Probiotic raw-material dossier for Bacillus: Freeze-drying, cryoprotection, and compliance with pharmacopoeial vietnam standards

Tóm tắt

This study investigated the impact of cryoprotectants on the quality and stability of freeze-dried powders of Bacillus subtilis M70 and Bacillus clausii M31, in compliance with the Vietnamese Pharmacopoeia V standards. Biomass was cultured, harvested, and suspended in protective media containing single (10% w/w) or binary (5% + 5% w/w) cryoprotectants. Samples were pre-frozen at -80 °C and lyophilized using a programmed cycle (-50 °C primary drying; 0.055 bar). Quality attributes, including macroscopic properties, moisture content, morphology, and viable cell count (CFU/g), were assessed immediately and after six months of storage at 4-6 °C. Among single agents, 10% mannitol produced the most favorable results, yielding dry, porous powders with consistent flowability and maintaining viability above 10¹⁰ CFU/g for both strains. Specifically, B. subtilis retained 10.27 log CFU/g after six months. In contrast, the binary combination of 5% trehalose + 5% maltodextrin exhibited a synergistic and strain-specific effect on B. clausii, ensuring the highest survival rate (10.54 log CFU/g) and superior powder morphology. All optimized formulations produced stable powders with low residual moisture and porous structures, meeting pharmacopoeial requirements while preserving probiotic viability above the threshold of 10¹⁰ CFU/g. Statistical analysis confirmed significant differences (p < 0.05) between optimized formulations and controls. These findings highlight the importance of tailored cryoprotectant strategies for spore-forming probiotics. Mannitol was identified as the optimal protectant for B. subtilis, while trehalose–maltodextrin combinations offered superior protection for B. clausii. The results provide a practical basis for the development of stable probiotic raw materials suitable for pharmaceutical and functional food applications.