An improved DNA extraction method for detecting Bacillus subtilis spores in spiked foods and beverages

Bacillus and Paenibacillus spp. are essential aerobic spoilage bacteria in various food industry sectors. Spoilage from microorganisms occurs at many points throughout food production systems. Due to their complex wall structures, spores can resist heat, radiation, chemical agents, and enzymatic treatments. An alkaline lysis and mechanical disruption combination method was developed and evaluated to counter this. This combination method effectively improved DNA extraction from B. subtilis spore cells spiked into food (solid) and beverages (liquid milk and coffee) at concentrations down to 10² CFU/mL or g when spiked into food matrices and drinks. Released DNA recoveries were 27 % and 25 % for potato salad and 38 % and 36 % for whole corn spiked at 10⁶ and 10³ CFU/mL concentrations. Conversely, there was a low recovery for wheat flour (10 % and 8.8 %) and milk powders (12 % and 25 %) at 10⁶ and 10³ CFU/mL spiked concentrations. The combination method provides rapid, specific, reliable, and accurate signature sequences identification for the detection and presence confirmation of psychrophilic and psychrotolerant spoilage spore cells, improving food spoilage assessments and food control applications.