1. Research Findings on the Application of Warming and Freezing Imme W. Kersten, Dr. Sita R. Tatini, Kaushik Subramanian

2. Increased research devoted to finding alternatives to pasteurization  isostatic high pressure  pulsed electric field  filtration  ozone  UV light  Freeze/thawing

3. Freeze/thawing as a viable method  little start-up capital compared to pasteurization  minimal to no nutritional loss  possibly no change in sensory characteristics i.e taste, smell, appearance

4. Contradictory  generally thought of as a method of preservation  presence of sugar as a cryoprotective agent Supportive  high acid, presence of preservative

5. Methods Preparation of inoculated cider Four strains of verotoxigenic Escherichia coli OD (O157:H7) 933(O157:H7) 406(O22:H6) O104:H21

6.  5 Minnesota Orchards  2 produce cider without sodium benzoate  pH range from 3.1 to 3.6 (early on in the season)  coliforms range from <10/ml to 50/ml  no E.coli detected Fresh, unpasteurizd, non-autoclaved cider

7. Test tube experiments (10ml)  test tubes inoculated with each strain (10 6 CFU/ml target level) frozen @ -16.8°C, removed on a weekly basis, thawed in a room temperature water bath, enumerated and held @ 4°C until gone

8.  test tubes subjected to 2 and 3 freeze/thaw cycles, then held @ 4°C until gone  test tubes frozen for 4 and 7 days, thawed, heated to 50°C

9. Large container experiments  similar experiment as with test tubes but with gallons and 1/2 gallons this time  also gallons and 1/2 gallons were inoculated with cells grown at a lower pH of 5.2

10. Survival of VTEC in Apple Juice of pH 3.4 at 4, 22, and 37°C Kaushik Subramanian M.S. Thesis

11. Influence of pH of apple cider on survival of freeze-thawed VTEC OD during storage at 4°C Kaushik Subramanian M.S. Thesis

12. Influence of frozen storage (-16.8ºC) at varying lengths of time on strain 933 in test tubes

13. Influence of frozen storage (  16.8  C), defrosting and holding at 4  C on verotoxigenic Escherichia coli in unpasteurized apple cider containing no preservative – test tubes 933OD406O104

14. Influence of frozen storage (  16.8  C), defrosting and holding at 4  C on survival of verotoxigenic Escherichia coli in unpasteurized apple cider containing sodium benzoate – test tubes 933OD406O104

15. Applying heat (50°C) to cider which had been frozen  freeze 7 days and then heat almost 5 log with sodium benzoate 4 log without preservative  hold for 1 or 2 days  raise temperature to 55°C

16. Use of 3 freeze/thaw cycles  with benzoate, 2 days holding = 6 days total  without benzoate, 8 days holding = 12 days total Time is shortened but….  energy expense  cycle time changes with larger sizes

17. Influence of frozen storage (-16.8°C) followed by thawing and holding @ 4°C on destruction of verotoxigenic Escherichia coli in unpasteurized apple cider without preservative Source Size StrainLength of time for 5 log destruction E 1 1 gallon933/406 2 wks, 13 days 1 gallonOD/O104 2 wks, 13 days A1 gallon933/406 4 wks, 5 days 1 gallonOD/O104 4 wks, 5 days 1/2 gallonall strains 4 wks, 7 days 1/2 gallonall strains 4 wks, 5 days

18. Influence of 1 week frozen storage (-16.8°C) followed by thawing and holding @ 4°C on destruction of verotoxigenic Escherichia coli in unpasteurized apple cider with sodium benzoate Source Size Strain Length of time @ 4°C for 5 log destruction E 2 1 gallon933/4069 days 1 gallonOD/O1049 days 1/2 gallonall strains9 days 1/2 gallonall strains14 days 1/2 gallonall strains14 days C1 gallon933/4063 days 1 gallon933/4067 days 1 gallonOD/O1043 days 1 gallonOD/O1047 days

19. Influence of 1 week frozen storage (-16.8°C) followed by thawing and holding @ 4°C on destruction of verotoxigenic Escherichia coli in unpasteurized apple cider with sodium benzoate (cells grown at pH 5.2) Source Size StrainLength of time @ 4°C for 5 log death D1 gallon 933/4061 day 1 gallon 933/4061 day 1 gallon OD/O1041 day 1 gallon OD/O1041 day C1/2 gallon all strains1 day 1/2 gallon all strains4 days E 2 1/2 gallon all strains3 days

20. Conclusion  sodium benzoate contributes to death of injured cells  behavior of E.coli in test tubes versus larger containers is not alike  cause of variability is unknown

21. Possible Explanations  variation in test tube versus gallon/ 1/2 gallon slow freezing/slow defrosting thought of as most damaging gallons/ 1/2 gallons are slow/slow test tubes are fast/fast

22.  Distribution of liquid water to ice?  Size and number of crystals formed? high cooling rates = small internal ice crystals slower cooling rates = external ice crystals leading to dehydration

23.  Freezing point of different apple cider?  Distribution of pectin and pulp in the test tubes? variation among orchards may be protective

24. More research needs to be conducted in various container sizes, involve cider with different pH’s, from different times of the season and from orchards in diverse agricultural areas.