Gelation of different phases of probiotic-loaded water-in-oil-in-water emulsion to enhance probiotic survival stability

Abstract This study aimed to develop water-in-oil-in-water (W1/O/W2) double emulsion gels (DEGs) with Lactiplantibacillus plantarum subsp. plantarum and Limosilactobacillus reuteri encapsulated within the inner aqueous phase (W1), with a focus on improving probiotic viability under adverse environmental conditions. The system incorporated gelling agents to enhance emulsion stability, including whey protein crosslinked with calcium chloride to gel the W2 phase and carnauba wax as an oleogelator to solidify the O phase. Three formulations were investigated: W2-gelled, O-gelled, and dual-phase gelled systems. Our study demonstrated that designed DEGs loaded with probiotics effectively maintained cell count in a sufficient amount (more than 6 log CFU/g) during 56-day storage, heat treatment (at 60 °C and at 72 °C for 1 hr), and four freezing–thawing cycles compared to free cells. During simulated digestion, free probiotic cells exhibited substantial cell reduction, particularly after intestinal digestion, with cell loss ranging from 3.00 to 3.50 lg colony forming unit (CFU)/g. However, encapsulation within DEGs effectively enhanced probiotic survival, minimising cell reduction throughout digestion with cell loss around 1 lg CFU/g. These findings highlight the practical application of W/O/W phase-specific gelling agents to enhance structural integrity and probiotic survival. The DEG matrix outperforms traditional encapsulation, providing superior probiotic stabilisation under stressors.