Nutrient-induced germination of Bacillus subtilis spores exhibiting shared metabolic profiles in both TSB and AGFK media

Abstract Introduction Bacillus subtilis is a spore-forming bacterium commonly implicated in food spoilage and foodborne illnesses due to its resistance to harsh conditions. Upon exposure to favorable environments, the spores germinate and resume metabolic activity and thus pose a risk to food safety. Detecting early germination stages is thus crucial for preventing contamination and subsequent illness outbreaks. Objectives This study evaluated the potential of nuclear magnetic resonance (NMR)-based metabolomics to identify significant metabolites released during Bacillus subtilis OSU 494 spore germination using two nutrient germinants. The goal was to uncover biomarkers that could support improved detection strategies in food safety applications. Methods The spores were induced to germinate using either tryptic soy broth (TSB) or L-asparagine, D-glucose, D-fructose, and potassium chloride (AGFK). The samples were collected hourly over 4 h and analyzed using an 850 MHz NMR spectrometer with a triple-resonance cryoprobe. 1D-¹H NOESY and 2D ¹H–¹³C HSQC spectra were obtained. Spectral binning and linear modeling were then applied to identify significant metabolic features. Results The AGFK-induced germination yielded dipicolinic acid (DPA), L-alanine, acetic acid, L-phenylalanine, and formic, succinic, and fumaric acids. The TSB-induced germination produced DPA, L-alanine, L-phenylalanine, acetic and fumaric acids. Conclusion Several metabolites were consistently released during germination in both nutrient conditions. These metabolites, particularly DPA and L-alanine, served as reliable biomarkers for the Bacillus subtilis spore germination. They then provided valuable insights for developing rapid detection tools to enhance food safety monitoring and contamination control.