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Multiple Hurdle Interventions Against E. coli O157:H7 in Apple Cider Steve Ingham University of Wisconsin-Madison Slide 2 Wisconsins Cider Safety Extension Team Teryl Roper - growers Steve Ingham - processors Barbara Ingham - consumers Slide 3 Apple Cider/E. coli O157:H7 Research Topics Lethality of freeze/thaw treatment Multiple hurdle interventions Other pathogens of concern Useful indicator organisms Slide 4 Lethality of Freeze/Thaw against E. coli O157:H7 in Apple Cider pH 3.5 cider, 10 ml/bottle, heat-sterilized two test strains -20 C, 24 h to freeze thaw at 4C, 23C, or 10 sec in microwave 0.69 - 3.43 log kill (TSA plating) 1.39 - 5.64 log kill (SMA plating) Slide 5 A New Intervention Paradigm Organic acids more lethal at warm temperatures Immediate refrigeration is NOT best for cider safety Short-term storage at 25 - 45C to kill pathogens Slide 6 Multiple Hurdle Protocol # 1 Heat-sterilized ciders; pH 3.3, 3.7, 4.1 7-log, cocktail inoculum enumeration of survivors by plating freeze/thaw sorbic acid = SA (0.1%) short-term storage at 4, 25, or 35C Slide 7 5-log kill of E. coli O157:H7 in pH 3.3 Cider 6h at 35C Freeze/thaw 4.0 ml, -20C for 48 h, then 4C for 4 h Slide 8 5-log kill of E. coli O157:H7 in pH 3.7 Cider 6 h at 4C + freeze/thaw 2 h at 25C + freeze/thaw 1 h at 35C + freeze/thaw 6 h at 35C 0.1% SA + 12 h at 25C Slide 9 5-log kill of E. coli O157:H7 in pH 4.1 Cider 6 h at 35C + freeze/thaw 0.1% SA + 12 h at 25C + freeze/thaw 0.1% SA + 4 h at 35C + freeze/thaw 0.1% SA + 6 h at 35C Slide 10 Taste Panels 6 h at 35C preferred over pasteurized freeze/thaw preferred over pasteurized 6 h at 35 C + freeze/thaw preferred over pasteurized Slide 11 Taste Panels 0.1% SA + 6 h at 35 C NOT preferred over pasteurized 0.1% SA + 6 h at 35 C + freeze/thaw NOT preferred over pasteurized Slide 12 Multiple Hurdle Protocol # 2 Irradiation-sterilized ciders (3), pH adjusted to 3.3, 3.5, 3.7, 3.9, 4.1 Potassium sorbate = SA (0.05, 0.1%) sodium benzoate = BA (0.05, 0.1%) Short-term storage at 5, 15, 25 35, 45C Freeze/thaw Growth/no growth broth assay for 5-log kill Slide 13 Protocol #2: 5-log Kill of E. coli O157:H7 pH 3.3 + 0.1% SA or BA + 6 h at 35C pH 3.3 - 3.7 + 0.1% SA or BA + 6 h at 45C pH 3.9 + 0.1% BA + 6 h at 45C pH 3.3 + 0.1% BA + 4 h at 25C + freeze/thaw pH 3.3 + 0.1% SA + 12 h at 25C + freeze/thaw Slide 14 Protocol #2: 5-log Kill of E. coli O157:H7 pH 3.3 + 0.1% SA + 4 h at 35C + freeze/thaw pH 3.3 + 0.1% BA + 2 h at 35C + freeze/thaw pH 3.3 + 0.05% SA or BA + 6 h at 35C + freeze/thaw pH 3.7 - 4.1 + 0.1% BA + 4 h at 45C + freeze/thaw Slide 15 Other Bacterial Pathogens of Possible Concern in Apple Cider Salmonella typhimurium DT104: intermediate survival in unpasteurized cider; will not survive treatments that are effective against E. coli O157:H7 Listeria monocytogenes: poor survival in unpasteurized cider; not an appropriate target pathogen Slide 16 Potential Indicator Organisms for Evaluating Sanitation Generic E. coli: survives well in refrigerated cider Coliforms: more prevalent, but problem with possible non-fecal origin Enterococcus spp.: survive poorly in refrigerated cider Slide 17 Testing for Indicator Organisms Drop apples more likely to contain generic E. coli; apple rinse testing may be useful Wash water can become a contamination step; testing here is appropriate Test cider quickly; neutralize or dilute it before testing Coliform kits vary widely in performance