In vitro evaluation of Bacillus subtilis isolated from Apis mellifera for potential use as a probiotic feed additive...

The honeybee Apis mellifera plays a crucial role in global agriculture but faces significant threats from pathogens, pesticides, and nutritional stress in the beekeeping industry. In this study, we evaluate the potential application of Bacillus subtilis MBLB2796 isolated from the honeybee gut. Solid-state fermentation of soybean meal with MBLB2796 reduced allergenic proteins and anti-nutritional factors, enhancing its nutritional quality. Strain MBLB2796 also exhibited high hydrolytic enzyme activities (15.5 mm of protease, 13.5 mm of amylase, and 15.0 mm of cellulase), strong antimicrobial activity (20.5 mm) against Paenibacillus larvae, and antioxidant activity against DPPH and ABTS radicals (66.2% and 62.3%, respectively). Strain MBLB2796 demonstrated a strong survival rate after freeze-drying up to 94.4%, excellent stability during long-term storage, and superior acid and bile salt tolerance compared to commercial strains. Adhesion to Caco-2 cells was also comparable to the probiotic reference and commercial strain. Optimization of culture conditions using a one-factor-at-a-time approach identified 2.0% of yeast extract, 1.0% of galactose, and 0.25% of K₂HPO₄ as major medium components, increasing about 2.6-fold of biomass production in the flask cultivation system. Batch fermentation in a 5-L bioreactor achieved an 11.8 g/L DCW, which was a 5.1-fold increase in biomass yield compared to basal flask cultivation. These results suggest that the industrial potential of MBLB2796 as a probiotic starter for honeybee feed additive, offering both functional and economic benefits.