ORIGINAL PAPER

Effects of gallotannin treatment on attachment, growth,and survival of Escherichia coli O157:H7 and Listeriamonocytogenes on spinach and lettuce

Christina Engels • Agnes Weiss • Reinhold Carle •

Herbert Schmidt • Andreas Schieber •

Michael G. Ganzle

Received: 14 January 2012 / Revised: 20 March 2012 / Accepted: 25 March 2012 / Published online: 12 April 2012

� Springer-Verlag 2012

Abstract Food-borne illness outbreaks are increasingly

associated with fresh produce. Their high prevalence may

reflect the lack of methods to effectively remove patho-

genic bacteria from the surface of fruits and vegetables.

This study evaluated the effect of antimicrobial gallotan-

nins on attachment, growth, and survival of food-borne

pathogens on green leafy vegetables. Spinach leaves and

interior leaves of lettuce harboring high and low cell counts

of background microbiota, respectively, were washed with

tap water with and without added gallotannins. To account

for the variability among organisms, green leafy vegetables

were inoculated with strain cocktails of Escherichia coli

O157:H7 and Listeria monocytogenes. Cell counts of

L. monocytogenes were significantly reduced by the gal-

lotannin treatment. Lower cell counts after storage for

8 days at 4 �C demonstrated antimicrobial effects of

gallotannins retained on the surface of green leafy vege-

tables. Gallotannin treatments with 1 g/L did not inhibit E.

coli O157:H7 but hindered their attachment to filter paper

by up to 94 %. The addition of gallotannin-containing

extracts from mango (Mangifera indica L.) kernels to the

washing water did neither alter color nor texture of bagged

fresh-cut products. In conclusion, gallotannin treatment

significantly reduced surface contamination of green leafy

vegetables with L. monocytogenes and reduced the

attachment of cells of E. coli O157:H7.

Keywords EHEC � Listeria � Gallotannins �Pentagalloylglucose � Lettuce � Spinach

Introduction

Fresh-cut produce has increasingly been identified as a

source of food-borne outbreaks, particularly with Listeria

monocytogenes and enterohemorrhagic Escherichia coli

(EHEC) [1]. Surveillance of retail products of leafy greens

showed a high prevalence of L. monocytogenes [2, 3].

L. monocytogenes survives in the food-processing environ-

ment and grows at refrigeration temperature. L. monocyt-

ogenes causes meningitis, septicemia, or fetal death in

pregnant women. Young, elderly, and immunocompromised

people as well as pregnant women are susceptible to infec-

tion. Serotypes of L. monocytogenes associated with human

listeriosis include 4b, 1/2a, and 1/2b [4]. L. monocytogenes

causes less than one percent of food-borne illness but liste-

riosis has a high hospitalization and mortality rate [5].

EHEC also caused major produce-associated outbreaks

[for reviews, see 2, 6]. In 2011, a severe outbreak of

hemolytic-uremic syndrome (HUS) caused by a Shiga

toxin-producing E. coli (STEC) was reportedly linked to

C. Engels � A. Schieber � M. G. Ganzle (&)

Department of Agricultural, Food and Nutritional Science,

University of Alberta, 4-10 Ag/For Centre, Edmonton,

AB T6G 2P5, Canada

e-mail: mgaenzle@ualberta.ca

A. Weiss � H. Schmidt

Institute of Food Science and Biotechnology, Food

Microbiology, Hohenheim University, Garbenstrasse 28,

70599 Stuttgart, Germany

R. Carle

Institute of Food Science and Biotechnology, Plant Foodstuff

Technology, Hohenheim University, Garbenstrasse 25,

70599 Stuttgart, Germany

Present Address:A. Schieber

Institut fur Universitat Bonn, Institut fur Ernahrungs- und

Lebensmittelwissenschaften, Universitat Bonn, Romerstrasse 16,

53117 Bonn, Germany

123

Eur Food Res Technol (2012) 234:1081–1090

DOI 10.1007/s00217-012-1727-6

contaminated Fenugreek sprouts [7]. Reported illness was

more frequent in females (68 %) than in males [8]. Because

there is no indication that gender influences the suscepti-

bility to EHEC, the high prevalence of female cases may

reflect gender-specific dietary habits. EHEC infections

cause symptoms ranging from mild diarrhea to hemor-

rhagic colitis and the life-threatening hemolytic-uremic

syndrome (HUS). The virulence of EHEC is attributed to a

combination of virulence factors such as adherence factors

and the production of Shiga-like toxins; EHEC has an

infectious dose of less than 50–100 cells [9]. Factors con-

tributing to the contamination of vegetables by EHEC were

reviewed by Brandl [10]. Although the surface of plants is

a non-host environment for E. coli, bacteria can survive the

harsh conditions on the field and during processing and

storage of produce [10].

Modifications in agronomic and processing practices,

increased international trade and distribution, changes in

consumer habits, and advances in epidemiological surveil-

lance and methodology contribute to the growing numbers of

food-borne illnesses linked to fresh fruits and vegetables [9–

11]. The sector of pre-washed, fresh-cut produce is growing

fast due to the convenience of such ready-to-use products,

reaching annual sales of about $5 billion [12]. The produc-

tion of minimally processed vegetables includes a washing

step to reduce contamination with soil, the microbial load,

and cellular fluids contributing to browning, a centrifugation

step to remove excess water and nutrients, and packaging

under modified atmosphere [13, 14]. By definition, minimal

processing does not include an intervention step to eliminate

primary contamination that occurred during cultivation or

secondary contamination during post-harvest handling,

processing, and storage [2, 10, 15]. The World Health

Organization (WHO) thus recommended research be con-

ducted on treatments for the decontamination of fruits and

vegetables that are consumed raw [16].

Gallotannins from mango kernels exhibit antibacterial

activity toward pathogenic food-borne bacteria [17].

L. monocytogenes is more sensitive to gallotannins than

E. coli [18]. It was the objective of this study to determine

whether gallotannins can reduce the cell counts of

L. monocytogenes and E. coli in lettuce and spinach, and to

investigate whether their selective activity is also observed

in a food matrix.

Materials and methods

Mango kernel extraction, methanolysis,

and quantification

Mango (Mangifera indica L.) kernels were extracted fol-

lowing the protocol outlined by Engels et al. [19]. Briefly,

defatted kernels were extracted with 80 % aqueous acetone

prior to liquid–liquid extraction of gallotannins with five to

twelve galloyl moieties using ethyl acetate [17]. This

extract is referred to as ‘crude extract’. The dry matter (dm)

content of the extract was determined after lyophilization.

To enable quantification, methanolysis, a stepwise

degradation of gallotannins with more than five galloyl

moieties, was applied [19]. Methanolysis yielded penta-O-

galloylglucose and methyl gallate, which were quantified

using ultra high-performance liquid chromatography-mass

spectrometry (UHPLC-MS) with UV detection at 280 nm.

To achieve complete breakdown of gallotannins with

degrees of galloylation higher than five, the extract was

subjected to methanolysis for 6 h. Crude and methanolyzed

extracts were used as aqueous solutions. Results of the

quantification of phenolic compounds using UHPLC-MS

are reported as means ± standard deviations of duplicate

independent experiments.

Bacterial strains

For the challenge studies, a Shiga toxin (Stx)-producing

E. coli O157:H7 strain (ATCC 700927) and five non-Stx-

producing E. coli O157:H7 strains (02-0627, 02-0628,

02-0304, 02-3581, and non-motile 02-1840, originally

isolated from clinical samples by Rafiq Ahmed, Public

Health Agency of Canada, National Microbiology Labo-

ratory, Winnipeg, MB) were used. The adhesion experi-

ments were performed with E. coli AW1.7 [20]. Strains of

L. monocytogenes included DSM20600T, CDC 7762, FS

15, Scott A, 08-5923, and 08-5578. Stock cultures were

maintained at -70 �C in 30 % glycerol. L. monocytogenes

CDC 7762 was associated with the listeriosis outbreaks in

the USA in 1999 linked to ready-to-eat meats. L. mono-

cytogenes 08-5923 and 08-5578 serotype 1/2a were iso-

lated from a human blood specimen associated with the

listeriosis outbreaks in Canada in 2008 linked to ready-to-

eat meats [21]. Strains were cultured in broth containing

5 g/L yeast extract, 5 g/L NaCl, and 5 g/L glucose for 19 h

at 250 rpm at 30 �C (L. monocytogenes) and 37 �C

(E. coli), respectively.

Determination of MICs

MICs (minimum inhibitory concentrations) were deter-

mined using a critical-dilution assay [22]. Twofold serial

dilutions of the crude extract and the methanolyzed extract

were inoculated with overnight cultures of E. coli and

L. monocytogenes strains to a cell count of about 7 log10

(CFU/mL) and incubated overnight at 37 and 30 �C,

respectively. MICs were defined as the lowest concentra-

tions of the substances that inhibited the growth of

microbial strains after 24 h. In cases where turbidity caused

1082 Eur Food Res Technol (2012) 234:1081–1090

123

by bacterial growth was obscured by precipitation of media

components with gallotannins, pH changes were measured

by the addition of bromocresol green. The MIC was

defined as the lowest gallotannin concentration that inhib-

ited the acidification of the medium and color change of the

pH indicator [18]. MICs are expressed as mean ± standard

deviation of three independent experiments.

Determination of the antibacterial effect of tannin

solutions against E. coli and L. monocytogenes on green

leafy vegetables

Preparation of green leafy vegetables

Head iceberg lettuce and spinach leaves were purchased at

a local supermarket in Edmonton, Canada, April to June.

Outer leaves, cores of the lettuce, and large rib tissues were

removed and discarded. Lettuce and spinach leaves were

cut into 3.0 ± 0.5 cm 9 3.0 ± 0.5 cm pieces using a

sterile surgical knife.

Inoculum preparation and experimental design

Overnight cultures were washed in tryptone water (1 g/L

tryptone, 8.5 g/L NaCl), re-suspended in tryptone water,

diluted with tryptone water, and combined to a two-strain

cocktail (E. coli ATCC 700927 and L. monocytogenes

DSM20600T) or ten-strain cocktail (E. coli O157:H7

strains and L. monocytogenes strains CDC 7762, FS 15,

Scott A, 08-5923, and 08-5578) to a cell density of about

7 log10 (CFU/mL) per strain.

In a preliminary experiment, lettuce samples were

inoculated with a two-strain mixture and treated with 0.1

and 1.0 g dm crude extract/L (T 0.1 and T 1.0). Each

9 cm2 piece of green leafy vegetables was spot-inoculated

with 100 lL of the strain cocktail and dried for 10 min in a

biosafety cabinet. Inoculated lettuce pieces were sub-

merged and stirred in sterile water containing varying

amounts of the tannin extracts at 4–10 �C for 10 min. As a

control, samples were treated in a similar way in sterile

water. Excess water was removed by placing green leafy

vegetables in sterile centrifuge beakers with tissue paper in

the bottom and centrifuged at 2009g for 3 min. Samples

were packed in sterile stomacher bags (17 9 30 cm2) and

stored at 4 ± 0.5 �C for up to 8 days. The background

microbiota of the samples was determined in non-inocu-

lated samples.

The ten-strain cocktail experiment was performed on

both lettuce and spinach using T 0.1 and T 1.0, and 0.1 and

1.0 g penta-O-galloylglucose/L (M 0.1 and M 1.0),

respectively. Samples of green leafy vegetables were

inoculated and washed following the same procedure as

described above. Excess water was removed by placing

green leafy vegetables on sterile tissue paper. The experi-

ment was performed in triplicate.

Microbiological characterization of samples

Samples of inoculated green leafy vegetables were tested

for microbial growth directly after washing and every

second day during storage. Samples were blended in

90 mL tryptone water (1 g/L tryptone, 8.5 g/L NaCl) in a

stomacher for 1 min prior to the determination of cell

counts. Appropriate dilutions were spread on Palcam agar

(agar base and selective supplement, Oxoid, Basingstoke,

England) and incubated for 48 h at 30 �C to determine

L. monocytogenes populations. Coliform bacteria, includ-

ing E. coli, were determined on SMAC (sorbitol Mac-

Conkey, Difco, Becton, Dickinson and Co., Sparks, MD,

USA) agar in preliminary study and on VRB agar (violet

red bile, Difco, Becton, Dickinson and Co., Sparks, MD,

USA) in the ten-strain experiment after 24-h incubation at

37 �C. Colonies of E. coli O157:H7 strains could be

readily differentiated from the background microbiota by

observation of the colony morphology. Total aerobic plate

counts were determined on agar plates containing 5 g/L

yeast extract, 5 g/L NaCl, 5 g/L glucose, and 15 g/L agar

for 48 h at 37 �C.

Anti-adhesive effect of gallotannins toward

E. coli AW1.7

Overnight cultures E. coli AW1.7 were diluted in tryptone

water (1 g/L tryptone, 8.5 g/L NaCl) to reach final con-

centrations of 7 log10 (CFU/mL). The crude extract and the

methanolyzed extract were added to reach the same final

concentrations as described above. Sterile pieces of filter

paper (2.0 ± 0.2 cm 9 2.0 ± 0.2 cm) were submerged for

15 min in the solutions, then removed and drained.

Attached bacteria were quantified by suspending the filter

paper in tryptone water, followed by thorough shaking with

glass beads using a vortex mixer for 1 min, and plating of

appropriate dilutions on nutrient agar plates containing 5 g/L

yeast extract, 5 g/L NaCl, 5 g/L glucose, and 15 g/L agar.

Cell counts were determined after overnight incubation at

37 �C. The experiment was repeated in triplicate.

Mean ± standard deviation of reductions when compared to

a control with water are shown.

Influence of gallotannins on quality and storage life

of non-inoculated lettuce samples

Lettuce processing, packaging, and storage

Endive lettuce heads (Cichorium endivia cv. Maruchka)

were directly purchased from a local grower in Stuttgart,

Eur Food Res Technol (2012) 234:1081–1090 1083

123

Germany, in July, cooled and processed the day after

harvest. After manually removing outer leaves and excision

of the cores, lettuce heads were shredded into 4.3-mm

strips using a stainless steel cutting machine. Potable tap

water was cooled to 4 �C with ice and mixed thoroughly

with the crude extract to reach final concentrations of 0.1

and 1.0 g dm/L (T 0.1 and T 1.0). Shredded lettuce was

kept in movement in the water using a stainless-steal

strainer. To remove excess water, the lettuce was centri-

fuged in a household salad spinner. Lettuce was packed

into film bags (Amcor Flexibles, Bristol, UK; 19.0 ±

0.5 cm 9 22.0 ± 0.5 cm, 35-lm antimist-coated-oriented

polypropylene, O2 and CO2 transmission rates at 23 �C and

0 % RH of *900 and 4,000 cm3 9 m-2 9 day-1 9 atm-1,

respectively) in 50.0 ± 0.2 g quantities and sealed under

ambient temperature using a R-25 packaging machine

(Boss, Friedrichsdorf, Germany). Bags were stored at 4 �C

for up to 1 week.

Microbial and headspace analysis, color,

and texture measurements

Microbiological analyses were carried out directly after

processing and on each day of storage following the pro-

cedure outlined above. In addition to the quantification of

total aerobic counts and coliforms, members of the Pseu-

domonadaceae were determined on GSP agar (glutamate

starch phenol red agar with 100,000 i.U/L potassium pen-

icillin G; Merck, Darmstadt, Germany). Plates were incu-

bated aerobically at 30 �C for 48 h.

Oxygen and carbon dioxide concentrations of samples

treated with different concentrations of gallotannins were

measured as described by Baur et al. [13] by analyzing the

headspace gas in unopened samples through an adhesive

septum by means of a hypodermic needle using a Check-

Mate 9900 O2/CO2 gas analyzer (PBI-Dansensor, Ringsted,

Denmark). O2/CO2 concentrations were repeatedly moni-

tored in three separate bags per treatment and sampling

day. The texture of lettuce samples was assessed using a

Kramer shear cell with a 10 blade probe attached to the

4301 test instrument of a Series IX automated materials

testing system (Instron, Canton, MA, USA). Specific force

(N/100 g) and specific energy (J/100 g) were calculated

using maximum peak force (N) and energy to breakpoint

(J) by the Series IX software, Version 5. Texture analysis

was performed with six replicates per treatment and sam-

pling day.

A CR-300 chroma meter (Minolta, Osaka, Japan) was

used to determine color values (L*a*b*) after calibration to

a standard white tile (Y-93.5, x-0.3155, y-0.3320). The

illuminant was the CIE (Commission International

de l’Eclairage) D65. Using the measured values, Chroma

(C, C = a*2 ? b*2)0.5, hue angle (H�, H� = 180 ? tan-1

(b*/a*), and total color difference (DE, DE = [(L* -

L0*)2 ? (a* - a0*)2 ? (b* - b0*)2]0.5, where L0*, a0*,

and b0* were values from day 0) were calculated. Lettuce

samples were spread out on a white plate to a 10 9 10 cm2

square prior to color value determination. Samples were

measured ten times per treatment and sampling day.

Data analysis

Statistical significance was evaluated by Student’s t test

using SigmaPlot software (Systat Software, San Jose, CA,

USA) at P \ 0.01.

Results

Quantification and inhibitory activity of phenolics

in the extracts

UHPLC-MS was used for qualitative and quantitative

analysis of phenolic compounds. The crude extract con-

tained gallotannins with four to twelve gallic acid moieties

(data not shown), in accordance with previous reports [17].

The dm content of the crude extract was 269.3 ± 5.2 g/L.

The methanolyzed extract contained methyl gallate and

penta-O-galloylglucose; gallotannins with degrees of gal-

loylation higher than five were not detected (data not

shown). The concentrations of methyl gallate and penta-O-

galloylglucose were 56.3 ± 3.9 and 103.3 ± 3.2 g/L,

respectively.

The MICs of the crude extract and the methanolyzed

extract are presented in Table 1. The crude extract, con-

taining gallotannins with various degrees of galloylation,

inhibited growth of L. monocytogenes strains with MICs

equal to or less than 0.02 g dm/L. MICs of strains of E. coli

were equal to or less than 1.4 g dm/L. The inhibitory

activity of penta-O-galloylglucose against S. aureus was

100-fold higher compared to methyl gallate (data not

shown). The MICs of the methanolyzed extract are thus

expressed relative to the concentration of penta-O-gal-

loylglucose. Growth of L. monocytogens was inhibited by

concentrations equal to or less than 0.02 g penta-O-gal-

loylglucose/L. E. coli O157:H7 strains were inhibited by

concentrations equal to or less than 0.9 g penta-O-gal-

loylglucose/L.

Preliminary investigations on the antibacterial effect

of tannin solutions of inoculated lettuce

The inhibitory activity of tannin-containing extracts in

vitro was validated using lettuce. Their influence on initial

reduction and growth during storage for 7 days of EHEC

and L. monocytogenes DSM20600T is shown in Fig. 1.

1084 Eur Food Res Technol (2012) 234:1081–1090

123

Results for samples from day 1, 3, and 5 were consistent

with the presented data (data not shown). Inoculated EHEC

counts were 4.4 ± 0.5 log10 (CFU/cm2). The addition of

crude extract to the washing water reduced after-washing

counts at higher concentrations (Fig. 1a). Despite increased

initial reduction, EHEC counts of water and tannin-solu-

tion-treated samples did not differ after the storage period.

Shredded lettuce was inoculated with L. monocytogenes

counts of 4.5 ± 0.2 log10 (CFU/cm2). Washing with water

reduced inoculated counts by about 90 %. L. monocyto-

genes did not grow on lettuce during storage (Fig. 1b).

Supplementation of washing water with 0.1 g/L of the

crude extract did not have an effect on survival during

storage. The addition of 1.0 g/L of crude extract to the

washing water, however, reduced Listeria counts below the

detection limit of 2.2 log10 (CFU/cm2) and Listeria counts

remained below the detection limit throughout storage.

Initial and final total aerobic counts of lettuce washed

with 0.1 and 1.0 g/L crude extract and control samples

washed with water were not different. Washing reduced

initial counts of 5.5 ± 0.1 log10 (CFU/cm2) to 4.9 ± 0.4

log10 (CFU/cm2) (water), 4.4 ± 0.2 log10 (CFU/cm2)

(T 0.1), and 4.3 ± 0.2 log10 (CFU/cm2) (T 1.0). Over

7 days of storage at 4 �C, the total aerobic count increased

about 1–2 log to reach final concentrations of 6.5 ± 0.4 log10

(CFU/cm2) (water), 6.8 ± 0.2 log10 (CFU/cm2) (T 0.1), and

6.9 ± 0.1 log10 (CFU/cm2) (T 1.0).

Anti-adhesive effect of gallotannins toward E. coli

AW1.7

The reduction of E. coli O157:H7 counts by gallotannin

treatment is inconsistent with the resistance of E. coli

strains toward gallotannins (Table 1 and [18]). Plate counts

on nutrient and VRB media indicated that E. coli cells in

the wash solutions were neither sub-lethally injured nor

dead (data not shown). Lower initial counts might be

caused by decreased bacterial adhesion. Therefore, anti-

adhesive effects of gallotannins toward cells of E. coli

AW1.7 on filter paper were investigated (Table 2). Both

extracts reduced the amount of adhered cells in a concen-

tration-dependent manner.

Table 1 MICs of a mango kernel crude extract and a methanolyzed extract against different strains of Escherichia coli and Listeriamonocytogenes

Strains Serotype MIC crude extracta MIC methanolyzed extractb

E. coli ATCC 700927 O157:H7 n.det. n.det.

E. coli 02-1840 O157:H7 1.40 ± 0.00 0.90 ± 0.31

E. coli 02-0627 O157:H7 0.70 ± 0.00 0.36 ± 0.16

E. coli 02-0628 O157:H7 0.70 ± 0.00 0.27 ± 0.00

E. coli 02-0304 O157:H7 0.47 ± 0.20 0.27 ± 0.00

E. coli 00-3581 O157:H7 0.70 ± 0.00 0.45 ± 0.16

L. monocytogenes DSM20600T 1/2a n.det. n.det.

L. monocytogenes CDC 7762 4b 0.01 ± 0.00 0.01 ± 0.00

L. monocytogenes FS 15 1/2b 0.02 ± 0.01 0.01 ± 0.00

L. monocytogenes Scott A 4b 0.02 ± 0.01 0.02 ± 0.00

L. monocytogenes 08-5923 1/2a 0.01 ± 0.01 0.01 ± 0.00

L. monocytogenes 08-5578 1/2a 0.01 ± 0.00 \0.01

Mean ± standard deviation of three independent experiments are shown

n.det. not determineda expressed in g dry matter/Lb expressed as g penta-O-galloylglucose/L

B

water T 0.1 T 1.0

Y D

ata

3

4

5

6A

water T 0.1 T 1.0

3

4

5

6

<2.2

cell

coun

ts [l

og(c

fu/c

m2 )]

Fig. 1 Cell counts of enterohemorrhagic Escherichia coli O157:H7

ATCC 700927 (a) and Listeria monocytogenes DSM20600T (b) on

inoculated lettuce directly after washing (black columns) and after

7 days of storage at 4 �C (gray columns). Wash water contained

0.1 g/L dm (T 0.1) or 1.0 g/L dm (T 1.0) mango kernel extract; water

was used as a control. Shown are mean ± standard deviation of

two independent experiments. Inoculation levels were 4.4 ± 0.5

log10 (CFU/cm2) and 4.5 ± 0.2 log10 (CFU/cm2) for E. coli and

L. monocytogenes, respectively

Eur Food Res Technol (2012) 234:1081–1090 1085

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Antibacterial effects of tannin solutions against

ten-strain cocktails of E. coli and L. monocytogenes

on green leafy vegetables

Cocktails of multiple strains were used in a second experi-

ment to account for the variability among strains of the same

species. Spinach and lettuce samples were investigated to

evaluate the influence of different levels of naturally

occurring background microbiota. Initial total aerobic counts

on unwashed spinach and center leaves of lettuce were

4.5 ± 1.6 and 2.2 log10 (CFU/cm2), respectively.

Cell counts of spinach samples inoculated with a ten-

strain mixture of E. coli and L. monocytogenes after

washing and after storage for 8 days at 4 �C are shown in

Fig. 2. Results for samples from day 2, 4, and 6 were

consistent with the presented data (not shown). After

inoculation, total aerobic counts on spinach were 6.9 ± 0.2

log10 (CFU/cm2). The washing step with water or tannin

solutions reduced total aerobic counts by about 99 %

(Fig. 2a). Similar results were obtained on lettuce (Fig. 3a).

Inoculation increased the bacterial load to 6.6 ± 0.0 log10

(CFU/cm2). The washing step with water or tannin solu-

tions reduced total aerobic counts by about 2 logs. Eight

days of storage at 4 �C led to a higher increase in total

aerobic counts on spinach than on lettuce irrespective of

the treatment (Figs. 2, 3a).

The lettuce and spinach used in this study were free of

L. monocytogenes (data not shown). After inoculation,

L. monocytogenes counts on spinach were 6.4 ± 0.1 log10

(CFU/cm2); counts were reduced by 1.9 log when washed

with water (Fig. 2b). Washing with tannin crude extract or

the methanolyzed extract reduced the listeria counts in a

concentration-dependent manner. When used at a concen-

tration of 0.1 g/L, both extracts reduced L. monocytogenes

counts by 2.8 log10 (CFU/cm2). A 10-fold increase in

tannin concentration reduced L. monocytogenes counts by

about 3.5 log10 (CFU/cm2). Over 8 days of storage at 4 �C,

L. monocytogenes counts remained unchanged on water-

washed spinach samples as well as samples treated with

0.1 g/L tannins. Tannins at a concentration of 1.0 g/L

decreased L. monocytogenes counts, reaching significantly

lower final counts after 8 days of storage.

Water washing of lettuce samples reduced inoculated

L. monocytogenes counts of 6.5 ± 0.0 log10 (CFU/cm2) by

1.9 log10 (CFU/cm2) (Fig. 3b). Both tannin extracts

reduced initial L. monocytogenes counts in a concentration-

dependent manner and led to significantly lower final

counts after 8 days of storage.

Prior to inoculation, E. coli O157:H7 was not detected

on lettuce and spinach samples. The total coliform counts

on uninoculated spinach and lettuce were 5.0 ± 0.9 and

3.9 ± 0.0 log10 (CFU/cm2), respectively. Counts of total

coliforms and E. coli O157:H7 after inoculation, washing,

and storage are presented in Figs. 2c, d (spinach) and 3c, d

(lettuce). Spinach and lettuce samples were inoculated to

cell counts of 6.4 ± 0.2 and 6.1 ± 0.1 log10 (CFU/cm2),

respectively. Inoculation levels of E. coli O157:H7 were

6.2 ± 0.3 and 6.0 ± 0.2 log10 (CFU/cm2) for spinach and

lettuce, respectively. The washing steps with water and

with tannin solutions reduced total coliform counts by

about 2 logs on both lettuce and spinach (Figs. 2, 3c).

Counts increased during the storage period, with a larger

increase observed for spinach samples. The washing step

with water and with tannin solutions also reduced E. coli

O157:H7 counts by about 2 logs for lettuce and spinach

(Figs. 2, 3d). E. coli O157:H7 counts decreased in the

course of the storage period, with a more pronounced

decline found for lettuce samples. No differences in initial

reduction and final E. coli O157:H7 counts were detected

between treatment with water and with tannin solutions for

lettuce and spinach, respectively.

Influence of gallotannins on quality and storage life

of non-inoculated lettuce samples

Microbiological analysis

Growth and survival of naturally occurring microflora was

evaluated on non-inoculated lettuce. Total aerobic counts

and total coliforms count on lettuce in the pilot plant

experiment followed the same trend as counts detected in

the laboratory scale experiment. Counts on plate count agar

after washing were 6.3 ± 0.2 log10 (CFU/cm2) for water,

6.1 ± 0.1 log10 (CFU/cm2) for T 0.1, and 6.2 ± 0.2 log10

(CFU/cm2) for T 1.0. In the course of storage, counts

increased about 2 logs for samples from all treatment types

(data not shown). Counts of total coliforms after washing

were 5.5 ± 0.1 log10 (CFU/cm2) for water, 4.7 ± 0.1 log10

Table 2 Cell counts on filter paper and adhesion of E. coli AW1.7 after washing with tryptone water (control) and tannin solutions

Control T 0.1 T 1.0 M 0.1 M 1.0

Counts (log) 6.3 ± 0.1 5.6 ± 0.2 5.3 ± 0.3 5.3 ± 0.1 5.1 ± 0.2

Reduction (%) - 79.6 ± 6.9 89.7 ± 5.1 89.7 ± 2.1 94.2 ± 1.3

Adhesion is expressed as percentage of the cell counts of the control treatment (washing with tryptone). Mean ± standard deviation of at least

three independent experiments are shown

1086 Eur Food Res Technol (2012) 234:1081–1090

123

3

4

5

6

7

8

**

*

* **

water T 0.1 T 1.0 M 0.1 M 1.0

cell

coun

ts [l

og (

cfu/

cm2 )]

3

4

5

6

7

8

water T 0.1 T 1.0 M 0.1 M 1.0

3

4

5

6

7

8

water T 0.1 T 1.0 M 0.1 M 1.0

3

4

5

6

7

8

A B

C D

Fig. 2 Total aerobic cell counts (a) and counts of Listeria monocyt-ogenes (b), total coliforms (c) and Escherichia coli O157 (d) on

inoculated spinach directly after washing (black columns) and after

7 days of storage at 4 �C (gray columns). Wash water contained

0.1 g/L dm (T 0.1) or 1.0 g/L dm (T 1.0) mango kernel extract; water

was used as a control. Shown are mean ± standard deviation of three

independent experiments. Asterisks mark significant differences from

control (p \ 0.01). Total aerobic and total coliforms counts after

inoculation were 6.9 ± 0.2 log10 (CFU/cm2) and 6.4 ± 0.2 log10

(CFU/cm2), respectively. Inoculation levels were 6.2 ± 0.3 log10

(CFU/cm2) and 6.4 ± 0.1 log10 (CFU/cm2) for E. coli O157 and

L. monocytogenes, respectively

cell

coun

ts [l

og (

cfu/

cm2 )]

3

4

5

6

7

3

4

5

6

7

* **

water T 0.1 T 1.0 M 0.1 M 1.0

3

4

5

6

7

A B

C

water T 0.1 T 1.0 M 0.1 M 1.0

3

4

5

6

7D

Fig. 3 Total aerobic cell counts (a) and counts of Listeria monocyt-ogenes (b), total coliforms (c) and Escherichia coli O157 (d) on

inoculated lettuce directly after washing (black columns) and after

7 days of storage at 4 �C (gray columns). Wash water contained

0.1 g/L dm (T 0.1) or 1.0 g/L dm (T 1.0) mango kernel extract; water

was used as a control. Shown are mean ± standard deviation of three

independent experiments. Asterisks mark significant differences from

control (p \ 0.01). Total aerobic and total coliforms counts after

inoculation were 6.6 ± 0.0 log10 (CFU/cm2) and 6.1 ± 0.1 log10

(CFU/cm2), respectively. Inoculation levels were 6.0 ± 0.2 log10

(CFU/cm2) and 6.5 ± 0.0 log10 (CFU/cm2) for E. coli and L. mon-ocytogenes, respectively

Eur Food Res Technol (2012) 234:1081–1090 1087

123

(CFU/cm2) for T 0.1, and 5.1 ± 0.5 log10 (CFU/cm2) for T

1.0 and essentially remained unchanged during storage

(data not shown). Tannin extracts in the wash water did not

significantly influence growth of Pseudomonadaceae.

Counts increased from 5.4 ± 0.1 to 7.5 ± 0.1 log10 (CFU/

cm2) for water, from 5.4 ± 0.1 to 8.0 ± 0.2 log10 (CFU/

cm2) for T 0.1, and from 5.4 ± 0.1 to 8.2 ± 0.1 log10

(CFU/cm2) for T 1.0.

Analysis of headspace, texture, and color

Consumers assess produce quality and freshness based on

texture and color. Green leafy vegetables are living plant

tissues and their respiratory activity alters the ratio of O2

and CO2 in bagged products. Gallotannin treatment affec-

ted the composition of headspace gas after 7 days of

storage at 4 �C. While oxygen levels in water-washed

lettuce samples decreased to 19.82 ± 0.15 %, the decrease

was significantly larger in samples washed with 0.1 g dm

tannin extract/L (19.21 ± 0.18 %). The addition of 1.0 g

tannin extract/L decreased oxygen levels even further,

making the result significantly different from water and T

0.1 (18.68 ± 0.19 %). Corresponding carbon dioxide lev-

els were significantly higher in tannin-treated samples

(water: 1.38 ± 0.12, T 0.1: 1.88 ± 0.16, and T 1.0:

2.36 ± 0.22 %).

The change of texture during storage was not signifi-

cantly different between treatments. The specific force at

the day of processing was 7,280 ± 200 N/100 g for water-

washed samples, 6,650 ± 170 N/100 g for samples treated

with 0.1 g tannin extract/L, and 6,870 ± 400 N/100 g for

samples treated with 1.0 g tannin extract/L. Normalized

values (N/N0) were 1.0 for all samples stored for a week.

Results for specific energy were in agreement with these

findings (data not shown). No significant differences in

color values (L*a*b*) and corresponding hue angles (H�),

chroma (C), and total color differences (DE) were found

(data not shown).

Discussion

Recently, produce, particularly salad, lettuce, melon, and

sprouts, caused the highest number of disease cases among

five major food categories (produce, beef, poultry, seafood,

and eggs) in the USA [9, 23]. These outbreaks reflect the

difficulties to effectively eliminate pathogenic bacteria

from fresh fruits and vegetables during processing [2, 15,

24]. Pathogens can reside within the plant where they are

protected from washing and antibacterial interventions.

Internalization through the root system or from inoculated

seeds has been shown for E. coli O157:H7 and L. mono-

cytogenes [25].

This study evaluated the use of antibacterial gallotannins

in washing water to reduce surface contamination of pro-

duce. Gallotannins and methanolyzed gallotannins led to a

similar reduction of the initial bacterial load as well as final

counts after storage. Methanolysis, however, simplifies

identification of compounds and standardization of extracts

with commercially available reference substances [19].

Spinach and lettuce samples exhibited a 2 log (CFU/cm2)

difference in initial bacterial counts, but inoculation,

washing, and storage led to similar results for both products.

In water-washed control samples, cell counts of L. mono-

cytogenes remained unchanged during storage at 4 �C.

Similarly, populations of L. monocytogenes on vegetables

remained unchanged over a 12-day storage period [26].

Growth of two strains of L. monocytogenes inoculated on

lettuce occurred only between 8 and 15 days of storage [27].

The use of gallotannins during washing significantly

reduced counts of L. monocytogenes after washing and

after a 7-day storage period, indicating that gallotannins

remaining on the product exhibited anti-listerial activity

during storage. The tannin treatment used in this study was

superior to commercially employed treatments, including

washing with chlorine solutions (200–250 mg/L) or mod-

ified atmosphere packaging (3 % O2, 97 % N2) that did not

influence growth of Listeria strains [27]. Mild heat treat-

ment even enhanced growth of Listeria during subsequent

storage at 5 �C [28]. The use of anti-listerial tannins in the

wash water may not eliminate bacterial cells that were

internalized through the root system, through stomata, or

from inoculated seeds, but will reduce cross-contamination

during processing.

Escherichia coli is more resistant toward gallotannins

when compared to L. monocytogenes [18, this study].

Tannin treatment did not significantly alter cell counts of

E. coli O157:H7 after storage. Cell counts of EHEC

increased during storage at 4 �C, but the cell counts of five

non-Shiga toxin-producing strains of E. coli O157:H7

remained unchanged. Growth and survival of E. coli at low

temperature are strain dependent: Two E. coli O157:H7

strains survived but did not grow during 12 days of storage

at 4 �C on lettuce [26]; however, cell counts of two dif-

ferent E. coli O157:H7 strains decreased by about 1 log

(CFU/g) under similar conditions [29]. Washing with tan-

nin solutions reduced attachment of E. coli cells to filter

paper and decreased EHEC counts on salad despite the lack

of antibacterial activity of gallotannins against E. coli.

Tannins denature proteins; this tanning property was sug-

gested to contribute to their antibacterial activity [18] and

may also explain decreased attachment. It remains uncer-

tain, however, if reduced adherence on filter paper corre-

sponds to reduced adherence to plant tissue. Moreover,

tannins failed to reduce the cell counts of a five-strain

cocktail of E. coli O157:H7 to plant surfaces.

1088 Eur Food Res Technol (2012) 234:1081–1090

123

Current processing of ready-to-eat produce includes a

washing step in cold chlorinated water, water removal by

centrifugation, and packaging. However, chlorination of

water in the washing of fresh-cut produce is still restricted

in Europe and does not conform to organic food labeling

[13, 14]. Addition of tannin-containing extracts to wash

water is compatible with conventional industrial processes,

and did not affect product quality. The faster decline in

oxygen levels with a corresponding large increase in car-

bon dioxide levels indicated increased respiration activity

of lettuce treated with tannin extracts that did, however, not

result in detectable changes in firmness or color of the

bagged products.

The composition of the crude extract used in this study

was similar to tannic acid, a commercial used mixture of

galloylglucoses that is generally recognized as safe

(GRAS) by the FDA [30]. Propyl gallate, which is closely

related to methyl gallate, also has GRAS status. Gallotan-

nins naturally occur in food and it is estimated that people

in the USA ingest 1 g of tannic acid each day [31]. Gal-

lotannins exhibit a distinct taste of astringency [32].

However, a tannin-rich witch hazel (Hamamelis virgini-

ana) extract added at a final concentration of 30 ppm did

not alter sensory properties of a fish meat product [33].

In conclusion, washing of lettuce and spinach samples

with gallotannin solutions failed to kill E. coli. However,

counts of L. monocytogenes after washing and storage at

4 �C were significantly reduced. Therefore, addition of

gallotannins to the wash water may reduce cross-contam-

inations during processing and eliminate L. monocytogenes

resident on the plant surface.

Acknowledgments Lynn McMullen of the University of Alberta

and Alex Gill from Health Canada are acknowledged for providing

bacterial strains. A.S. and M.G.G. acknowledge funding from the

Canada Research Chair Program.

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