when stored at 4 °C, with GABA encapsulation remaining above 70% for 60 days. Importantly, the encapsulated bacteria were viable (10⁵–10⁷ CFU/mL) and remained entrapped after exposure to sequential digestion, whereas free bacteria died (0 CFU/mL). GABA was stable during sequential digestion regardless of encapsulation. GABA was released slowly from dextran capsules during sequential digestion (<15% release) as compared to whey protein capsules (~80% release). Ultrasonically produced microcapsules have promise for targeted intestinal delivery of probiotic bacteria and bioactive compounds in food formulations.