Evaluation of Microbial Safety of Dairies using Bacterial Proteomic Profiling <em>via</em> MALDI Approach

We present a protocol using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, hereinafter referred to as "MS") identification method for monitoring the presence of microorganisms in different dairy products (butter, mozzarella cheese, milk, cream) using a proteomic approach, modern culturomics, and various sample preparation procedures that could be incorporated to the food safety quality control laboratories as a fast, reliable, and robust tool for monitoring microbial safety. It includes a microbial isolation step, picking grown colonies, sample preparation procedures (intact cells, on-spot and in-tube extraction), setting MS analysis parameters, generating and collecting MS spectra (protein fingerprints), and finally getting taxonomy using the MS platform, followed by data interpretation and visualization. This method, validated by numerous studies, offers a more reliable and cost-effective identification of microorganisms from milk compared to commercial biochemical test systems. It stands out as a promising advancement in food microbiology, meeting the diverse requirements of laboratories through its simple protocols and significantly reduced analysis time. The presented protocol addresses the needs of the food industry by providing reliable microbial identification in a proper turnaround time (TAT) that is well-suited to manufacturing processes.