Lactic acid

Individual inoculums of each pathogen type were added to 100 mL of TSB at a 1:1000 dilution in 500 mL flasks. Bacterial cultures were grown to early stationary phase without shaking at 37 °C. When the pre-growth condition was either 14 or 23 °C, then the cells were grown at those respective temperatures. Growth curves were generated for each strain at each condition to determine the optical density for each growth phase at 600 nm using a UV/V is spectrophotometer (Biospectrometer, Eppendorf™, Hamburg, Germany). The cells were then subjected to six different pre-growth conditions that were found relevant to food processing and/or fresh produce environment by following Harrand et al. (2019) with modifications: (i) Acid stress: The pH of growth media was adjusted to 5 to 5.5 using Lactic acid (Fisher™, Fair Lawn, NJ, USA); (ii) Salt stress: High salt environment was generated with additional 4% NaCl (w/v); (iii) Desiccation stress: water activity was adjusted using glycerol (Fisher™, Fair Lawn, NJ, USA) at 15.6% (v/v) and 13% (v/v) to achieve an a_w of 0.95 ± 0.01; and (iv). Temperature stress: The cells were grown at three different temperatures (14, 23, and 37 °C). All the cells in different stress conditions were grown until the cell density reached to 10⁶⁻⁷ log CFU/mL and confirmed by the optical density measurement.

The overnight culture of E. coli was 10-fold diluted in LB broth and was used for acid treatment. Acid treatment was carried out in LB containing 5.5% (vol/vol) of 90% lactic acid (Sigma-Aldrich, Oakville, ON, CA) with the pH adjusted to around 2.9, followed by sterilization using 0.02-μm filtration units (Nalgene, VWR, Edmonton, AB, CA). Aliquots of 100 μL of cell suspension in LB broth of each strain were added to 0.9 mL acidified LB broth and LB broth without the addition of lactic acid or adjustment of pH (control), followed by incubation in ice water for 1 h. At the end of the treatment, 100 μL of treated and control samples were transferred into 9.9 mL of phosphate-buffered saline solution (PBS) (Fisher Scientific, Ottawa, ON, Canada) to neutralize the solution and stop the treatment. After neutralization, the pH of the cell suspension in PBS ranged from 6.5 to 6.8 as measured by a pH meter (Fisherbrand accumet AP115, Fisher Scientific). Cell suspensions in PBS solution were serial-diluted in PBS, and then 1 mL of appropriate dilutions was plated onto Aerobic 3M Petrifilm Aerobic Count Plates (3M Corp., St. Paul, MN, USA). Plates were incubated at 35°C for 18 h. The colonies were then counted following the manufacturer’s instructions and regarded as surviving E. coli.