Treatment of Alfalfa Sprout Seeds Contaminated with Escherichia coli O157:H7 Using Supercritical Carbon Dioxide and Peracetic Acid

Introduction: Recent sprout-implicated outbreaks, relative to their consumption levels, suggest that both sprout production and consumption present challenges for ensuring consumer safety.  Seeds are a source of contamination and 30°C and humid conditions required for germination are conducive to growth of low levels of pathogens.

Purpose: In an attempt to suggest a method for producing safe sprouts for human consumption, supercritical carbon dioxide (SC-CO₂) was applied in combination with a food grade sanitizer, peracetic acid (PAA), to alfalfa sprout seeds.  

Methods: Alfalfa seeds inoculated with a five strain cocktail of Escherichia coli O157:H7 were exposed to treatments of SC-CO₂ at 500, 630, 710, 740, and 780 kg/m³ with and without pre-soaking in 80 parts per million PAA.  Inoculum was recovered using TSA with ampicillin and confirmed with UV fluorescence of GFP.  Following treatments, percent germination was determined by incubating seeds at 30°C in the dark for 2 days, watering twice daily and counting the number of seeds with the appearance of at least 2 mm of visibly protruding radicle.  All experiments were run in biological triplicate. 

Results: SC-CO₂ treatment alone had less than 1 log reduction of E. coli O157:H7.  Similarly, treatment with 80 ppm PAA for 15 min achieved less than 1-log reduction.  The most effective treatment was the use of 80 ppm PAA for 5 min with SC-CO₂ at 780 kg/m³ for 15 min, achieving greater than 4-log CFU/g reduction. The most effective decontamination treatment above decreased percent germination of alfalfa seeds to less than 60%.

Significance: While the combined use of SC-CO₂ and surface sanitizer can result in significant log reduction of E. coli O157:H7 on alfalfa seeds, the recommended safety level of 5-log reduction is not met and the negative effect on the utility of the seed remains a limitation for practical implementation.