Development of Probiotic Dark Chocolate Enriched with Encapsulated Saccharomyces boulardii: Storage Stability and In Vitro Gastrointestinal Survival

This study investigated the development of a sugar-free prebiotic and probiotic dark chocolate formulated with 39.76% cocoa mass, 41.856% cocoa butter, 14.8% inulin, and 1.6% stevioside. To enhance the stability of the probiotic yeast Saccharomyces boulardii, a microencapsulation technique using a cocoa powder, Na-alginate, and fructooligosaccharides (10:1:1 ratio) matrix was employed. The physicochemical properties and probiotic viability were monitored over a 120-day storage period at 25 °C. A significant increase in pH values was observed during storage (p < 0.05), while water activity (aw) values significantly decreased (p < 0.05). Encapsulation provided superior protection, with encapsulated samples retaining 2.51 log CFU/g more probiotics than unencapsulated samples after 120 days. Furthermore, in vitro gastrointestinal digestion assays conducted after one day of storage demonstrated the protective efficacy of the matrix; while unencapsulated probiotics suffered a drastic reduction of 6.42 log CFU/g under gastric conditions (pH 3, 2 h), the encapsulated probiotics showed a significantly lower reduction of only 3.99 log CFU/g. These results confirm that the Na-alginate/FOS-based encapsulation significantly improves the resilience of S. boulardii against both storage conditions and gastrointestinal stress, making this sugar-free prebiotic chocolate an effective delivery vehicle for probiotic yeasts.