U.S. Food & Drug Administration
Center for Food Safety & Applied Nutrition
APPLE CIDER FOOD SAFETY
CONTROL WORKSHOP
July 15-16, 1999

Research on Decontamination of Apples by Washing with Detergents and Sanitizing Agents

Gerald M. Sapers, Ph.D.

Eastern Regional Research Center, Agricultural Research Service

U.S. Department of Agriculture

600 E. Mermaid Lane, Wyndmoor, PA 19038

gsapers@arserrc.gov

Introduction (S-1)
1. Overview of research on microbiological safety of fruits and vegetables at Eastern Regional Research Center (S-2).
2. Research objectives (S-3).
  1. Compare effectiveness of conventional and experimental washing/sanitizing agents.
  2. Determine efficacy of promising washing treatments applied in commercial brush washer.
  3. Identify factors limiting efficacy of washing.
Comparison of Commercial and Experimental Washing Agents in Laboratory Studies

Methodology (S-4)

Apples inoculated by immersion in suspension of non-pathogenic E. coli to give 10,000-100,000 CFU/g.

Inoculated apples washed by immersion in solution of cleaning or sanitizing agent at 20° or 50°C with agitation for 1 min (S-5).

200 ppm chlorine as sodium hypochlorite (pH 6.5).

Acidic and alkaline detergent formulations.

Trisodium phosphate.

Peroxyacetic acid formulations.

Washed apples and inoculated controls homogenized and plated on BHIA for enumeration of surviving bacteria.

Effectiveness of commercial washing and sanitizing agents (S-6).

Commercial washing agents and chlorine (sodium hypochlorite, at pH 6.5) achieve 1-2 log reduction (90-99%).

Small improvement (less than 1 log) if solutions applied at 50°C.

Efficacy of hydrogen peroxide in decontaminating apples (S-7).

5% hydrogen peroxide at 50°C superior to conventional agents.

Combinations of hydrogen peroxide and conventional agents can achieve 3-4 log reductions (99.9-99.99%) on inoculated apple halves.

Population reductions of approx. 3 logs (99.9%) can be obtained with whole apples.

Washing Trials in a Commercial Cider Mill (Placerville, California)

Methodology (S-9)

Apples inoculated on day before trials.

Apples held 15 min in dump tank, then washed in flatbed brush washer, ground, and pressed.

Most promising washing formulations compared.

Bacterial population on apples and in cider determined.

Efficacy of washing treatments (S-10).

No reduction in dump tank.

None of washing treatments achieved even 1 log reduction (90%) in bacterial population.

Cross-contamination.

No cross contamination in dump tank.

Major cross contamination in hammermill or press.

Factors Limiting Efficacy of Washing (laboratory studies)

E. coli attaches to apple surface within 24 hr and cannot be rinsed of with water (S-11).

E. coli in inaccessible stem and calyx regions survives wash (S-12).

E. coli in skin punctures can grow in puncture and survive wash (S-13).

E. coli in contaminated water (drench water, dump tank, flume?) might infiltrate through calyx into apple core under suitable conditions (S-14-Photo).

New Approaches (S-15)

Targeted scrubbing/pressure washing, sonication.

Targeted abrasion, peeling/coring.

Surface pasteurization.

Combination treatments (hurdle principle) -- novel agents.

Defect detection and sorting.

Conclusions (S-16)

Commercial washing formulations tested and 200 ppm Cl₂ (pH 6.5) cannot reduce bacterial population on apples by more than 1-2 logs (90-99%) when apples are washed in laboratory by immersion in solution.

Hydrogen peroxide solutions can reduce bacterial population on apples by 3 logs (99.9%) when apples are washed in laboratory by immersion.

Washing apples in a flatbed washer will not reduce bacterial population on apples, even with effective anti-microbial agents.

Efficacy of population reduction by washing may be limited by bacterial adhesion to apple surface, attachment in inaccessible areas of apple (calyx and core), presence in punctures, and infiltration within apple core.

Code	Composition tested	Concentration (%)	pH
A	Acid anionic surfactant	1	2.4
B	Acid soap	5	3.4
C	Phosphoric acid + surfactant	1	2.1
D	Phosphoric acid + surfactant	1-2	1.7-1.8
E	Phosphoric acid + surfactant	1.6	1.9
F	Citric acid + surfactant	3.2	2.3
G	NaOH + surfactant	0.66-1	11.9-12.2
H	Trisodium phosphate	1-8	11.8-12.4
I	Surfactant	1	9.3
J	Peracetic acid + H₂O₂ + acetic acid	0.01-0.1	3.3-3.9

Composition of Sanitizing Agent in Wash^(b)	Concn Tested	pH	Log₁₀ Reduction
Cl₂ (pH 6.5)	200 ppm	6.5	2.07±0.31
Surfactant	1%	9.3	0.98±0.07
Phosphoric acid + surfactant	1%	1.7	1.90±0.11
Phosphoric acid + surfactant at 50°C	1%	1.7	2.61±0.11
Trisodium phosphate	4%	12.4	2.36±0.08
Trisodium phosphate at 50°C	4%	12.4	2.45±0.08
Peracetic acid + H₂O₂ + acetic acid	1000 ppm	3.3	2.05±0.48
Peracetic acid + H₂O₂ + acetic acid at 50°C	1000 ppm	3.3	2.58±0.22
^(a)Apples halves immersed 5 min in E. coli inoculum containing 2.0 x 10⁷ cfu/mL.
^(b)Washed for 1 min.

Washing Treatment^(b)	Log₁₀Reduction
200 ppm Cl₂ (pH 6.5)	2.01±0.17
5% H₂O₂	3.39±0.39
5% H₂O₂ at 50°C	3.82±0.82
5% H₂O₂ + 1% Surfactant	3.22±0.20
5% H₂O₂ + 1% H₃PO₄/surfactant	3.27±0.21
5% H₂O₂ + 1% H₃PO₄/surfactant at 50°C	4.20±0.56
5% H₂O₂ + 2% Trisodium phosphate	3.27±0.29
5% H₂O₂ + 2% Trisodium phosphate at 50°C	3.55±1.67^(c)
^(a)Apples halves immersed 5 min in E. coli inoculum containing 2.0 x 10⁷ cfu/mL.
^(b)Washed for 1 min.
^(c)Variable response due to decomposition of heated alkaline H₂O₂.

Treatment^(b)	n	Log₁₀ Reduction^(c)
5% H₂O₂ at 50°C	2	2.67±0.10
5% H₂O₂ + 1% Sanitizer C at 50°C	2	2.82±0.11
1% Sanitizer C at 50°C	2	1.53±0.33
^(a)For each treatment, 9 whole apples inoculated by immersion for 5 min in 3L diluted E. coli inoculum containing approx. 1.3 x 10⁷ CFU/mL.
^(b)1 min wash.
^(c)Based on log₁₀(CFU/g) of corresponding inoculated controls (mean=4.16±0.17); data from BHIA plate counts.

Treatment^(b)	Before Dump Tank	After Dump Tank	After Brush Washer	In Cider^(c)	In Dump Tank Water^(c)
Water, 20C	5.49±0.09	4.92±0.37	4.81±0.26	3.83±0.15	0.00
Water, 50C	5.49±0.09	5.03±0.15	4.59±0.08	3.73±0.15	0.00
200 ppm Cl₂, 20C	5.87±0.07	5.45±0.05	5.64±0.23	4.30±0.10	0.00
Cross Contam.	0.69	0.00	0.50	3.07±0.03	--
5% H₂O₂, 22C	5.87±0.07	5.46±0.40	5.27±0.09	3.83±0.05	0.00
5% H₂O₂, 54C	5.87±0.07	5.54±0.31	5.49±0.10	4.30±0.60	0.00
^(a)Mean of 2 determinations ± SD.
^(b)200 ppm Cl₂ prepared from sodium hypochlorite adjusted to pH 6.4 with citric acid.
^(c)Log₁₀ CFU/mL.

	Log₁₀CFU/g^(a)
Time after inoculation (hr)	Inoculated control	After wash
0.5	4.35^(bc)	3.38^(d)(*)
24	4.80^(b)	4.33^(bc)(*)
48	4.06^(c)	4.65^(b)
72	4.18^(bc)	3.88^(cd)
^(a)Mean of duplicate trials.
^(b)-(d)Within the same column, means with no letter in common are significantly different (p<0.05) by Bonferroni LSD.
^{(*)Log₁₀CFU/g reduction significant at p<0.01) by ANOVA.}

	Log₁₀(CFU/cm²)^(a)
Location	Inoculated	Washed^(b)
Skin on wedges	4.77^(d)	2.05^(d)
Skin at calyx end of core	7.26^(c)	5.20^(c)
Skin on stem end of core	6.63^(c)	5.06^(c)
^(a)24 h after inoculation; based on calculated surface area of skin.
^(b)Washed 1 min in 5% H₂O₂ at 50°C.
^(c)-(d)Within the same column, means with no letter in common are significantly different (p<0.05) by Bonferroni LSD.

No. of punctures	Inoculum strength (log₁₀ CFU/mL)	Log₁₀ CFU/g^(a) Time after inoculation (hr)
0.5	24	48
4^(b)	7.24	4.85	6.03	ND^(d)
1^(c)	6.40	3.53	4.85	4.96
1	6.37	4.42	5.09	5.24
^(a)Based on weight of whole apple; mean of duplicate trials.
^(b)Four 1-cm deep punctures made with sterile nail on opposite sides of apple.
^(c)Single 1-cm deep puncture made with sterile nail 2-3 cm from stem.
^(d)ND=not determined.

S-1

Research on Decontamination of Apples by Washing with Detergents and Sanitizing Agents

Gerald M. Sapers

Eastern Regional Research Center
Agricultural Research Service
U.S. Department of Agriculture
600 E. Mermaid Lane, Wyndmoor, PA 19038

gsapers@arserrc.gov

S-2

Research on Microbiological Safety of Fruits and Vegetables at Eastern Regional Research Center, ARS, USDA

Environmental sources of microbial contamination
State of microbial contaminants on produce

Microbial ecology
Factors affecting resistance to anti-microbial treatments

Interventions to improve microbiological safety

Exclusion of contaminated produce

Decontamination by washing or other means

Suppression of bacterial growth

Commodities currently under investigation

Apples

Potatoes

Fresh-cut fruits and vegetables

Sprouts

S-3

Research Objectives

Compare effectiveness of conventional and experimental washing/sanitizing agents.

Determine efficacy of promising washing treatments applied in commercial brush washer.

Identify factors limiting efficacy of washing.

S-4
Methodology for Comparison of Commercial and Experimental Washing Agents

Apples -- unwaxed Golden Delicious: whole, punctured, or cut in half.

Inoculation - 5 min in suspension of E. coli ATCC 25922 (non-pathogenic) to give 10⁴-10⁵ CFU/g.

Washing - 1 min in washing solution at 20° or 50°C with agitation on shaker; then drained and rinsed.

Microbiological evaluation - composites (6-8 apples) homogenized, diluted, and plated on BHIA.

S-5
Characteristics of Commercial Sanitizing Washes for Apples

Code Composition tested Concentration
(%) pH

A Acid anionic surfactant 1 2.4

B Acid soap 5 3.4

C Phosphoric acid + surfactant 1 2.1

D Phosphoric acid + surfactant 1-2 1.7-1.8

E Phosphoric acid + surfactant 1.6 1.9

F Citric acid + surfactant 3.2 2.3

G NaOH + surfactant 0.66-1 11.9-12.2

H Trisodium phosphate 1-8 11.8-12.4

I Surfactant 1 9.3

J Peracetic acid + H₂O₂ + acetic acid 0.01-0.1 3.3-3.9

S-6
Effect of Commercial Sanitizing Agents on E. coli (ATCC 25922) in Inoculated Golden Delicious Apple Halves^(a)

Composition of
Sanitizing Agent in Wash^(b) Concn
Tested pH Log₁₀
Reduction

Cl₂ (pH 6.5) 200 ppm 6.5 2.07±0.31

Surfactant 1% 9.3 0.98±0.07

Phosphoric acid + surfactant 1% 1.7 1.90±0.11

Phosphoric acid + surfactant at 50°C 1% 1.7 2.61±0.11

Trisodium phosphate 4% 12.4 2.36±0.08

Trisodium phosphate at 50°C 4% 12.4 2.45±0.08

Peracetic acid + H₂O₂ + acetic acid 1000 ppm 3.3 2.05±0.48

Peracetic acid + H₂O₂ + acetic acid at 50°C 1000 ppm 3.3 2.58±0.22

^(a)Apples halves immersed 5 min in E. coli inoculum containing 2.0 x 10⁷ cfu/mL.

^(b)Washed for 1 min.

S-7

Efficacy of Washes Containing Hydrogen Peroxide and Commercial Sanitizing Agents For Decontamination of Golden Delicious Apple Halves Inoculated with E. coli (ATCC 25922) ^(a)

Washing Treatment^(b) Log₁₀Reduction

200 ppm Cl₂ (pH 6.5) 2.01±0.17

5% H₂O₂ 3.39±0.39

5% H₂O₂ at 50°C 3.82±0.82

5% H₂O₂ + 1% Surfactant 3.22±0.20

5% H₂O₂ + 1% H₃PO₄/surfactant 3.27±0.21

5% H₂O₂ + 1% H₃PO₄/surfactant at 50°C 4.20±0.56

5% H₂O₂ + 2% Trisodium phosphate 3.27±0.29

5% H₂O₂ + 2% Trisodium phosphate at 50°C 3.55±1.67^(c)

^(a)Apples halves immersed 5 min in E. coli inoculum containing 2.0 x 10⁷ cfu/mL.

^(b)Washed for 1 min.

^(c)Variable response due to decomposition of heated alkaline H₂O₂.

S-8

Efficacy of Washes Containing Hydrogen Peroxide and Commercial Sanitizing Agent in Decontaminating Whole Golden Delicious Apples Inoculated with E. coli (ATCC 25922)^(a)

Treatment^(b)
n Log₁₀ Reduction^(c)

5% H₂O₂ at 50°C 2 2.67±0.10

5% H₂O₂ + 1% Sanitizer C at 50°C 2 2.82±0.11

1% Sanitizer C at 50°C 2 1.53±0.33

^(a)For each treatment, 9 whole apples inoculated by immersion for 5 min in 3L
diluted E. coli inoculum containing approx. 1.3 x 10⁷ CFU/mL.

^(b)1 min wash.

^(c)Based on log₁₀(CFU/g) of corresponding inoculated controls
(mean=4.16±0.17); data from BHIA plate counts.

S-9

Methodology for Washing Trials in Placerville Cider Mill

Apples - unwaxed Golden Delicious (inoculated) and Fuji (not inoculated).

Inoculation - 20 lb portions of Golden Delicious apples immersed 5 min in suspension of E. coli K-12 (non-pathogenic) to give 10⁵ CFU/g. Apples held overnight at 10°C before washing.

Cider mill unit operations:

Dump tank - 40 lb Golden Delicious apples (inoculated) mixed with approx. 250 lb Fuji apples (not inoculated) in 350 gal water at 20°C for 15 min.

Brush washer - sprayed with wash solution at 20° or 50°C during 25 sec transit over brushes, then rinsed with water on exit conveyor.

Hammermill

Press

Cider collection tank

Microbiological evaluation - duplicate 6-apple samples (Golden Delicious and Fuji) obtained before and after inoculation, after dump tank, and after brush washer; samples homogenized in 1 gal blender. Samples of dump tank water and cider also obtained. Samples diluted and plated on BHIA containing streptomycin (20mg/L).

S-10

Decontamination of Apples Inoculated with E. coli (Strain K12) with Sanitizing Washes in a Flat-Bed Brush Washer
E. coli (log₁₀CFU/g)^(a)

Treatment^(b) Before Dump Tank After Dump Tank After Brush Washer In Cider^(c) In Dump
Tank Water^(c)

Water, 20C 5.49±0.09 4.92±0.37 4.81±0.26 3.83±0.15 0.00

Water, 50C 5.49±0.09 5.03±0.15 4.59±0.08 3.73±0.15 0.00

200 ppm Cl₂, 20C 5.87±0.07 5.45±0.05 5.64±0.23 4.30±0.10 0.00

Cross Contam. 0.69 0.00 0.50 3.07±0.03 --

5% H₂O₂, 22C 5.87±0.07 5.46±0.40 5.27±0.09 3.83±0.05 0.00

5% H₂O₂, 54C 5.87±0.07 5.54±0.31 5.49±0.10 4.30±0.60 0.00

^(a)Mean of 2 determinations ± SD.

^(b)200 ppm Cl₂ prepared from sodium hypochlorite adjusted to pH 6.4 with citric acid.

^(c)Log₁₀ CFU/mL.

S-11

Attachment of E. coli (ATCC 25922) to apple surfaces at 20°C

Log₁₀CFU/g^(a)

Time after
inoculation (hr) Inoculated
control After
wash

0.5 4.35^(bc) 3.38^(d)(*)

24 4.80^(b) 4.33^(bc)(*)

48 4.06^(c) 4.65^(b)

72 4.18^(bc) 3.88^(cd)

^(a)Mean of duplicate trials.

^(b)-(d)Within the same column, means with no letter in common
are significantly different (p<0.05) by Bonferroni LSD.

^{(*)Log₁₀CFU/g reduction significant at p<0.01) by ANOVA.}

S-12

Distribution of E. coli (ATCC 25922) on surface of inoculated apples before and after washing with 5% H₂O₂ at 50°C

Log₁₀(CFU/cm²)^(a)

Location Inoculated Washed^(b)

Skin on wedges 4.77^(d) 2.05^(d)

Skin at calyx end of core 7.26^(c) 5.20^(c)

Skin on stem end of core 6.63^(c) 5.06^(c)

^(a)24 h after inoculation; based on calculated surface area of skin.

^(b)Washed 1 min in 5% H₂O₂ at 50°C.

^(c)-(d)Within the same column, means with no letter in common
are significantly different (p<0.05) by Bonferroni LSD.

S-13

Growth of E. coli in Punctures on Inoculated Golden Delicious Apples

No. of
punctures Inoculum strength
(log₁₀ CFU/mL) Log₁₀ CFU/g^(a)
Time after inoculation (hr)

0.5 24 48

4^(b) 7.24 4.85 6.03 ND^(d)

1^(c) 6.40 3.53 4.85 4.96

1 6.37 4.42 5.09 5.24

^(a)Based on weight of whole apple; mean of duplicate trials.

^(b)Four 1-cm deep punctures made with sterile nail on opposite sides of apple.

^(c)Single 1-cm deep puncture made with sterile nail 2-3 cm from stem.

^(d)ND=not determined.

S-14

Photo not available at this time.

S-15

New Approaches to Produce Decontamination

Targeted scrubbing/pressure washing, sonication

Removal of contaminated surface by targeted abrasion, peeling, or coring

Surface pasteurization with hot water or steam

Combination treatments, novel anti-microbial agents, gas-phase treatments

Defect detection and sorting

S-16

Conclusions

Commercial washing formulations tested and 200 ppm Cl₂ (pH 6.5) cannot reduce bacterial population on apples by more than 1-2 logs (90-99%) when apples are washed in laboratory by immersion in solution.

Hydrogen peroxide solutions can reduce bacterial population on apples by 3 logs (99.9%) when apples are washed in laboratory by immersion.

Washing apples in a flatbed washer will not reduce bacterial population on apples, even with effective anti-microbial agents. This is probably due to short exposure and ineffective brushing.

Efficacy of population reduction by washing may be limited by bacterial adhesion to apple surface, attachment in inaccessible areas of apple (calyx and core), presence in punctures, and infiltration within apple core.

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Research on Decontamination of Apples by Washing with Detergents and Sanitizing Agents

Research on Decontamination of Apples by Washing with Detergents and Sanitizing Agents

Gerald M. Sapers

Eastern Regional Research Center
Agricultural Research Service
U.S. Department of Agriculture
600 E. Mermaid Lane, Wyndmoor, PA 19038

Research on Microbiological Safety of Fruits and Vegetables at Eastern Regional Research Center, ARS, USDA

Efficacy of Washes Containing Hydrogen Peroxide and Commercial Sanitizing Agents For Decontamination of Golden Delicious Apple Halves Inoculated with E. coli (ATCC 25922) ^(a)

Efficacy of Washes Containing Hydrogen Peroxide and Commercial Sanitizing Agent in Decontaminating Whole Golden Delicious Apples Inoculated with E. coli (ATCC 25922)^(a)

Methodology for Washing Trials in Placerville Cider Mill

Decontamination of Apples Inoculated with E. coli (Strain K12) with Sanitizing Washes in a Flat-Bed Brush Washer

Attachment of E. coli (ATCC 25922) to apple surfaces at 20°C

Distribution of E. coli (ATCC 25922) on surface of inoculated apples before and after washing with 5% H₂O₂ at 50°C

Growth of E. coli in Punctures on Inoculated Golden Delicious Apples

New Approaches to Produce Decontamination

Conclusions

Research on Decontamination of Apples by Washing with Detergents and Sanitizing Agents

Research on Decontamination of Apples by Washing with Detergents and Sanitizing Agents

Gerald M. Sapers

Eastern Regional Research Center Agricultural Research Service U.S. Department of Agriculture 600 E. Mermaid Lane, Wyndmoor, PA 19038

Research on Microbiological Safety of Fruits and Vegetables at Eastern Regional Research Center, ARS, USDA

Efficacy of Washes Containing Hydrogen Peroxide and Commercial Sanitizing Agents For Decontamination of Golden Delicious Apple Halves Inoculated with E. coli (ATCC 25922) (a)

Efficacy of Washes Containing Hydrogen Peroxide and Commercial Sanitizing Agent in Decontaminating Whole Golden Delicious Apples Inoculated with E. coli (ATCC 25922)(a)

Methodology for Washing Trials in Placerville Cider Mill

Decontamination of Apples Inoculated with E. coli (Strain K12) with Sanitizing Washes in a Flat-Bed Brush Washer

Attachment of E. coli (ATCC 25922) to apple surfaces at 20°C

Distribution of E. coli (ATCC 25922) on surface of inoculated apples before and after washing with 5% H2O2 at 50°C

Growth of E. coli in Punctures on Inoculated Golden Delicious Apples

New Approaches to Produce Decontamination

Conclusions

Eastern Regional Research Center
Agricultural Research Service
U.S. Department of Agriculture
600 E. Mermaid Lane, Wyndmoor, PA 19038

Efficacy of Washes Containing Hydrogen Peroxide and Commercial Sanitizing Agents For Decontamination of Golden Delicious Apple Halves Inoculated with E. coli (ATCC 25922) ^(a)

Efficacy of Washes Containing Hydrogen Peroxide and Commercial Sanitizing Agent in Decontaminating Whole Golden Delicious Apples Inoculated with E. coli (ATCC 25922)^(a)

Distribution of E. coli (ATCC 25922) on surface of inoculated apples before and after washing with 5% H₂O₂ at 50°C