Effectiveness of Calcium Hypochlorite on Bacterial and Viral Contamination of Alfalfa Seeds

Introduction: Alfalfa sprouts have been involved in numerous foodborne outbreaks increasing awareness for seed and sprout safety. FDA recommends soaking seeds in calcium hypochlorite (Ca(ClO)₂) solution before sprouting to reduce pathogens.  

Purpose: The objective of this study was to determine the effectiveness of Ca(ClO)₂ on inactivation of bacteria and viruses on alfalfa seeds. The efficacy of Ca(ClO)₂in the presence of a simulated organic load during disinfection was also considered.

Methods: Alfalfa seeds were inoculated with human norovirus (hNoV) GII, Tulane virus (TV), Escherichia coli O104:H4, and Salmonella Agona. Seeds were air-dried for 60 min and treated with Ca(ClO)₂ (2000 ppm or 20,000 ppm, pH 7.00) for 20 min at 22°C. Similar treatments were conducted in the presence of artificial organic material (10%, 30%, or 50% fetal bovine serum) in Hanks Balanced Salt Solution. Pathogens recovered from seeds were quantified by real-time RT-PCR, plaque assay, or bacterial enumeration. For each organism and Ca(ClO)₂concentration, three treated seed samples and one recovery/negative control were prepared for each of three trials. One seed sample was also included without virus inoculation for a neutralization/cytotoxicity control.

Results: Significant log reductions were observed in the order of hNoV < TV < Salmonella Agona < E. coli O104:H4 for both concentrations of Ca(ClO)₂; data ranged from 0.86 ± 0.18 to 3.37 ± 0.37 at 2000 ppm, and from 1.66 ± 0.41 to 5.47 ± 0.33 at 20,000 ppm. Ca(ClO)₂ at 20,000 ppm was more effective than 2000 ppm for all the organisms tested. This trend was also observed in samples containing an artificial organic material load. Ca(ClO)₂ activity on virus inactivation substantially decreased as the organic load increased, especially at FBS levels ≥ 30%; however, little difference were observed on bacteria, as Ca(ClO)₂inactivated all the bacteria completely in the presence of organic loads. Reduction was greater in FBS-containing samples compared to alfalfa seeds, indicating a close relationship between the organisms and alfalfa seeds.

Significance: Results obtained in this research reveal more significant inactivation of bacteria rather than viruses on alfalfa seeds by calcium hypochlorite; indicating the potential risk of virus transmission via sprouted seeds.