Milkfat influences thermal tolerance and biofilm formation of Salmonella Typhimurium during pasteurization

The bacterial contamination and failure of pasteurization are leading threats to the safety of dairy productions. Milk contains a large quantity of fat which supports bacterial growth but its influence on pasteurization efficacy and biofilm formation remains poorly understood. In this study, we first demonstrate that the inactivation of Salmonella during pasteurization is significantly less effective in whole milk compared to skim milk, with the D-value increasing from 0.60 min in skim milk to 0.67 min in whole milk ( P  = 0.0047). In whole milk, Salmonella biofilms form greater biomass and display a distinct architecture from biofilms in skim milk. Specifically, Salmonella in whole milk was embedded within the extracellular polymeric substances (EPS), while cells in skim milk biofilms were sparsely distributed. Raman spectroscopy showed that biofilms formed in whole milk possess a distinctly higher lipid content. Therefore, we hypothesized that the thermal tolerance of Salmonella in whole milk was directly linked to its physical interaction with milkfat. Confocal laser scanning microscopy showed an increase from 26.1 % to 86.1 % of the fraction of Salmonella co-localizing with milkfat after 12 h of incubation in whole milk. Lipase treatment (200 units/mL) on whole milk reduced biofilm formation and improved the efficacy of thermal inactivation. Our findings demonstrate the milkfat acts as a physical scaffold, facilitating bacterial aggregation and the formation of a protective, lipid-rich biofilm matrix enhancing Salmonella survival during pasteurization. These insights should inform pasteurization strategies of high-fat dairy products to ensure food safety.