Efficacy of novel organic acid and hypochlorite treatments for eliminating Escherichia coli O157:H7 from alfalfa seeds prior to sprouting

Abstract

This study investigated novel two-step organic acid/hypochlorite treatments as alternatives to 20 000 ppm active chlorine (from calcium hypochlorite) for eliminating Escherichia coli O157:H7 from alfalfa seeds prior to sprouting. Commercially available alfalfa seeds were inoculated with a five-strain E. coli O157:H7 mixture and dried to attain ca. 10 6 CFU/g of seeds. Seeds then underwent one of several soak treatments including: (1) 5% (v/v) lactic acid for 10 min at 42°C; (2) 5% acetic acid (v/v) for 10 min at 42°C; (3) 2.5% lactic acid for 10 min at 42°C followed by 2000 ppm active chlorine (from calcium hypochlorite) for 15 min at 25°C; (4) 5% lactic acid for 10 min at 42°C followed by 2000 ppm active chlorine for 15 min at 25°C; or (5) 20 000 ppm active chlorine for 15 min at 25°C. Each treatment reduced numbers of inoculum cells by about 6.0 log 10 CFU/g as determined by plating on Sorbitol MacConkey agar (SMac). Plating on non-selective brain heart infusion agar (BHI) showed that treatments 1–4 reduced counts by 2.3–4.1 log 10 CFU/g, thus indicating a large proportion of injured cells. Successive lactic acid and hypochlorite treatments (3 and 4) were more lethal than either organic acid alone (1 and 2). No surviving cells were detected on SMac or BHI following treatment with 20 000 ppm active chlorine (treatment 5). Regardless of the previous treatment, E. coli O157:H7 counts increased to 10 7–10 8 CFU/g during sprouting. Germination of seeds was not adversely affected by any of the treatments (germination >90%). Results of this study show that: (a) non-lethal cell injury must be considered when evaluating intervention treatments against E. coli O157:H7 on alfalfa seeds; (b) reductions of 2–4 log 10 CFU/g can be attained without using 20 000 ppm active chlorine; (c) successive lactic acid and hypochlorite treatments have greater lethality than organic acid treatments alone; and (d) none of the treatments tested can prevent regrowth of surviving E. coli O157:H7 during sprouting.