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Restaurants used to prepare tenderloin beef medallions using different frying parameters in order to satisfy their customers. However, some of those cooking methods are not in accordance with food safety recommendations, because they do not reach internal temperatures recognized as save, in countries such as US, Canada and Brazil. This study aimed to assess the microbial inactivation in tenderloin beef medallions fried to five pre-determined degrees of doneness. Two different frying techniques were investigated, allowing beef pieces to reach the following degrees of doneness: rare, medium-rare, medium, medium-well and well-done. Before thermal processing, tenderloin beef medallions were artificially contaminated with approximately 106 CFU/g of Escherichia coli and the survival of this microorganism and mesophilic total counts were measured using VRBA + MUG and BHI plates, respectively. Bacterial colonies grew on BHI plates were also analyzed by Matrix-Assisted Laser and Time-Of-Flight desorption/ionization mass spectrometry (MALDI-TOF/MS) in order to identify survivors. Results indicated that complete E. coli inactivation was only observed on tenderloin beefs subjected to well-done degree established by both techniques, given that Technique 1 recommended a final heating inside oven after frying. Without this final oven processing, which is not always done in restaurants, more than 2 log CFU/g of viable E. coli were detected. Modeling of the kinetic parameters by GInaFIT revealed a D value (time needed to reduce 90% of the bacterial population) of 1.67 min for Technique 1 and D = 3.81 min for Technique 2 and this difference was associated to the difference of temperatures of olive oil. MALDI-TOF analyses demonstrated high diversity in natural microbiota of tenderloin beef pieces and among the main identified survivor genera was Citrobacter, Enterobacter, Hafnia, Serratia, Raoultella, Bacillus, Lactobacillus, Micrococcus, Pseudomonas, Serratia and Staphylococcus. Pathogens such as Salmonella and Listeria monocytogenes were not found. Based on the results, we recommend the use of high microbiological quality beef for the preparation of tenderloin beef medallions and the validation of frying process considering specific conditions of restaurants, because different degrees of fried may result in different counts and types of bacterial survivors. • E. coli was not observed only on well-done tenderloin beefs. • Temperature control was more effective than time control in reducing E. coli. • E. coli was not found when the center of medallions reached 70 °C. • Predicted D value of technique 1 was lower than D value of technique 2. • MALDI-TOF showed high diversity of survivors after all degrees of doneness. [ABSTRACT FROM AUTHOR]