1. Multiple Hurdle Interventions Against E. coli O157:H7 in Apple Cider Steve Ingham University of Wisconsin-Madison

2. Wisconsin’s Cider Safety Extension Team Teryl Roper - growers Steve Ingham - processors Barbara Ingham - consumers

3. Apple Cider/E. coli O157:H7 Research Topics Lethality of freeze/thaw treatment Multiple hurdle interventions Other pathogens of concern Useful indicator organisms

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 -20º C, 24 h to freeze thaw at 4ºC, 23ºC, or 10 sec in microwave 0.69 - 3.43 log kill (TSA plating) 1.39 - 5.64 log kill (SMA plating)

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 - 45ºC to kill pathogens

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 35ºC

7. 5-log kill of E. coli O157:H7 in pH 3.3 Cider 6h at 35ºC Freeze/thaw 4.0 ml, -20ºC for 48 h, then 4ºC for 4 h

8. 5-log kill of E. coli O157:H7 in pH 3.7 Cider 6 h at 4ºC + freeze/thaw 2 h at 25ºC + freeze/thaw 1 h at 35ºC + freeze/thaw 6 h at 35ºC 0.1% SA + 12 h at 25ºC

9. 5-log kill of E. coli O157:H7 in pH 4.1 Cider 6 h at 35ºC + freeze/thaw 0.1% SA + 12 h at 25ºC + freeze/thaw 0.1% SA + 4 h at 35ºC + freeze/thaw 0.1% SA + 6 h at 35ºC

10. Taste Panels 6 h at 35ºC preferred over pasteurized freeze/thaw preferred over pasteurized 6 h at 35 C + freeze/thaw preferred over pasteurized

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

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, 45ºC Freeze/thaw Growth/no growth broth assay for 5-log kill

13. Protocol #2: 5-log Kill of E. coli O157:H7 pH 3.3 + 0.1% SA or BA + 6 h at 35ºC pH 3.3 - 3.7 + 0.1% SA or BA + 6 h at 45ºC pH 3.9 + 0.1% BA + 6 h at 45ºC pH 3.3 + 0.1% BA + 4 h at 25ºC + freeze/thaw pH 3.3 + 0.1% SA + 12 h at 25ºC + freeze/thaw

14. Protocol #2: 5-log Kill of E. coli O157:H7 pH 3.3 + 0.1% SA + 4 h at 35ºC + freeze/thaw pH 3.3 + 0.1% BA + 2 h at 35ºC + freeze/thaw pH 3.3 + 0.05% SA or BA + 6 h at 35ºC + freeze/thaw pH 3.7 - 4.1 + 0.1% BA + 4 h at 45ºC + freeze/thaw

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

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

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