Sanitizing action of triple-strength vinegar against Escherichia coli on lettuce

Abstract

Vegetable sanitization protocols recommend the use of chlorine, which has adverse effects on the environment and carcinogenic effects on humans. Acetic acid is an interesting alternative to chlorine because it possesses no risk to human health and is widely available in the form of vinegar. This study aimed to evaluate the sanitizing action of vinegar, 130 g L-1 total titratable acidity expressed as acetic acid, on lettuce. Vinegar was chosen because it is a low-cost product widely available in the Brazilian market. The minimum inhibitory concentration and minimum bactericidal concentration of vinegar against Escherichia coli were 2.5 and 15 g L-1 total acidity, respectively. Lettuce leaves artificially contaminated with E. coli or naturally contaminated with total coliforms were washed with water and immersed in vinegar solution (15 g L-1 total acidity) for 15 min. This period was sufficient to reduce E. coli counts in artificially contaminated samples and total coliforms in naturally contaminated samples. There were no visual changes in lettuce leaves, which indicates that vinegar at 15 g L-1 total acidity can be used to sanitize vegetables without affecting their appearance.

Full text

414
Scientic communication
Hortic. bras., Brasília, v.36, n.3, July - September 2018
Foodborne diseases are still a public
health problem, and the supply of
safe food products has been a constant
concern for government health agencies,
epidemiological surveillance agencies,
and the food industry.
Lettuce (Lactuca sativa) is one of
the most consumed vegetables in salads
in Brazil. Chlorinated compounds are
widely used to sanitize lettuce due
to low cost and recommendations
of the National Agency of Sanitary
Surveillance (ANVISA). The formation
of trihalomethanes from chlorinated
sanitizers, however, is a cause for
concern, as these compounds are
carcinogenic. Sanitization alternatives
that provide economic and safety
benets by replacing chlorine are of
interest.
Acetic acid, produced by
fermentation of ethanol by acetic acid
bacteria, may be an alternative to
chlorine in food sanitization. Acetic
acid is the main component of vinegar
and can be produced by Acetobacter
spp. fermentation of fruit must, honey,
cereals, hydroalcoholic mixtures, and
vegetables (Spinosa et al., 2015). An
important characteristic of vinegar is
its low toxicity. Moreover, its use as
sanitizer does not alter the taste or smell
of foods (Nascimento et al., 2003).
According to Brazilian legislation,
the product of acetic fermentation of
a hydroalcoholic mixture containing
potable ethanol of agricultural origin is
denominated alcohol vinegar. Vinegar
with volatile acidity over 12 g of acetic
acid per 100 mL is classied as triple
and called triple-strength vinegar. The
product of acetic fermentation of ethanol
produced from one or more cereals is
denominated cereal vinegar (BRASIL,
2012).
SOUZA, GC; SPINOSA, WA; OLIVEIRA, TCRM. 2018. Sanitizing action of triple-strength vinegar against Escherichia coli on lettuce. Horticultura
Brasileira 36: 414-418. DOI - http://dx.doi.org/10.1590/S0102-053620180321
Sanitizing action of triple-strength vinegar against Escherichia coli on
lettuce
Giovanna C Souza; Wilma A Spinosa; Tereza CRM Oliveira1
lUniversidade Estadual de Londrina (UEL), Londrina-PR, Brazil; giovannacavagnari@hotmail.com; wilma.spinosa@gmail.com;
terezaoliveira@yahoo.com.
ABSTRACT
Vegetable sanitization protocols recommend the use of chlorine,
which has adverse eects on the environment and carcinogenic
eects on humans. Acetic acid is an interesting alternative to chlorine
because it possesses no risk to human health and is widely available
in the form of vinegar. This study aimed to evaluate the sanitizing
action of vinegar, 130 g L-1 total titratable acidity expressed as
acetic acid, on lettuce. Vinegar was chosen because it is a low-cost
product widely available in the Brazilian market. The minimum
inhibitory concentration and minimum bactericidal concentration of
vinegar against Escherichia coli were 2.5 and 15 g L-1 total acidity,
respectively. Lettuce leaves articially contaminated with E. coli or
naturally contaminated with total coliforms were washed with water
and immersed in vinegar solution (15 g L-1 total acidity) for 15 min.
This period was sucient to reduce E. coli counts in articially
contaminated samples and total coliforms in naturally contaminated
samples. There were no visual changes in lettuce leaves, which
indicates that vinegar at 15 g L-1 total acidity can be used to sanitize
vegetables without aecting their appearance.
Keywords: Lactuca sativa, acetic acid, sanitization.
RESUMO
Ação sanitizante de vinagre triplo sobre Escherichia coli em
alface
Protocolos de sanitização de hortaliças utilizam cloro, que
apresenta efeitos adversos sobre o meio ambiente e na produção de
compostos cancerígenos. Uma alternativa para substituir o cloro é o
uso do ácido acético, devido à disponibilidade na forma de vinagre
e por não apresentar riscos à saúde humana. O trabalho teve como
objetivo avaliar a ação do fermentado acético de álcool com 130 g
L-1 de acidez total, expressa em ácido acético, para uso na saniti-
zação de alfaces. A escolha deste fermentado é justicada por ser
um produto de baixo preço e disponível no mercado brasileiro. A
concentração inibitória mínima e a concentração bactericida mínima
(concentração que eliminou a bactéria) para Escherichia coli foi de
2,5 e 15 g L-1 de acidez total, respectivamente. Amostras de alface
articialmente contaminadas com suspensões de E. coli e amostras
com contaminação de origem foram lavadas com água e imersas em
solução com 15 g L-1 de ácido acético, por 15 minutos. Esse tempo
foi adequado para a redução da contagem de E. coli nas amostras
articialmente contaminadas e na contagem de coliformes totais nas
amostras com contaminação de origem. Nenhuma alteração visual
das folhas de alface foi observada, indicando que a concentração de
15 g L-1 de acidez total pode ser utilizada na sanitização da hortaliça
sem prejuízo à sua aparência.
Palavras-chave: Lactuca sativa, ácido acético, sanitização.
Received on April 4, 2017; accepted on May 9, 2018

415Hortic. bras., Brasília, v.36, n.3, July - September 2018
Brazilian industries have sucient
capacity to meet the demand for
diversified products based on
vinegar. Triple-strength vinegar is
commercialized in the Brazilian market
for $ 0.15/L (FOB, Free On Board).
Noteworthy, vinegar production
facilities have a low impact on the
environment (ANAV, 2015).
The aim of this study was to
evaluate the eciency of triple-strength
vinegar for sanitization of articially
contaminated lettuce with Escherichia
coli and naturally contaminated lettuce
with total coliforms and E. Coli
determining the minimum inhibitory
concentration (MIC) and the minimum
bactericidal concentration (MBC) using
the macrodilution method.
MATERIAL AND METHODS
Twenty-seven crisphead lettuce
heads were randomly obtained and
purchased at different commercial
establishments in Londrina, in dierent
weeks from October to December
2014. The vegetables were marketed
as products grown by conventional
agricultural methods.
Triple-strength alcohol vinegar was
provided by Tecnologia em Saúde,
Indústria e Comércio de Alimentos
Ltda., Assis, São Paulo, Brazil. The
vinegar was produced by biological
fermentation of alcohol. Its total
titratable acidity expressed as acetic
acid was 130 g L-1, and its pH was 2.50.
Alcohol vinegar and cereal vinegar
were purchased from commercial
establishments in Londrina. Alcohol
vinegar and cereal vinegar presented
pH 2.3 and 2.6, respectively, and total
titratable acidity expressed as acetic acid
was 40 g L-1.
Production of E. coli suspension
E. coli ATCC 25922 cephae was
inoculated into brain heart infusion
(BHI) and incubated for 24 h at 35±2°C.
Serial dilutions were performed in 1
g L-1 peptone water (Himedia). and
put on MacConkey agar (Himedia) to
determine bacterial counting (log CFU/
mL).
Determination of the minimum
concentration to inhibit (MIC) E.
coli growth
The MIC of triple-strength alcohol
vinegar, and cereal vinegar against E.
coli was determined by microdilution
in tubes. Aliquots of 100 μL of E. coli
suspension containing approximately
6 log CFU/mL were added to tubes
containing BHI and vinegar at final
concentrations of 0, 2.5, 5.0, 10.0,
15.0, 20.0, or 40.0 g L-1 total acidity in
a nal volume of 5 mL. After 15 min,
5 mL of BHI was added to all tubes.
Tubes were incubated at 35°C for 24
h. MIC was determined in the tube
with no turbidity and with the lowest
concentration of acetic acid, that is, the
minimum concentration that did not
allow bacterial growth.
Determination of the minimum
bactericidal concentration (MBC) of
vinegar
The determination of MBC of
vinegar against E. coli was performed
by plate counting using MacConkey
solid medium. Subsequent to the
determination of MIC, 10 μL aliquots
from the test tubes containing E. coli and
vinegar at dierent concentrations were
inoculated onto MacConkey agar plates.
After 24 h of incubation, MBC was
determined as the lowest concentration
of acetic acid that completely inhibited
E. coli growth.
Efficiency evaluation of the
sanitization procedure using triple-
strength vinegar on artificially
contaminated lettuce leaves with E.
coli
Six lettuce heads were analyzed
individually, representing six replicates
of the sanitization procedure. From each
lettuce head, the inner and outer leaves
were discarded and two 25 g portions of
leaves were weighed. The two portions,
identied as A and B, were contaminated
with 4 mL each of E. coli suspension
containing approximately 6 log CFU/
mL. The suspension was randomly
distributed over the leaves using a
pipette. After the leaves had dried at
room temperature for 3 to 4 h, the units
of E. coli on portion A were counted.
Portion B leaves were immersed for
15 min in a triple-strength vinegar
solution containing 15 g L-1 acetic acid,
considered the MBC. Portion B leaves
were washed with sterile distilled water
and centrifuged in a centrifuge suitable
for handling leafy vegetables. E. coli
counts were then determined.
Portions A (not sanitized) and B
(sanitized) were homogenized separately
in 50 mL of 1 g L-1 buered peptone
water, and serial dilutions from 10-2 to
10-4 were carried out in 1 g L-1 buered
peptone water. Subsequently, 100 μL of
each dilution was plated on MacConkey
agar medium in duplicate. Colonies
were counted after 24 h of incubation
at 35±2°C.
Evaluation of the efficiency of
sanitization procedure using triple-
strength vinegar on naturally
contaminated lettuce leaves
For this analysis, 31 lettuce heads
were purchased commercially in
Londrina, and analyzed individually.
Three 25 g portions of leaves were
collected from each lettuce head and
labelled as A, B, and C. Portions B
and C were washed under chlorinated
running water. Portion C was immersed
in a triple-strength vinegar solution (15
g L-1 acetic acid) for 15 min; the leaves
were then washed in sterile distilled
water and centrifuged in a centrifuge
suitable for handling leafy vegetables.
The most probable numbers (MPN) of
total coliforms and E. coli on portions
A, B, and C were determined by the
multiple-tube fermentation technique
(Silva et al., 2007).
Determination of total titratable
acidity of vinegar
To determine total titratable acidity,
1 mL of triple-strength vinegar and two
drops of a 10 g L-1 phenolphthalein
solution were added to a 100 mL
Erlenmeyer flask. Titration was
performed with 0.1 M sodium hydroxide
until the solution turned red. Each mL of
sodium hydroxide solution corresponded
to 6 g L-1 total titratable acidity expressed
as acetic acid (H3CCOOH) (Spinosa et
al., 2015).
Statistical analysis
E. coli counts on the six articially
contaminated lettuce samples were
converted into decimal logarithms
(log CFU/mL). Data were submitted
to statistical analysis in Microsoft
Excel 2013. Student’s t-test was used
Sanitizing action of triple-strength vinegar against Escherichia coli on lettuce

416 Hortic. bras., Brasília, v.36, n.3, July - September 2018
to determine whether E. coli counts
before and after sanitization with
triple-strength vinegar diered at 5%
signicance level.
RESULTS AND DISCUSSION
The concentrations of triple-strength
vinegar, alcohol vinegar, and cereal
vinegar used for determining MIC and
MBC were 0, 2.5, 5.0, 10.0, 15.0, 20.0,
and 40.0 g L-1 total titratable acidity
expressed as acetic acid. The addition of
5 mL of BHI after 15 min of exposure
of the E. coli inoculum to vinegar was
essential for the determination of MIC
and MBC values. Without this step, it
was not possible to recover viable E. coli
cells, independently of the concentration
of acetic acid used in the experiment.
This procedure allowed the recovery
of E. coli cells damaged by the action
of vinegar after 15 min sanitization
probably because of dilution of the
acetic acid present in the medium at the
end of the experiment. The MIC and
MBC values of the vinegars against E.
coli were respectively 2.5 and 15.0 g
L-1 total titratable acidity expressed as
acetic acid.
A signicant reduction was observed
in the mean E. coli count of articially
contaminated lettuce leaves after
sanitization using 15 g L-1 acetic acid
solution prepared with triple-strength
vinegar (Table 1).
Nascimento et al. (2003) reported
similar results to those of the present
study. The authors compared the
eciency of acetic acid, chlorine, and
peracetic acid for sanitizing lettuce
and recommended the use of 20 g L-1
acetic acid for 15 min by immersion.
Other studies have also evaluated the
bactericidal action of acetic acid on
E. coli using articially contaminated
lettuce, but assays were performed with
dierent concentrations from those used
in the present study. Park et al. (2011)
tested the antimicrobial eciency of
different organic acids and obtained
a 1.57-log CFU/g reduction in E. coli
count using 20 g L-1 acetic acid for 5
min, a result similar to that reported
by Akbas & Olmez (2007), a 1.5-log
CFU/g reduction after 2 min of exposure
of lettuce leaves to 10 g L-1 acetic acid
solution. Vijayakumar & Wolf-Hall
(2002) evaluated the effect of apple
vinegar, white vinegar, chlorine, and
lemon juice against E. coli on lettuce.
The most eective result was achieved
with white vinegar at 19 g L-1 total
titratable acidity expressed as acetic
acid. There was a 5-log reduction in
E. coli counts when samples were
immersed in white vinegar solution
for 5 min under shaking or for 10 min
without shaking.
Bjornsdottir et al. (2006) stated that
many factors aect the antimicrobial
activity of organic acids, such as pH,
acid concentration, bacterial species,
and the environment of bacterial
cultures. Several studies have been
carried out on the bactericidal action
of acetic acid against other pathogenic
bacteria, using varied vegetables and
acetic acid concentrations. Reductions
of up to 1.4 log CFU/g of Listeria
monocytogenes were observed in
artificially contaminated lettuce and
leafy salads sanitized with acetic acid
solutions at concentrations ranging
from 5 to 10 g L-1 (Porto & Eiroa,
2006; Samara & Koutsoumanis, 2009;
Nastou et al., 2012; Ramos et al.,
2014). A reduction of 1.74 log CFU/g in
Salmonella spp. count was obtained by
sanitizing alfalfa and lettuce with 50 and
20 g L-1 acetic acid solution for 10 min,
respectively (Weissinger & Beuchat,
2000; Park et al., 2011). Karapinar &
Gonul (1992) achieved a 7-log CFU/
mL reduction in Yersinia enterocolitica
counts using 20 g L-1 acetic acid.
Other important factors to be
considered in studies that evaluate food
sanitization using vinegar are the food’s
inherent characteristics. Dierences in
bacterial microenvironments formed
on the surfaces of dierent vegetables
should be taken into consideration when
extrapolating the ndings of this study
to other foods. Washing processes may
not be as ecient when there are cracks,
crevices, or interstices on food surfaces
(Ramos et al., 2014).
Studies that tested the performance
of 50 g L-1 acetic acid in the sanitization
of vegetables reported negative changes
to the sensorial characteristics of the
product (Chang & Fang, 2007; Wu
et al., 2000). The results obtained in
the present study using triple-strength
vinegar containing 15 g L-1 acetic acid
are satisfactory from an application point
of view, as the higher the concentration
of acetic acid in the sanitizing solution,
the greater the probability of sensorial
changes in vegetables.
Most of the studies on the ecacy
of sanitizing agents on fruits and
vegetables were carried out with
articially contaminated products. In the
present study, in addition to the analysis
of lettuce articially contaminated with
E. coli, naturally contaminated lettuce
leaves were analyzed. Table 2 shows the
results of log MPN/g of total coliforms
on lettuce samples. Contamination
ranged from 1.63 to 3.38 log MPN/g.
Brazilian legislation does not establish
limits for total coliforms, and their
presence is natural in fresh vegetables
because of the type of cultivation.
Berbari et al. (2001) stated, however,
that total coliform counts can be used
Table 1. E. coli counts in articially contaminated lettuce leaves before and after sanitization
with vinegar (15 g L-1 total titratable acidity expressed as acetic acid). Londrina, UEL, 2014.
Sample E. coli count before sanitization
(log CFU/g)
E. coli count after sanitization
(log CFU/g)
1 2.99a 2.11b
2 3.00a 0.60b
3 3.04a 0.78b
4 3.36a 0.00b
5 3.36a 0.78b
6 3.88a 0.30b
Mean 3.27 ± 0.31a0.76 ± 0.66b
Counts and mean counts followed by dierent letters dier signicantly (p≤0.05).
GC Souza et al.

417Hortic. bras., Brasília, v.36, n.3, July - September 2018
as an indicator of hygiene and are
considered high when above 5.0 log
MPN/g in foods. All samples analyzed
in this study were contaminated with
total coliforms but none showed counts
greater than 5.0 log MPN/g, as opposed
to other studies, in which high counts
of total coliforms were identied in the
samples (Cabrini et al., 2002; Oliveira
& Figueiredo, 2006).
The results of the sanitization of
naturally contaminated lettuce using
the triple-strength vinegar solution
containing 15 g L-1 acetic acid are in
Table 2. A reduction of 1.03 to 3.38 log
MPN/g in total coliform bacteria was
observed. This reduction was greater
than that obtained by washing samples
in running chlorinated water (0-2.5 log
MPN/g).
Statistical analysis was performed
only on the results of E. coli counts
of the six artificially contaminated
lettuce samples before and after vinegar
sanitization (Table 1). The results
of total coliform counts in naturally
contaminated lettuce before and after
sanitization (Table 2) were not submitted
to statistical analysis because of the
great variation between initial and nal
bacterial counts. The purpose was to
present the reduction in bacterial count
promoted by the treatments as raw data.
Other studies also evaluated the
performance of acetic acid solutions
in the sanitization of naturally
contaminated vegetables. Oliveira et
al. (2012) reported a reduction of 2.09
log CFU/g in total coliforms on lettuce
using 8 g L-1 acetic acid. Nascimento
et al. (2003) used 20 g L-1 acetic
acid and obtained a 3.57-log CFU/g
reduction in mesophilic aerobes on
lettuce samples. Fantuzzi et al. (2004)
obtained a reduction of 1.8 log CFU/g
in mesophilic aerobes after sanitizing
cabbage samples for 10 min with 10 g
L-1 acetic acid at room temperature.
A total of 23 (74.2%) of the 31
naturally contaminated lettuce samples
analyzed in this study were contaminated
with E. coli before washing with
water. All counts were below the limit
established by Brazilian legislation and
ranged from 0.4 to 46 log MPN/g. E.
coli was isolated from nine of the 23
contaminated samples after sanitization
with running chlorinated water and
from one sample after sanitization
using triple-strength vinegar solution
containing 15 g L-1 acetic acid.
Washing lettuce leaves under running
chlorinated water and immersing them
in 15 g L-1 acetic acid solution for 15 min
promoted a reduction in E. coli counts
on articially contaminated samples and
a reduction in total coliform counts in
naturally contaminated samples. There
were no visual changes in treated lettuce
leaves, which indicates that vinegar
at 15 g L-1 total titratable acidity can
be used to sanitize vegetables without
aecting their appearance.
ACKNOWLEDGMENTS
The authors thank the Coordination
for the Improvement of Higher
Table 2. Total coliform counts in naturally contaminated lettuce leaves2 and count reductions
after washing samples under running chlorinated water3 and after washing samples under
running chlorinated water and sanitizing with vinegar solution (15 g L-1)4 (total titratable
acidity expressed as acetic acid). Londrina, UEL, 2014.
Sample1Total coliform
count (log MPN/g)2
Total coliform count
after washing (log
MPN/g)3
Total coliform count
after washing and
sanitizing (log MPN/g)4
1 2.38 0.00 2.38
2 2.38 1.75 2.38
3 2.38 1.41 2.02
4 2.38 1.93 2.02
5 1.66 1.49 1.66
6 2.38 1.41 2.38
7 2.38 1.41 1.75
8 2.38 0.34 2.02
9 2.38 0.72 1.20
10 2.38 2.20 2.38
11 2.38 1.20 1.41
12 1.66 1.03 2.06
13 2.04 1.07 2.04
14 2.38 1.41 1.75
15 2.38 1.06 2.38
16 1.63 0.79 1.03
17 1.97 1.37 1.97
18 2.66 1.34 2.66
19 3.38 2.54 2.78
20 3.04 2.44 3.04
21 2.32 0.69 1.72
22 3.04 2.09 3.04
23 3.04 0.87 2.44
24 3.38 1.41 3.38
25 2.32 1.00 1.72
26 3.04 0.00 2.09
27 3.38 0.73 2.02
28 3.04 0.39 1.41
29 3.38 0.73 3.38
30 3.04 1.68 3.04
31 3.38 1.00 2.78
1Samples purchased at dierent commercial establishments in Londrina, Paraná, Brazil.
Sanitizing action of triple-strength vinegar against Escherichia coli on lettuce

418 Hortic. bras., Brasília, v.36, n.3, July - September 2018
Education Personnel (CAPES) for
granting a master’s scholarship to
Giovanna Cavagnari de Souza and the
National Council for Scientific and
Technological Development (CNPq) for
the nancial support provided.
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