lable at ScienceDirect

Food Microbiology 27 (2010) 210–219

Contents lists avai

Food Microbiology

journal homepage: www.elsevier .com/locate/ fm

Alternative disinfection techniques to extend the shelf life of minimallyprocessed iceberg lettuce

Angeline Gopal a, John Coventry b, Jason Wan b, Hubert Roginski c, Said Ajlouni c,*

a Product Design Manager, Fonterra Brands Innovation, Fonterra Australia Corporate Centre. 327 FernTree Gully Road, Mt Waverley, Victoria 3149, Australiab Food Science Australia, Private Bag 16, Werribee, Victoria, Australiac Department of Food Science and Agribusiness, Melbourne School of Land and Environment, Parkville, Vic, Australia

a r t i c l e i n f o

Article history:Received 16 June 2009Received in revised form5 October 2009Accepted 6 October 2009Available online 13 October 2009

Keywords:SilverHydrogen peroxideDisinfectionChlorinationTotal plate countPseudomonasEnterobactericeaeYeast and moulds

* Correspondence to: Said Ajlouni, Department of AMelbourne School of Land and Environment, The Unville, Vic 3010, Australia. Tel.: þ61 3 83448620; fax: þ

E-mail addresses: angeline.achariya@fonterra.comedu.au (S. Ajlouni).

0740-0020/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.fm.2009.10.006

a b s t r a c t

Chlorination continues to be widely used by the fresh-cut industry for washing and disinfecting mini-mally processed fruits and vegetables. However, as chlorination has been reported to produce unhealthyby-products such as, chloramines and trihalomethanes, many alternative methods have been reported inthe literature. This study examined the use of silver and hydrogen peroxide as possible alternative tochlorination. The results revealed an obvious bactericidal effect of hydrogen peroxide, silver and theircombination on spoilage organisms. Combination of electrochemically generated silver (5 ppm) andhydrogen peroxide (0.4 ppm) caused significant (P < 0.05) reduction in the total plate count (0.87 log),Pseudomonas (2.66 logs), Enterobactericeae (1.61 logs) and yeast and mould (1.60 logs) immediately afterwashing in comparison to water washed shredded lettuce. However, washing with chlorinated water(5 ppm) under same conditions revealed insignificant reduction in TPC (0.17 log), Pseudomonas (0.60 log),Enterobactericeae (0.15 log) or yeast and mould (0.81 log) counts. Results revealed also that both sourcesof silver (electrochemical silver and silver nitrate) have similar disinfecting effects, however, electro-chemical silver maintained the quality of washed lettuce.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Silver has been largely documented for its use as a therapeuticagent and for the sterilisation of potable water (Beveridge andDoyle, 1989; Russell and Hugo,1994). The therapeutic application ofsilver dates back to the early Arabs and Egyptians, and has also beenused in ancient Chinese and Indian Ayurvedic preparations(Schmid, 1957). Low concentrations of silver and copper-basedsystems (0.05–0.1 ppm) have been used for many years to disinfectdrinking water, swimming pools and cooling systems (Just andSzniolis, 1936; Landeen et al., 1989). Moreover, there has beenincreasing interest in the use of silver and copper-based systems forwater sanitation as an alternative to chlorine. The interest inchlorine alternatives has largely been due to recent reports sug-gesting possible production of carcinogenic by-products duringchlorination (Richardson et al., 1998).

griculture and Food Systems,iversity of Melbourne, Park-61 3 83445037.

(A. Gopal), said@unimelb.

All rights reserved.

Medicinal uses of silver for treatment and control of burnwounds, prevention of infections along with coating applicationson nylon fabrics, catheters, lozenges and breath mints are available(Modak and Fox, 1973; Ahearn et al., 1995). It is also used for ster-ilisation of recycled water on the MIR Space station and is beingmarketed as health food alternatives by various companies (Guptaand Silver, 1998). Various commercial silver systems are currentlyavailable for potable and pool water disinfection (Etris, 1997;Simons and Sanguansri, 1997). Other prospective applications ofsilver have been in postharvest cleaning of seafood, fruits andvegetables (Ajlouni, 2003).

The effectiveness of hydrogen peroxide as a disinfectant andantimicrobial agent has been also well established (Bayliss andWaites, 1980; Christensen et al., 1990; Olmez & Kretzschmar, 2009;Pietersen et al., 1996). Pedahzur et al. (1997) noticed an increase inthe bactericidal effect of hydrogen peroxide and silver on Escher-ichia coli when applied together, suggesting that such increment inthe bactericidal effect of these two disinfectants might be due to thesynergism between the two chemicals. Silver and hydrogenperoxide disinfectants have been approved for use as a drinkingwater disinfectant in Australia, Switzerland and Germany(Batterman et al., 2000).

A. Gopal et al. / Food Microbiology 27 (2010) 210–219 211

The proposed silver and hydrogen peroxide system was evalu-ated for its disinfection effects against total plate count (TPC),Ps. fluorescens, Enterobactericeae and yeast and moulds in freshlettuce. The enhanced bactericidal effect of silver in the presence ofhydrogen peroxide indicates a potential for its application for waterdisinfection, particularly in the food industry. The current system(silver:hydrogen peroxide) was evaluated for its possible applica-tion as an alternative treatment to chlorination for washing anddisinfecting minimally processed vegetables.

2. Materials and methods

2.1. Preparation of fresh-cut lettuce

Iceberg lettuce was purchased from a local vegetable grower(Werribee, Victoria) on the morning of each testing day. The outerleaves and core were removed using a knife and discarded. Theremaining leaves were then shredded into small pieces (1 cm wide)using the same knife. All vegetables were kept at 2–5 �C betweenthe time of purchase and sample preparation.

2.2. Preparation of disinfectants

Chemically prepared silver (from silver nitrate) and chlorine,chemically prepared silver and hydrogen peroxide, and electro-chemically generated silver and hydrogen peroxide were the keytreatments in sub-blot 1, 2, and 3, respectively. All hydrogenperoxide solutions were freshly prepared from a 30% hydrogenperoxide solution (Ajax Chemicals, Ajax Finechem Pty Ltd,Australia) at either 0.4 or 4 ppm final concentration using steriledeionised water. Silver solutions from silver nitrate (chemical silver,100% pure, Ajax Finechem Pty Ltd, Australia,) and electrochemicallygenerated silver were prepared following the method of (Gopal,2003). A special apparatus developed by Home Pure water treat-ment systems, AusTec Pty. Ltd, Australia, was used for electro-chemical silver generation (Fig. 1). Various concentrations ofchemical silver were prepared in the dark using sterile deionisedwater and stored in sterile polypropylene containers (Techno-Plas,South Australia). All silver solutions were tested for total silverusing silver DR 100 calorimeter (HACH, Loveland, USA) prior to useand checked against results of silver analysis conducted by StateChemistry Laboratories (DPI, Werribee. Vic). Chlorine solutions

Power-pack

Hydrogen

peroxide canister

Water in

Hydrogen

peroxide pump

control

Ampere

control

Water out

Silver

electrodes

Fig. 1. Electrochemical silver generation unit (AusTec Pty. Ltd, Australia).

were prepared using 10% sodium hypochlorite solution (AjaxChemicals). Total chlorine in the final solution for washing waschecked using an ion specific meter for total and free chlorinemeasurements (Hanna Instruments, Model HI 93711, USA).

2.3. Washing procedure for fresh-cut lettuce

Shredded lettuce was weighed and divided into 1 kg batches foreach. One batch was left as control (unwashed), while each of theremaining was subjected to one of various washing treatments(water, hydrogen peroxide, chemical silver solution, electrochemi-cally generated silver, chlorine and the combination of silver andhydrogen peroxide). Washing solutions (10 L) were prepared usingcold tap water (4 �C). Shredded lettuce was immersed into thetreatment solution for 2 min with a continual manual agitation(30–40 round/min). The washed product was then removed fromthe treatment solution using strainers, rinsed twice for 30 s using2.5 L of cold tap water and spin-dried manually for 1 min usinga vegetable spinner (90–100 RPM). The spin-dried shredded lettucewas then randomly packaged (30 g each) in permeable poly-propylene pouches (PD961, Cryovac, Australia) and heat-sealedusing a polythene sealer (Venus Packaging machines, Aust.). Allsamples were stored in a refrigerated incubator (Thermoline,Australia) at 12 �C for 7 days.

2.4. Visual examination of minimally processed lettuce

Triplicate samples were randomly selected from each batch onthe day of measurement. Samples were visually assed for changesin physical characteristics and appearance (colour, texture anddeterioration) before doing the microbial analysis.

2.5. Microbiological analysis

Triplicate samples of packaged lettuce were randomly selectedfrom the refrigerated incubator for microbiological analysis on day0, 2, 4 and 7 of storage. Samples (20 g portions) were stomached in180 mL of 0.1% bacteriological peptone (Oxoid, Australia), and spiralplated (WASP spiral plater, Don Whitely Scientific Limited, York-shire, England) into various media that were bought from Oxoid,Australia. Plate Count Agar (PCA), Pseudomonas agar with CFCsupplement, Voilet Red Bile Glucose Agar (VRBGA) and DichloranRose-Bengal Chloroamphenicol with Chloroamphenicol supple-ment (DRBC), and were used for enumeration of total viable count,presumptive Pseudomonas, Enterobactericeae and yeast andmoulds, respectively.

2.6. Quantitation of silver in washing waterand washed iceberg lettuce

Testing for the amount silver in washing water and residualsilver on lettuce samples was performed by State ChemistryLaboratories (DPI, Werribee. Vic) using ICP-MS (Varian Ultra MassICP mass spectrometer, Vic., Australia). Water samples were alsotested in our laboratory using the Silver DR 100 silver meter (HACH,Loveland, USA) for verification.

2.7. Statistical analysis

A spilt blot design was used in this study, and triplicate sampleswere used for each measurement within a blot. Data were statis-tically analysed using Statistical Package for Social Science (SPSS)software licensed through the University of Melbourne computernetwork. One way analysis of variance was conducted and the LSDwas estimated to separate the means at 95% confidence level. The

A. Gopal et al. / Food Microbiology 27 (2010) 210–219212

reported values represented the mean of six measurements,generated from two independent trials and three replicates withineach trial.

3. Results

3.1. Effect of silver (from silver nitrate) and chlorineon fresh-cut lettuce

A wide range of silver concentrations (0, 0.1, 0.5, 5, 50 and125 ppm) with comparable concentrations of chlorine were used inthis trial to determine the relationship between silver concentra-tion and its disinfection effects. Fresh-cut lettuce samples werewashed with tap water containing silver or chlorine at each of theseconcentrations. All samples were stored at 12 �C to mimic fresh-cutabuse temperatures, and examined for viable count, presumptivePseudomonas, yeast and moulds and Enterobactericeae over a 7-daystorage trial.

Washing shredded Iceberg lettuce with water containing silverwas more effective than washing with chlorinated water (Fig. 2).Silver at 50 ppm caused significant (P � 0.05) reduction in viablecount in the same day of washing. The effect of silver was moresevere at higher concentration (125 ppm) and caused 49% reduc-tion in viable counts compared to water washed samples. The samedata showed also that chlorine was not effective at all at compa-rable concentrations. The disinfection effect of silver was imme-diate and appeared on the same day of washing only.

Data in Fig. 3 revealed that silver at concentrations as low as0.5 ppm caused 0.7 log reduction in Pseudomonas counts. However,no significant effects of chlorine treatment were recorded even atconcentrations as high as 125 ppm, when compared to sampleswashed with tap water.

Enterobactericeae appeared to be very sensitive to silver even atlow concentrations. Data in Fig. 4 indicated that washing shreddedlettuce with water containing only 0.1 ppm silver caused a 50%reduction in Enterobactericeae counts on day zero of measurement.Similar results were also recorded using 0.5–125 ppm silver.Chlorine was less effective than silver. A high chlorine concentra-tion (125 ppm) showed insignificant (P > 0.05) bactericidal effect

0

1

2

3

4

5

6

7

8

9

10

unwashed waterwashed

0.1ppmsilver

0.1ppmchlorine

0.5ppmsilver

0.5ppmchlorin

Trea

Via

ble

c

ou

nt (

lo

g c

fu

/g

)

Fig. 2. Total viable count on shredded lettuce washed with tap water containing various con4 ( ) and 7 ( ) days. Values represent the mean of 6 measurements � standard deviation

against Enterobactericeae (0.46 log reduction after 4 days of storageat 12 �C) compared to water washed samples. However the effect ofsilver on Enterobactericeae was more severe at such high concen-tration (125 ppm) and revealed a significant reduction (P � 0.05) inEnterobactericeae counts (3.72 logs) after 4 days of storage at 12 �C(Fig. 4) in comparison to water washed samples.

Yeast and mould were very easily removed from shreddedlettuce by washing with plain tap water or with water containinglow concentration of silver or chlorine (0.1 ppm). However, silverwas more effective than chlorine at high concentration (125 ppm)and caused more than 5.9 logs reduction of yeasts after 7 days ofstorage at 12 �C in comparison to water washing (Fig. 5). Visualobservations of washed shredded lettuce indicated some dis-colouration with a brown tinge colour on tissue washed with silverfrom silver nitrate at concentrations higher than 50 ppm. However,washing with electrochemically generated silver at similarconcentrations had no discolouration effect, which may suggestthat such discolouration was due to the nitrate ions and not to thesilver itself.

3.2. Effect of silver (from silver nitrate) and hydrogenperoxide on fresh-cut lettuce

Unsimilar to the 1st trial, smaller concentrations (0.05 and0.1 ppm) of silver from silver nitrate along with hydrogen peroxide(0.4 and 4 ppm) and their combinations were used in theseexperiments. These two selected silver concentrations (0.05 and0.1 ppm) are approved and permitted levels for use in drinkingwater (Simons and Sanguansri, 1997).

The total viable count of shredded lettuce washed with watercontaining low concentrations of silver (0.05 and 0.1 ppm) in thepresence and absence of hydrogen peroxide (0.4 and 4 ppm) wasnot affected. Data in Fig. 6 showed only 0.5–1 log reduction in thetotal counts in shredded lettuce washed with water containing0.1 ppm silver and 4 ppm hydrogen peroxide after 7 days storage at12 �C. No significant differences (P > 0.05) in the total counts oflettuce treated with silver or silver and hydrogen peroxide weredetected. Similar observations were noted when the effect ofwashing treatments on Pseudomonas and Enterobactericeae were

e5ppm silver 5ppm

chlorine50ppmsilver

50ppmchlorine

125ppmsilver

125ppmchlorine

tments

centrations of chlorine or silver (from silver nitrate) and stored at 12 �C for 0 ( ), 2 ( ),.

0

1

2

3

4

5

6

7

8

9

10

unwashed waterwashed

0.1ppmsilver

0.1ppmchlorine

0.5ppmsilver

0.5ppmchlorine

5ppm silver 5ppmchlorine

50ppmsilver

50ppmchlorine

125ppmsilver

125ppmchlorine

Treatments

Ps

. flu

ore

sc

en

s (lo

g c

fu

/g

)

Fig. 3. Total Pseudomonas count on shredded lettuce washed with tap water containing various concentrations of chlorine or silver (from silver nitrate) and stored at 12 �C for 0 ( ),2 ( ), 4 ( ) and 7 ( ) days. Values represent the mean of 6 measurements � standard deviation.

A. Gopal et al. / Food Microbiology 27 (2010) 210–219 213

examined (Figs. 7 and 8). Less than 1 log reduction in Pseudomonasand about 1.5 log reduction in Enterobactericeae were detected inshredded lettuce washed with silver:hydrogen peroxide(0.1:0.4 ppm) and stored at 7 days at 12 �C (Figs. 7 and 8).

As shown previously (Fig. 5), yeast and mould appear to behighly sensitive not only to silver and chlorine, but also to allsilver and hydrogen peroxide washing treatments. Data in Fig. 9revealed 50% elimination of yeast and mould immediately afterwashing with silver:hydrogen peroxide at all ratios. However,yeasts recovered very quickly after 2 days of storage at 12 �C andmaintained high counts (7 log) throughout the storage period(Fig. 9).

0

1

2

3

4

5

6

7

8

9

unwashed waterwashed

0.1ppmsilver

0.1ppmchlorine

0.5ppmsilver

0.5ppmchlorine

Trea

En

tero

bactericeae (lo

g cfu

/g

)

Fig. 4. Total Enterobactericeae count on shredded lettuce washed with tap water containing0 ( ), 2 ( ), 4 ( ) and 7 ( ) days. Values represent the mean of 6 measurements � stand

3.3. Effect of electrochemically generated silver and hydrogenperoxide on fresh-cut lettuce

Based on results from the previous trials, and because low silverconcentration (0.1 ppm) did not show significant disinfectioneffects, this trial included a larger concentration (5 ppm) of elec-trochemically generated silver, hydrogen peroxide (0.4 ppm), sil-ver:hydrogen peroxide combination (5:0.4 ppm) and chlorine(5 ppm). Shredded lettuce samples were prepared, washed andstored following the same previously outlined procedures.Unwashed and water washed samples were also included ascontrols.

5ppm silver 5ppmchlorine

50ppmsilver

50ppmchlorine

125ppmsilver

125ppmchlorine

tments

various concentrations of chlorine or silver (from silver nitrate) and stored at 12 �C forard deviation.

0

1

2

3

4

5

6

7

8

9

unwashed waterwashed

0.1ppmsilver

0.1ppmchlorine

0.5ppmsilver

0.5ppmchlorine

5ppm silver 5ppmchlorine

50ppmsilver

50ppmchlorine

125ppmsilver

125ppmchlorine

Treatments

Ye

as

t (lo

g c

fu

/g

)

Fig. 5. Total Yeast count on shredded lettuce washed with tap water containing various concentrations of chlorine or silver (from silver nitrate) and stored at 12 �C for 0 ( ), 2 ( ),4 ( ) and 7 ( ) days. Values represent the mean of 6 measurements � standard deviation.

A. Gopal et al. / Food Microbiology 27 (2010) 210–219214

Data from these experiments clearly revealed that washingfresh-cut lettuce with water containing both silver (5 ppm) andhydrogen peroxide (0.4 ppm) was more effective than chlorinatedwater (5 ppm). Washing with silver and hydrogen peroxide solu-tion showed 0.87 and 1.21 log reduction in the total count after0 and 7 days of storage at 12 �C, respectively. While washing with

0

1

2

3

4

5

6

7

8

9

10

unwashed waterwashed

0.05ppmsilver

0.1ppmsilver

0.4ppmhydrogeperoxid

Treatme

Via

ble

c

ou

nt (lo

g c

fu

/g

)

Fig. 6. Total viable count on shredded lettuce washed with tap water containing low concenstorage at 12 �C for 0 ( ), 2 ( ), 4 ( ) and 7 ( ) days. Values represent the mean of 6 me

chlorinated water (5 ppm) under same conditions showed 0.28 logincrement on day zero and only 0.28 log reduction on day 7(Fig. 10). These results are different from those recorded frompseudomonas counts. Significant (P � 0.05) reduction in Pseudo-monas counts were observed immediately after treatment withsilver and hydrogen peroxide alone and in combination when

ne

4.0ppmhydrogenperoxide

0.05 +0.4ppm

0.05 +4.0ppm

0.1 +0.4ppm

0.1 +4.0ppm

nts

trations of silver (from silver nitrate), hydrogen peroxide and their combinations duringasurements � standard deviation.

0

1

2

3

4

5

6

7

8

9

10

unwashed waterwashed

0.05ppmsilver

0.1ppmsilver

0.4ppmhydrogenperoxide

4.0ppmhydrogenperoxide

0.05 +0.4ppm

0.05 +4.0ppm

0.1 +0.4ppm

0.1 +4.0ppm

Treatments

Ps

eu

do

mo

na

s (lo

g c

fu

/g

)

Fig. 7. Total Pseudomonas count on shredded lettuce washed with tap water containing low concentrations of silver (from silver nitrate), hydrogen peroxide and their combinationsduring storage at 12 �C for 0 ( ), 2 ( ), 4 ( ) and 7 ( ) days. Values represent the mean of 6 measurements � standard deviation.

A. Gopal et al. / Food Microbiology 27 (2010) 210–219 215

compared to chlorine and water washed samples. Pseudomonascounts immediately after washing with silver (5 ppm), hydrogenperoxide (0.4 ppm), silver:hydrogen peroxide (5:0.4 ppm) andchlorine (5 ppm) were 2.36, 2.22, 2.6 and 0.6 log cfu/g, respectivelyin comparison to the pseudomonas counts of 5.56 log cfu/g forsamples washed with water (Fig. 11). These results clearly indicated

0

1

2

3

4

5

6

7

8

9

10

unwashed waterwashed

0.05ppmsilver

0.1ppmsilver

0.4ppmhydrogenperoxide

Treat

En

tero

bacteriaceae (lo

g cfu

/g

)

Fig. 8. Total Enterobacteriaceae count on shredded lettuce washed with tap water contaicombinations during storage at 12 �C for 0 ( ), 2 ( ), 4 ( ) and 7 ( ) days. Values repres

that silver and silver:hydrogen peroxide treatments were moreeffective than chlorine against pseudomonas immediately afterwashing.

As observed in previous washing trials (Figs. 8 and 9), washingfresh-cut lettuce with water containing silver:hydrogen peroxide(5:0.4 ppm) has been proven again to have significant effects

4.0ppmhydrogenperoxide

0.05 +0.4ppm

0.05 +4.0ppm

0.1 +0.4ppm

0.1 +4.0ppm

ments

ning low concentrations of silver (from silver nitrate), hydrogen peroxide and theirent the mean of 6 measurements � standard deviation.

0

1

2

3

4

5

6

7

8

9

10

unwashed waterwashed

0.05ppmsilver

0.1ppmsilver

0.4ppmhydrogenperoxide

4.0ppmhydrogenperoxide

0.05 +0.4ppm

0.05 +4.0ppm

0.1 +0.4ppm

0.1 +4.0ppm

Treatments

Yeasts (lo

g cfu

/g

)

Fig. 9. Total Yeast count on shredded lettuce washed with tap water containing low concentrations of silver (from silver nitrate), hydrogen peroxide and their combinations duringstorage at 12 �C for 0 ( ), 2 ( ),4 ( ) and 7 ( ) days. Values represent the mean of 6 measurements � standard deviation.

A. Gopal et al. / Food Microbiology 27 (2010) 210–219216

against Enterobactericeae (Fig. 12) and yeasts and moulds (Fig. 13).The combined treatment of silver:hydrogen peroxide in compar-ison to water washing reduced Enterobactericeae by 1.61 log cfu/g,while treatments with chlorinated water caused only 0.15 logreduction immediately after washing (Fig. 12). Yeast population onfresh-cut lettuce was also decreased at similar rate after washingwith water containing both silver and hydrogen peroxide. Thecombined treatment of silver:hydrogen peroxide reduced the yeastpopulation by 1.60 log, while samples treated with chlorine showed0.81 log immediately after washing (Fig. 13).

0

1

2

3

4

5

6

7

8

9

10

Unwashed Water washed 5 ppm silver

Treat

Via

ble

c

ou

nt (lo

g c

fu

/g

)

Fig. 10. Total viable count on shredded lettuce washed with tap water containing low connations during storage at 12 �C for 0 ( ), 2 ( ), 4 ( ) and 7 ( ) days. Values represent th

3.4. Residual silver uptake by washed lettuce

Results from silver analysis in washed lettuce tissues revealedthat the highest uptake of silver ions was 0.13 ppm in sampleswashed with silver:hydrogen peroxide (0.05:0.4 ppm). However,washing the samples with larger concentrations of silver:hydrogenperoxide (5:0.4 ppm) showed smaller silver uptake (0.042 ppm)(Table 1). These discrepancies were attributed to the low sensitivityof the instrument (0.1 ppm). It should be mentioned here thatconcentrations of silver in water and on vegetable tissue were

0.4 ppm hydrogenperoxide

Silver + hydrogenperoxide

5ppm chlorine

ments

centrations of electrochemically generated silver, hydrogen peroxide and their combi-e mean of 6 measurements � standard deviation.

0

1

2

3

4

5

6

7

8

9

10

Unwashed Water washed 5 ppm silver 0.4 ppm hydrogenperoxide

Silver + hydrogenperoxide

5ppm chlorine

Treatments

Pseu

do

mo

nas (lo

g cfu

/g

)

Fig. 11. Total Pseudomonas count on shredded lettuce washed with tap water containing low concentrations of electrochemically generated silver, hydrogen peroxide and theircombinations during storage at 12 �C for 0 ( ), 2 ( ), 4 ( ) and 7 ( ) days. Values represent the mean of 6 measurements � standard deviation.

A. Gopal et al. / Food Microbiology 27 (2010) 210–219 217

initially measured using Atomic Absorption Spectroscopy (AAS),(Barwon Water Authority, Geelong, Australia) and the InductivelyCouple Plasma-Mass Spectrometer (ICP-MS), (Department ofNatural Resources and Environment, State Chemistry Laboratory,Werribee, Victoria). However results from those measurementwere insignificantly different (P > 0.05) from those performedusing a silver DR 100 calorimeter meter (HACH, Loveland.

Although these data indicate possible silver uptake by tissueduring washing, it should be noted that lettuce samples werecollected before rinsing, which can reduce silver uptake

0

1

2

3

4

5

6

7

8

9

10

Unwashed Water washed 5 ppm silver

Trea

En

tero

bacteriaceae (lo

g cfu

/g

)

Fig. 12. Total Enterobactericeae count on shredded lettuce washed with tap water containingcombinations during storage at 12 �C for 0 ( ), 2 ( ), 4 ( ) and 7 ( ) days. Values repres

significantly. Additionally, the highest reported silver uptake(0.13 ppm) is very close to the recommended levels of silver indrinking water (0.05–0.1 ppm) (World Health Organization, 2003).

4. Discussion

Mesophilic and psychrotrophic gram-negative rods arepredominant microorganims on fresh-cut vegetables with thegenus Pseudomonas being the most numerous (Adams et al., 1989;Garg et al., 1990; King et al., 1991). In this study, Ps. fluorescens were

0.4 ppm hydrogenperoxide

Silver + hydrogenperoxide

5ppm chlorine

tments

low concentrations of electrochemically generated silver, hydrogen peroxide and theirent the mean of 6 measurements � standard deviation.

0

1

2

3

4

5

6

7

8

9

10

Unwashed Water washed 5 ppm silver 0.4 ppmhydrogenperoxide

Silver +hydrogenperoxide

5ppm chlorine

Treatments

Ye

as

ts

(lo

g c

fu

/g

)

Fig. 13. Total Yeast count on shredded lettuce washed with tap water containing low concentrations of electrochemically generated silver, hydrogen peroxide and their combi-nations during storage at 12 �C for 0 ( ), 2 ( ), 4 ( ) and 7 ( ) days. Values represent the mean of 6 measurements � standard deviation.

A. Gopal et al. / Food Microbiology 27 (2010) 210–219218

significantly (P � 0.05) reduced when combination treatments ofsilver and hydrogen peroxide at 5:0.04 ppm ratio was used onfresh-cut lettuce. Washing Iceberg lettuce with silver/hydrogenperoxide revealed stronger disinfection effect on Pseudomonas,Enterobacteriaceae and yeast and mould than chlorine. Althoughtreatment with silver at low concentrations (0.05–5 ppm) didn’tachieve better elimination of the microflora from fresh produce,such disinfection effects were better than treatments with chlorine(Figs. 11, 12 and 13).

The results showed also a positive relationship between thedisinfecting effect of silver and its concentration. Various lowconcentration of (0.1–125 ppm) were included in this study todetermine if the recommended doses of silver in drinking water(0.1 ppm) (World Health Organization, 2003) is good enough forwashing fresh produce. Low concentrations of silver (0.05–5 ppm)caused immediate reduction in the microbial counts in treatedproduce. However, when a more concentrated silver (125 ppm)solution was used, more severe and continuous killing effects wererecorded after 4 and 7 days of storage at 12 �C (Figs. 2–5). Thecombined effect of silver (0.05 and 0.1 ppm) and hydrogenperoxide (0.4 and 4 ppm) reduced populations of Enterobactericeaeand yeast in shredded lettuce. The microbial counts were muchlower during storage of lettuce treated with silver/hydrogenperoxide than compared with water washed and unwashedtreatments (Figs. 8 and 9).

Table 1Summary of residual silver (ppm) analysis in wash water and lettuce tissue.

Sample Water before washing Water afte

Unwashed <0.004 <0.004Water washed <0.004 <0.0040.05 ppm silver wash 0.28 0.260.1 ppm silver Wash 1.3 10.5 ppm silver wash 5 5.10.05 ppm silver þ 0.4 ppm H2O2 wash 0.71 0.580.1 ppm silver þ 4.0 ppm H2O2 wash 1.1 1.10.4 ppm H2O2 þ 5 ppm silver 5.5 2.1

Visual examination of washed fresh produce revealed somedeterioration and discolouration after 4 days of storage at 12 �C.However, samples treated with electrochemically generated silverwere better than those treated with chlorinated water with lessbrowning and softening. Washing shredded lettuce with highconcentration of silver (>50 ppm) sourced from silver nitratecaused more deterioration and browning than chlorinated water.However, that browning didn’t happen when silver (>50 ppm) waselectrochemically generated. Consequently, browning of washedtissues caused by silver nitrate was more likely due to the nitrateions and not the silver itself.

Combination of electrochemically generated silver (5 ppm) andhydrogen peroxide (0.4 ppm) caused more significant reduction inthe number of microorganisms on shredded lettuce whencompared to chlorine washed samples at 5 ppm. Significantreductions in Enterobactericeae (1.18 log) and yeast numbers (1.16log) were observed during the 7-day shelf life trial at 12 �C, whencompared to water washed samples. Samples washed with sil-ver:hydrogen peroxide combinations (5:0.4 ppm) were visuallyacceptable with little browning and soft-rot spoilage, whilesamples washed in chlorinated water displayed severe browningand spoilage. All washing trials of fresh lettuce revealed that theantimicrobial effect of silver and chlorine at low concentrations(0.05–5 ppm) was immediate as indicated by several log reductionin microbial counts during the first 24 h of storage. However, the

r washing Shredded lettuce before washing Silver uptake by lettuce

<0.003 00.003 00.036 0.020.039 0.030.033 �0.10.033 0.130.039 00.03 0.042

A. Gopal et al. / Food Microbiology 27 (2010) 210–219 219

microbial population appeared to increase quickly starting from the2nd day of storage to match the original counts. These observationshave led to the conclusion that some partially damaged microbialcells have recovered during storage. This recovery process wasfaster in samples washed with chlorinated water in comparison tosilver at same concentrations. It was evident from these data thatsilver is not only better disinfectant than chlorine, but has alsostronger residual effects after treatment. A positive relationshipbetween silver concentration in washing water and silver residualeffect was also recorded (Figs. 2–5). The silver residue levels onshredded lettuce washed with 0.05, 0.1 or 5 ppm were comparablewith the maximum permitted level of silver (0.1 ppm) for drinkingwater (World Health Organization, 2003). Consequently, thecombined treatment of silver:hydrogen peroxide (5:0.4 ppm) canbe proposed as a good alternative treatment to washing withchlorinated water.

5. Conclusion

The application of the silver/hydrogen peroxide system forwashing minimally processed vegetables appears to have a greatpotential for application in the minimally processed food industry.Combination of electrochemically generated silver (5 ppm) andhydrogen peroxide (0.4 ppm) can be recommended as an alterna-tive treatment to chlorination. The silver:hydrogen peroxidetreatment is anticipated to provide a safer fresh produce withextended shelf life and better quality. However, appropriate regu-lations and consumer acceptance is highly critical in the potentialapplication of this novel technology, and must be further evaluated.

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