Levulinic acid plus SDS treatment to inactivate E. coli O157:H7 and Salmonella on Plant Seeds

Summary

Studies were done to determine the best concentration and exposure time for treatment of the alfalfa seeds with levulinic acid plus sodium dodecyl sulfate (SDS) to inactivate E. coli O157:H7 and Salmonella and not adversely affect seed germination. Alfalfa seeds inoculated with a 5-strain mixture of E. coli O157:H7 or S. Typhimurium were dried in a laminar flow hood at 21°C for up to 72 h. Inoculated alfalfa seeds were dried for 4 hours then treated for 5 min at 21°C with 0.5% levulinic acid and 0.05% SDS reduced the population of E. coli O157:H7 and S. Typhimurium by 5.6 and 6.4 log CFU/g, respectively. Treatment of 72-h dried seeds with 0.5% levulinic acid and 0.05% SDS for 20 min at 21ºC reduced E. coli O157:H7 and S. Typhimurium populations by 4 log CFU/g. Germination rates of alfalfa seeds treated with 0.5% levulinic acid plus 0.05% SDS for up to 1 h at 21°C were compared with a treatment of 20,000 ppm calcium hypochlorite or tap water only. Best germination rates were for seeds treated with levulinic acid plus SDS or distilled water. Results revealed that treating alfalfa seeds with 0.5% levulinic acid plus 0.05% SDS for 5 min at 21°C inactivated >3.0 log E. coli O157:H7 and Salmonella /g.

Situation

Alfalfa sprouts have for more than decade been associated with outbreaks of foodborne illness (13). Salmonellosis was associated with consumption of alfalfa sprouts in two large outbreaks in 1994; 282 cases were reported in Sweden and 210 cases were documented in Finland (9). Salmonella Bovismorbificans was the causative agent and the vehicle was Australian alfalfa seeds which did receive an antimicrobial treatment of 0.5% NaOCl for 45 min. S. Bovismorbificans was isolated from germinated sprouts but not from seeds. Since 1995, raw and lightly cooked sprouts have been implicated in outbreaks of foodborne illness in the United States (5). Most involved alfalfa sprouts, but cress, mung bean, and clover sprouts were also implicated. Thirteen Salmonella serotypes were isolated from the clinical cases (10). Examples include S. Newport associated with alfalfa sprouts in an outbreak in Oregon and British Columbia in 1995 (8), S. Stanley from alfalfa sprouts in Finland and the United States (5), and S. Senftenberg from an alfalfa and clover sprout mixture in northern California. Most recently a S. Saintpaul outbreak, initially among Nebraska residents with 228 cases in 13 states in 2009, was associated with eating alfalfa sprouts produced at multiple facilities from seeds that likely originated from a common grower (13). Seeds were considered the principal source of contamination for these outbreaks. In 1999, the U.S. Food and Drug Administration recommended (1) that sprout growers sanitize seeds with 20,000 ppm calcium hypochlorite. Studies of alfalfa seed coats have revealed that seed coats can be wrinkled or cracked and bacteria can become trapped under the coat during processing (11). When bacteria are entrapped under the seed coat, sanitizers cannot contact the bacteria to inactivate them. A variety of interventions, including heating and chemical treatments (e.g., NaOCl, Ca(OCl)2, acidified NaCIO2, acidified ClO2, Na3PO4, peroxyacetic acid, Tsunami™, Vortexx™, and H2O2), have been evaluated for their efficacy for reducing of E. coli O157:H7 and Salmonella contamination of alfalfa seeds (18). Results revealed that none of the above chemical treatments was able to eliminate E. coli O157:H7 and Salmonella contamination of alfalfa seeds and sprouts, although use of 1% peroxyacetic acid had promising results in reducing bacterial pathogens on seeds (12).

Response

Our previous studies revealed that a combination of levulinic acid plus SDS could effectively inactivate a variety of different bacterial pathogens within a short period of time while retaining the quality of the treated foods (21). The purpose of this study was to determine the antimicrobial activity of a combination of 0.5% levulinic acid plus 0.05% sodium dodecyl sulfate (SDS) at 21° C to reduce E. coli O157:H7 and Salmonella by >3 log CFU/g of alfalfa seeds without adversely affecting seed germination.

Impact

Results revealed that the population of E. coli O157:H7 on alfalfa seeds after inoculation and drying in a laminar flow hood for 4 h was consistently at 108 CFU/g for PBS-treated seeds. Seeds treated with 20,000 ppm calcium hypochlorite or 0.5% levulinic acid plus 0.05% SDS for up to 60 min reduced E. coli O157:H7 populations by greater than 6 and 5 log CFU/g, respectively. The population of E. coli O157:H7 was reduced by ca. 3 log CFU/g during drying for 24 h. Treatment with calcium hypochlorite or 0.5% levulinic acid plus 0.05% SDS for 5 min inactivated most E. coli O157:H7, with the pathogen only detectable on seeds by selective enrichment culture. Similar results, including survival and inactivation characteristics, were obtained with seeds dried for 48 or 72 h. Results revealed that the population of S. Typhimurium DT 104 on alfalfa seeds after drying in a laminar flow hood for 4 h was at 106 to 107 CFU/g for PBS-treated seeds. Both 20,000 ppm calcium hypochlorite and 0.5% levulinic acid plus 0.05% SDS reduced Salmonella on inoculated alfalfa seeds to levels only detectable by selective enrichment culture after 5 min. Drying inoculated Salmonella on alfalfa seeds for 24, 48, or 72 h decreased the population of Salmonella by ca. 2 log CFU/g. Both treatments of 20,000 ppm calcium hypochlorite and 0.5% levulinic acid plus 0.05% SDS on inoculated alfalfa seeds for 5 min reduced Salmonella to levels only detectable by selective enrichment culture. All samples of calcium hypochlorite and levulinic acid solutions used for treatment were negative for E. coli O157:H7 or Salmonella by enrichment culture after use. Inoculated seeds treated with 20,000 ppm calcium hypochlorite or 0.5% levulinic acid and 0.05% SDS in a glass beaker for 10 min then transferred with treatment solution to a stomacher bag and pummeled at 230 rpm for 10 min were negative for E. coli O157:H7 and Salmonella by the direct plating method and 8 of 10 treated samples with 0.5% levulinic acid and 0.05% SDS were E. coli O157:H7-positive by enrichment culture. Germination results revealed that treatment with 0.5% levulinic acid plus 0.05% SDS for 1 h at 21°C (80% germination rate) did not adversely affect alfalfa seed germination compared to the control treatment with tap water, whereas treatment with 20,000 ppm calcium hypochlorite for 1 h substantially reduced germination percentage (47% with sodium hypochlorite vs. 71% with tap water, P<0.05).

State Issue

Food Safety

Details

• Year: 2010
• Geographic Scope: International
• County: Spalding
• Program Areas:
  • Agriculture & Natural Resources