Design and development of nanostructured multilayer Water-In-Oleogel-In-Water (W/O/W) emulsions supported by ultrasound and nanotechnology for optimizing protection, stability, and targeted release of probiotics in oleogel matrices to enhance therapeutic and biological performance

This study presents an advanced investigation into the formulation and stabilization of probiotic-loaded Water-In-Oleogel-In-Water (W/O/W) double emulsion gels (DEGs), employing Lactiplantibacillus plantarum subsp. plantarum and Limosilactobacillus reuteri encapsulated within the innermost aqueous phase. The research strategically integrates dual gelation mechanisms: whey protein crosslinked with calcium chloride to form a hydrogel network in the external aqueous phase and carnauba wax-induced oleogelation to solidify the intermediate oil phase, thus fabricating a robust multilayered emulsion matrix. Three gelation configurations-external aqueous phase gelled, oil phase gelled, and dual-phase gelled systems-were scrutinized for their effects on emulsion structural integrity and probiotic viability under thermally and mechanically stressful conditions (heat exposure at ۶۰ °C and ۷۲ °C for ۱ hour, freeze-thaw cycling) as well as during prolonged refrigerated storage (up to ۵۶ days). Quantitative microbiological analyses indicated that the DEG systems preserved probiotic viability above the critical threshold of ۶ log CFU/g, significantly outperforming free-cell controls, which exhibited substantial viability loss. Encapsulation within these DEGs dramatically mitigated the deleterious effects of simulated gastrointestinal digestion, reducing viable cell loss to approximately ۱ log CFU/g, compared to ۳-۳.۵ log CFU/g decline in free probiotics, attributable to protective barriers formed by the gelled hydrophilic and lipophilic phases. The oleogel network surrounding the inner aqueous compartment confers a fortified shield against environmental insults, such as pH fluctuations, enzymatic hydrolysis, and bile salt-induced membrane disruption. Emulsification and gelation parameters were optimized using ultrasound-assisted homogenization and nanostructuring techniques, enhancing droplet size homogeneity and interfacial film strength that prevent phase coalescence and creaming, thus ensuring colloidal stability and uniform probiotic distribution. These findings elucidate the synergistic protective effects of composite DEG matrices, emphasizing the pivotal role of phase-specific gelation in augmenting mechanical resilience and sustaining probiotic bioactivity during food processing and gastrointestinal transit. The methodological approach highlights the utility of integrating oleogelators with protein-based hydrogels to engineer advanced functional food delivery systems with enhanced therapeutic efficacy and targeted release profiles. This research offers a comprehensive framework for the rational design of probiotic carriers leveraging nanotechnology and physicochemical gelation, with implications for industrial-scale functional food formulation and tailored microbial therapeutics.

کلیدواژه ها:

Probiotics ، Water-In-Oleogel-In-Water (W/O/W) emulsions ، nanostructured emulsions ، oleogel ، ultrasound-assisted homogenization ، nanotechnology ، gelation ، whey protein hydrogel ، carnauba wax oleogelation ، double emulsion gels (DEGs)