Escherichia coli outbreaks linked to wheat flour consumption have kept emerging in recent years, which necessitated an antimicrobial step being incorporated into the flour production process. The objectives of this in vivo study were to holistically evaluate the sanitizing efficacy of thermal treatment at 60 and 70°C against the "big six" E. coli strains (O26:H11, O45:H2, O103:H11, O111, O121:H19, and O145) in wheat flour and to assess the strain-specific metabolic responses using nuclear magnetic resonance (NMR) spectroscopy. The 70°C treatment temperature indiscriminatingly inactivated all strains by over 4.3-log CFU/g within 20 min, suggesting the high sanitization effectiveness of this treatment temperature, whereas the treatment at 60°C inactivated the strains to various degrees during the 1-h process. The most resistant strains at 60°C, O26 and O45, were characterized by amino acid and sugar depletion, and their high resistance was attributed to the dual effects of activated heat shock protein (HSP) synthesis and promoted glycolysis. O121 also demonstrated these metabolic changes, yet its thermal resistance was largely impaired by the weakened membrane structure and diminished osmotic protection due to phosphorylcholine exhaustion. In contrast, O111, O145, and O103 presented a substantial elevation of metabolites after stress at 60°C; their moderate thermal resistance was mainly explained by the accumulation of amino acids as osmolytes. Overall, the study enhanced our understanding of the metabolic responses of big six E. coli to heat stress and provided a model for conducting NMR-based metabolomic studies in powdered food matrices. IMPORTANCE "Big six" Escherichia coli strains have caused several outbreaks linked to wheat flour consumption in the last decade, revealing the vital importance of adopting an antimicrobial treatment during the flour production process. Therefore, the present study was carried out to evaluate the efficacy of a typical sanitizing approach, thermal treatment, against the big six strains in wheat flour along with the underlying antimicrobial mechanisms. Findings showed that thermal treatment at 60 and 70°C could markedly mitigate the loads of all strains in wheat flour. Moreover, activated heat shock protein synthesis combined with expedited glycolysis and enhanced osmotic protection were identified as two major metabolic alteration patterns in the E. coli strains to cope with the heat stress. With the responses of big six in wheat flour to thermal treatment elucidated, scientific basis for incorporating a thermal inactivation step in wheat flour production was provided.
Keywords: Escherichia coli; NMR; flour; foodomics.