Inactivation of Escherichia coli O157:H7 on inoculated alfalfa seeds with ozonated water and heat treatment

Alfalfa seeds inoculated with a five-strain mixture of Escherichia coli O157:H7 were immersed in water containing 4, 8, 10, and 21 ppm of ozone for 2, 4, 8, 16, 32, and 64 min at 4°C. Direct ozone sparging of seeds in water was used as an alternative mode of ozone treatment. Ozone-sparged seeds were also subsequently exposed to heat treatment at 40, 50, and 60°C for 3 h. Populations of E. coli O157:H7 on untreated and treated seeds were determined by spread plating diluted samples on tryptic soy agar supplemented with 50 μg/ml of nalidixic acid. Since E. coli O157:H7 was released from inoculated seeds during treatment with ozone, the rate of release of cells from inoculated seeds soaked in 0.1% peptone water for up to 64 min was also determined. The overall reduction of E. coli O157:H7 on seeds treated with ozonated water without continuous sparging ranged from 0.40 to 1.75 log₁₀ CFU/g (59.6 to 98.2%), whereas reductions for control seeds were 0.32 to 1.03 log₁₀ CFU/g (51.7 to 90.5%). Treatment with higher ozone concentrations enhanced inactivation, but contact time longer than 8 min did not result in significantly higher reductions (P > 0.05). For seeds treated by ozone sparging, a 1.12-log₁₀ CFU/g (92.1%) reduction was achieved using a 2-min contact time, and a 2.21-log₁₀ CFU/g (99.4%) reduction was achieved with a 64-min contact time. The corresponding reductions for control seeds were 0.71 log₁₀ CFU/g (79.5%) and 2.21 log₁₀ CFU/g (99.4%), respectively. Treatment of ozone-sparged seeds at 60°C for 3 h reduced the population to an undetectable level by direct plating (4 to 4.8 log₁₀ CFU/g), although survivors were detected by enrichment. Ozone did not have a detrimental effect on seed germination percentage.