Host-Adapted Apilactobacillus kunkeei and Yeast Co-Fermentation Improves Fermented Bee Pollen Quality and Physiological Performance in Heterotrigona itama

Host-adapted probiotics offer a promising strategy for improving stingless bee nutrition and colony sustainability. In this study, gut-derived lactic acid bacteria (LAB) isolated from Heterotrigona itama were evaluated for probiotic potential and used to develop fermented bee pollen. Of 37 presumptive LAB isolates, three strains (BP-2, BP-3, and BPW-B1) exhibited strong tolerance to simulated gastrointestinal conditions, favorable adhesion-related properties, and acceptable safety profiles. Phylogenetic and biochemical analyses identified the selected isolates as Apilactobacillus kunkeei. The LAB strains were co-cultured with the osmophilic yeasts Zygosaccharomyces bailii TSU_YK2 and Starmerella meliponinorum TSU_YP10 to establish a host-associated LAB–yeast co-fermentation model that mimics stingless bee pollen fermentation. Co-fermentation significantly improved protein digestibility, organic acid production, antioxidant activity, and microbial viability relative to spontaneous fermentation controls (p < 0.05). Feeding experiments demonstrated that probiotic-fermented pollen increased feed intake, body weight, abdominal lipid reserves, hypopharyngeal gland development, and survival among H. itama workers. In addition, probiotic supplementation was associated with shifts in the dominant gut-associated bacterial taxa, including Lactobacillus, Bifidobacterium, and Snodgrassella. This study demonstrates the potential of combining gut-derived A. kunkeei with osmophilic yeasts as a functional fermentation starter culture to develop biologically relevant probiotic feed supplements for stingless bees.