Research Findings on the Application of Warming and

FreezingImme W. Kersten, Dr. Sita R. Tatini,

Kaushik Subramanian

Increased research devoted to finding alternatives to pasteurization

isostatic high pressure

pulsed electric field

filtration

ozone

UV light

Freeze/thawing

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

Contradictory

generally thought of as a method of preservation

presence of sugar as a cryoprotective agent

Supportive

high acid, presence of preservative

Methods

Preparation of inoculated cider

Four strains of verotoxigenic Escherichia coli

OD (O157:H7)933(O157:H7)406(O22:H6)O104:H21

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

Test tube experiments (10ml)

test tubes inoculated with each strain (106 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

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

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

Survival of VTEC in Apple Juice of pH 3.4 at 4, 22, and 37°C

Kaushik Subramanian M.S. Thesis

Influence of pH of apple cider on survival of freeze-thawed VTEC OD during storage at 4°C

Kaushik Subramanian M.S. Thesis

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

933(O157:H7)

0

2

4

6

week

0

week

1

week

2

week

3

week

4

week

6

week

8

Weeks Frozen at -16.8C

Lo

g C

FU

/ml

TSAYE

Influence of frozen storage (16.8C), defrosting and holding at 4C on verotoxigenic Escherichia coli in

unpasteurized apple cider containing no preservative – test tubes

01234567

Log

CFU/

ml Sample A

Sample B

933 OD 406 O104

Influence of frozen storage (16.8C), defrosting and holding at 4C on survival of verotoxigenic

Escherichia coli in unpasteurized apple cider containing sodium benzoate – test tubes

01234567

Log

CFU/

ml Sample C

Sample D

933 OD 406 O104

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

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

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 Strain Length of time for 5 log destruction

E1 1 gallon 933/406 2 wks, 13 days1 gallon OD/O104 2 wks, 13 days

A 1 gallon 933/406 4 wks, 5 days1 gallon OD/O104 4 wks, 5 days1/2 gallon all strains 4 wks, 7 days1/2 gallon all strains 4 wks, 5 days

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

E2 1 gallon 933/406 9 days1 gallon OD/O104 9 days1/2 gallon all strains 9 days1/2 gallon all strains 14 days1/2 gallon all strains 14 days

C 1 gallon 933/406 3 days1 gallon 933/406 7 days1 gallon OD/O104 3 days1 gallon OD/O104 7 days

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 Strain Length of time @ 4°C for

5 log death

D 1 gallon 933/406 1 day1 gallon 933/406 1 day1 gallon OD/O104 1 day1 gallon OD/O104 1 day

C 1/2 gallon all strains 1 day1/2 gallon all strains 4 days

E2 1/2 gallon all strains 3 days

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

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

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

Freezing point of different apple cider?

Distribution of pectin and pulp in the test tubes?

• variation among orchards• may be protective

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.