Matrix Matters: Context-Driven Metabolic Shifts in Bacillus cereus and Bacillus subtilis

Spore-forming Bacillus species, including pathogenic Bacillus cereus and spoilage-associated Bacillus subtilis, are major contributors to foodborne illness and product degradation. Understanding their metabolic behaviour in diverse food matrices is essential for improving risk assessment, spoilage prediction, and fermentation control. This study integrates isothermal microcalorimetry and targeted metabolomics to characterize the metabolic activity of B. cereus and B. subtilis in five nutrient sources: Brain Heart Infusion (BHI) medium, oat drink, milk, pea hydrolysate, and a combined oat-pea matrix. Metabolic heat production was monitored for 24 hours at 30 °C. In BHI, B. cereus exhibited a shorter lag phase (mean ± sd: 4.3 hours ± 0.8) than B. subtilis (7.9 hours ± 1.0) but produced less total heat. Across all food matrices, B. subtilis consistently generated more heat. The oat–pea matrix supported the highest growth rates, surpassing oat or pea alone, and showed sugar depletion and accumulation of organic acids, indicating enhanced carbohydrate metabolism. Free amino acid release was matrix– and species–specific: B. subtilis had increased levels in oat, while B. cereus did so in pea. While B. cereus was metabolically active in all matrices, cereulide levels were matrix-dependent: 47.3  ± 1.7 ng/mL in oat, 3.0  ± 0.1 ng/mL in oat–pea, and undetectable in pea. These findings reveal clade-specific and matrix–driven metabolic strategies. This is the first study to combine calorimetry and metabolomics to evaluate Bacillus activity in plant-based and dairy matrices. This approach enhances our understanding of microbial physiology in complex food systems and provides a foundation for developing targeted strategies to improve food safety, stability, and product design.