Implications of Sample Preparation Methods on the MALDI-TOF MS Identification of Spore-Forming Bacillus Species from Food Samples: A Closer Look at Bacillus licheniformis, Peribacillus simplex, Lysinibacillus fusiformis, Bacillus flexus, and Bacillus marisflavi

[Image: see text] This research underscores the criticality of tailored culture conditions and incubation periods for effective and accurate identification of spore-forming bacteria: Bacillus licheniformis, Peribacillus simplex, Lysinibacillus fusiformis, Bacillus flexus, and Bacillus marisflav, isolated from food samples, utilizing the MALDI-TOF MS technique. All isolated strains were confirmed as Gram-positive bacteria from diverse genera through 16S rDNA gene sequencing. To enhance the accuracy of the identification process, the study employed an optimization strategy involving a varied incubation time (ranging from 1 to 48 h) and two distinct sample preparation approaches—direct transfer facilitated by formic acid and protein extraction via ethanol. It was observed that matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) could successfully identify approximately 47% of the samples following a 24 h incubation period. The study emphasizes the critical role of sample preparation methods in enabling precise bacterial identification. Our findings reveal the necessity of tailoring the incubation time for each sample, as the optimum period for accurate identification fluctuated between 1 and 12 h. Further demonstrating the interplay between incubation time and spore quantity, our study used the Schaeffer–Fulton staining method to show that the lowest spore counts were detected between 5 and 8 h of incubation. This provides evidence that spore formation impacts bacterial identification. Our research thus deepens the understanding of spore-forming bacteria identification using MALDI-TOF MS and illuminates the various factors affecting the dependability and accuracy of this technique. Future research may explore additional variables, such as the effect of varying culture media, to further augment identification accuracy and gain a holistic understanding of spore-forming bacterial behavior in food samples. By enhancing our knowledge, these findings can substantially contribute to improving food safety and quality assurance strategies by enabling the more accurate and efficient identification of spore-forming bacteria in the food industry, thereby elevating the standards of food safety.