Food Microbiology 73 (2018) 61e66

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

Use of acetic and citric acids to inhibit Escherichia coli O157:H7,

Salmonella Typhimurium and Staphylococcus aureus in tabbouleh salad
Walid M. Al-Rousan a, *, Amin N. Olaimat b, Tareq M. Osaili c, d, Anas A. Al-Nabulsi d,
Radwan Y. Ajo a, Richard A. Holley e
a
Department of Nutrition and Food Processing, Al-Huson University College, Al-Balqa Applied University, PO Box 50, 21510 Al-Huson, Jordan
b
Department of Clinical Nutrition and Dietetics, Faculty of Allied Health Sciences, The Hashemite University, PO Box 150459, Zarqa 13115, Jordan
c
Department of Clinical Nutrition and Dietetics, Research Institute of Medical and Health Sciences, College of Health Sciences, University of Sharjah,
Sharjah, United Arab Emirates
d
Department of Nutrition and Food Technology, Faculty of Agriculture, Jordan University of Science and Technology, Irbid 22110, Jordan
e
Department of Food and Human Nutritional Sciences, Faculty of Agriculture and Food Sciences, University of Manitoba, Winnipeg, MB R3T 2N2 Canada

a r t i c l e i n f o a b s t r a c t

Article history: The objective of the current study was to evaluate the antimicrobial action of different concentrations of
Available online 2 January 2018 acetic (0.3% and 0.4%) or citric (1% and 1.4%) acids and their combinations (1% citric acid plus 0.4% acetic
acid and 1.4% citric acid plus 0.3% acetic acid) against Salmonella Typhimurium, Escherichia coli O157:H7
Keywords: and Staphylococcus aureus in tabbouleh salad stored at 21, 10 and 4
C. Acetic acid was more inhibitory
Tabbouleh salad toward S. Typhimurium and E. coli O157:H7 than citric acid at 21
C; S. Typhimurium and E. coli O157:H7
Acetic acid
cells were not detected in tabbouleh treated with 0.4% acetic acid after 5 and 7 days, respectively. The
Citric acid
combined effect of acetic and citric acid was synergistic against S. Typhimurium, and E. coli O157:H7, but
E. coli O157:H7
Salmonella
not against S. aureus. The combinations of acetic and citric acids reduced S. Typhimurium, and E. coli
S. aureus O157:H7 to below the detection levels after 2 and 3 days at 21
C, respectively. However, these treat-
ments significantly reduced S. aureus numbers compared to the control at tested temperatures by the
end of storage. Acetic and citric acids have the potential to be used in tabbouleh salad to reduce the risk
from S. Typhimurium, E. coli O157:H7 and S. aureus.
© 2018 Published by Elsevier Ltd.

1. Introduction
Tabbouleh is a traditional popular Middle Eastern salad
consumed as an appetizer, and recently its popularity has grown to
the extent that it has now become an international dish (Khiyami
et al., 2011; Zubaida, 2000). It has been reported that tabbouleh
along with other Arabic cuisine like hummus, baba ghanouj and
pita have become popular “American ethnic foods” (Zelinsky,
2001). Tabbouleh is composed of soaked bulgur (coarse ground
wheat), chopped parsley, tomato, cucumber, onion, herb mint, olive
oil, citric acid (lemon juice) and salt. However, the recipe in some
countries may include additional ingredients such as garlic and
lettuce (Zubaida, 2000).
Tabbouleh can become contaminated by foodborne pathogens
from its fresh ingredients, or during its preparation and some
* Corresponding author.
E-mail address: Walid.al-rousan@bau.edu.jo (W.M. Al-Rousan).
contaminants may grow during storage. Recently, the demand for
fresh produce has increased because of its convenience and nutri-
tional value; however, foodborne outbreaks associated with it have
increased worldwide (Olaimat and Holley, 2012). Several undesir-
able microorganisms such as Escherichia coli, Enterobacter aero-
genes, Shigella spp., and Salmonella spp. have occasionally been
isolated from tabbouleh (Al-Jobori and Al-Bakri, 2015; Khiyami
et al., 2011). This type of salad can support microbial growth
because it is a good source of nutrients, has high water activity (aw)
and is often prepared without controlling the pH (Al-Jobori and Al-
Bakri, 2015; Khiyami et al., 2011). Moreover, it is not consistently
refrigerated and may be held at room temperature for several
hours. During such periods and because there is inadequate
awareness of the sanitary needs for its preparation, large numbers
of microorganisms, along with pathogenic bacteria, can be exposed
to the food. Among these microorganisms are Escherichia coli
O157:H7, Salmonella and Staphylococcus aureus which have
emerged as major foodborne pathogens and have been implicated
in illness outbreaks that have involved a variety of salads (Olaimat

https://doi.org/10.1016/j.fm.2018.01.001
0740-0020/© 2018 Published by Elsevier Ltd.
62 W.M. Al-Rousan et al. / Food Microbiology 73 (2018) 61e66
and Holley, 2012).
The interest in applying natural antimicrobial compounds to
food products has increased because of health concerns. Organic
acids are natural food additives that can be incorporated either by
direct addition or through microbiological fermentation of food
products in order to enhance their flavor, taste, quality and/or
safety. Organic acids are considered as generally recognized as safe
(GRAS) by the United States Food and Drug Administration (Doores,
2005; Mani-Lo pez et al., 2012). The antimicrobial activity of organic
acids can be affected by the acid type, exposure time, food com-
ponents and temperature. The antimicrobial activities of citric and
acetic acids against pathogens have been studied in a variety of
food products such as moutable (eggplant dip) (Osaili et al., 2015;
Skandamis and Nychas, 2000), salads (Alali et al., 2012; Tassou
et al., 2009), hummus (Al-Holy et al., 2006) and tahini (Al-Nabulsi
et al., 2014). However, no information is available about the anti-
microbial activity of citric and acetic acids against foodborne
pathogens in tabbouleh. Therefore, the objective of the current
study was to assess the antimicrobial activity of different concen-
trations of acetic and citric acids or their combination against Sal-
monella spp., E. coli O157:H7 and S. aureus in tabbouleh stored at 4,
10 and 21
C.
2. Materials and methods
2.1. Preparation of tabbouleh
Tabbouleh was prepared with fresh bunches of parsley, to-
matoes, onions, food grade salt, bulgur wheat and olive oil that
were purchased from a local market. All other chemicals, media and
reagents used in the study were from Oxoid Ltd. (Basingstoke, UK).
The fresh flat-leaf parsley and tomatoes were washed twice, first by
soaking in 0.1% chlorine solution as sanitizer for 10 min at room
temperature, then washed carefully with distilled water. The sani-
tized tomatoes and parsley leaves were dried using a sterile cloth.
The parsley and peeled onions, which were washed thoroughly
with potable water that had been chlorinated, were chopped with a
food processor (Conti. Model No.: FP-666 made in P. R. China.),
while tomatoes were cut into small cubes using a sterile knife. After
that, bulgur wheat was placed into a small bowl, covered with
sterile distilled water and held at room temperature until the
bulgur wheat had absorbed all of the water. All ingredients were
tested for the presence of Salmonella spp., E. coli O157:H7 and
Staphylococcus aureus and were found free from these pathogens.
Because there is no official or standard recipe specifying the
percentage of specific ingredients for tabbouleh, we prepared
several batches containing different ingredients and then per-
formed sensory evaluation. The product best ranked in sensory
analysis was selected for further testing. The tabbouleh was pre-
pared by mixing 100 g chopped parsley (35.7% w/w), 100 g chopped
tomatoes (35.7% w/w), 10 g chopped onions (3.6% w/w), 5 g salt
(1.8% w/w), 30 g olive oil (10.7% v/w) and 35 g pre-soaked bulgur
wheat (12.5% w/w). A sterile spoon was used to mix the ingredients
for 5e10 min in a sterile bowl, until they were combined well.
Traditionally, tabbouleh is acidified with lemon juice (which con-
tains mainly citric acid) to achieve an acceptable tabbouleh taste,
and should be sufficiently acidic to decrease the risk of foodborne
illnesses. In the present test series, acetic acid in addition to
different concentrations of citric acid were combined to evaluate
the added potential safety of the combined acids.
Tabbouleh was acidified with the following compounds (w/w):
1% or, 1.4% citric acid; 0.3% or, 0.4% acetic acid and combinations of
1% citric acid plus 0.4% acetic acid or, 1.4% citric acid plus 0.3% acetic
acid.
2.2. Culture preparation
S. Typhimurium (ATCC 14028 and 127) and S. aureus (ATCC
25923) used in this study were provided by the Jordan Food and
Drug Administration. E. coli O157:H7 strains 00e3581 and 02e0304
were provided by Rafiq Ahmed, National Microbiology Laboratory,
Public Health Agency, Canadian Science Centre for Human and
Animal Health, Winnipeg, MB, Canada. S. Typhimurium and E. coli
O157:H7 strains were grown individually in 10 ml sterile Tryptone
Soy Broth (TSB) tubes which were incubated at 37
C for 24 h.
S. aureus strains were grown individually in 10 ml sterile TSB tubes
containing 0.6% (w/v) yeast extract and incubated at 37
C for 24 h.
2.3. Culture inoculation of the tabbouleh
One milliliter of the S. Typhimurium cocktail, E. coli O157:H7
cocktail or S. aureus culture was individually mixed drop-wise with
50 g of the tabbouleh (without acetic or citric acids) and acidified
tabbouleh to yield an initial inoculums level of ~7 log CFU/g.
Inoculated tabbouleh samples were placed in sterile air-tight con-
tainers with covers (one container/sampling time) and stored at 4,
10 and 21
C for 7 days.
2.4. Microbiological enumeration
Tabbouleh in each container was mixed by sterile spatula before
sampling of 10 g into a sterile stomacher bag (Seward Co., Worth-
ing, UK), mixed with 90 ml of sterile 0.1% peptone water and ho-
mogenized with a stomacher (Easy Mix, AES Laboratoire, Bruz,
France) for 2 min. Appropriate decimal dilutions using sterile
peptone water were made. Enumeration of S. Typhimurium, E. coli
O157:H7 and S. aureus cells was determined by spread plating 1 ml
or 0.1 ml of diluted samples onto Xylose Lysine Deoxycholate (XLD)
agar for S. Typhimurium; Sorbitol MacConkey (SMAC) agar for
E. coli O157:H7, and Baird-Parker agar supplemented with egg yolk
tellurite (BP) agar for S. aureus. Inoculated plates were incubated
under aerobic conditions at 37
C for 24 h. A sample without acids
as a control was monitored with each test organism, and all ex-
periments and tests were conducted in duplicate. Microbiological
analyses were conducted at the following intervals: 0, 0.33, 1, 2, 3, 4,
5, 6, and 7 days for all samples stored at 4, 10, or 21
C.
2.5. pH measurements
The initial and final pH values of tabouleh salads were measured
by inserting the glass probe directly in the samples using a pH
meter (Cyberscan 500, Eutech Instr., Singapore).
3. Statistical analysis
The effect of acetic or citric acid and their combinations on the
viable numbers of S. Typhimurium, E. coli O157:H7, and S. aureus
were analyzed with Tukey’s test using JMP 12.0 software (SAS Inst.
Inc., Cary, N.C., U.S.A.) and differences were considered significant
when P < .05. All tests were performed in two experiments with
duplicate analyses and results were expressed as means ± SD.
4. Results
4.1. Antimicrobial activity of acetic or citric acid and their
combinations against E. coli O157:H7
Tabbouleh salad samples treated with the selected concentra-
tions of citric and acetic acids or their combination used during
microbiological tests in the current study were analyzed for the
 W.M. Al-Rousan et al. / Food Microbiology 73 (2018) 61e66 63

consumer acceptability. It was obvious that the organic acids at the
used concentrations enhanced the sensory characteristics (except
1.4% citric acid plus 0.3% acetic acid showed a lower appearance
ranking) of salads and all treated samples had an average rating
close to like moderately (Results not shown).
The inhibitory effects of acetic or citric acid and their combi-
nations against E. coli O157:H7 are presented in Table 1. E. coli
O157:H7 numbers were gradually reduced in control samples
(without organic acids) during storage by 3.6 log CFU/ml at 21
C
after 7 days. However, there was no obvious reduction at 10 or 4
C.
At 21
C, citric acid at 1e1.4% was not inhibitory against E. coli
O157:H7, while at 10 or 4
C it caused additional reduction in
numbers of E. coli along with possible inhibitory effects of olive oil
and salt during incubation. On the other hand, acetic acid was more
active at 21
C than 4
C or 10
C where viable E. coli O157:H7 were
not detected when tabbouleh was treated with 0.40% acetic acid at
21
C. However, slight reductions in E. coli O157:H7 numbers were
observed at 10 or 4
C compared to the control. Acetic acid at 0.3%
gradually reduced E. coli O157:H7 numbers up to 6 days, after that
the pathogen increased in a manner similar to the control. Com-
binations of acetic and citric acids were most effective, with viable
E. coli O157:H7 not being detected after 2 days at 21
C when
treated with 1.4% citric acid and 0.3% acetic acid, or after 3 days
when treated with 1% citric acid and 0.4% acetic acid. At 10 or 4
C,
the latter combination was the most effective against E. coli
O157:H7, where numbers were reduced by 1.9 and 1.4 log 10 CFU/g,
respectively.
4.2. Antimicrobial activity of acetic or citric acid and their
combinations against S. Typhimurium
Table 2 shows the inhibitory effects of acetic or citric acid and
their combinations against S. Typhimurium. Numbers of S. Typhi-
murium were reduced in control samples by 4.1 log CFU/g at 21
C
after 7 days, but this was not observed at refrigerator temperatures
where the numbers were reduced by only 0.4 log CFU/g. Similar to
E. coli O157:H7, S. Typhimurium was more sensitive to acetic than
citric acid. S. Typhimurium numbers were not detected after 5 days
in tabbouleh samples treated with 0.4% acetic acid or after 6 days in
samples treated with 0.3% acetic acid at 21
C. However, these
treatments were less inhibitory at lower temperatures where
numbers of S. Typhimurium were reduced by 1.4 and 1.1 log CFU/g
at 10
C or by 0.9 and 1 log CFU/g at 4
C, compared to the control.
On the other hand, citric acid did not cause any additional inhibi-
tion with salt and olive oil against S. Typhimurium at 21
C, while its
activity was evident at 4 and 10
C. Numbers of S. Typhimurium
were reduced by 2.3 and 3.4 log CFU/g at 10
C or by 1.5 and 4.1 log
CFU/g at 4
C in the samples treated with 1 and 1.4% citric acid,
respectively. The combined effect of acetic and citric acid against S.
Typhimurium was synergistic at all temperatures used. At 21
C S.
Typhimurium cells were not detected after 2 days in tabbouleh
treated with 1.4% citric acid and 0.3% acetic acid or after 3 days in
tabbouleh treated with 1% citric acid and 0.4% acetic acid. At 10
C,
both acid combinations reduced the S. Typhimurium numbers to
below detection levels after 4 days, while at 4
C S. Typhimurium
was not detected in samples treated with 1% citric acid and 0.4%
acetic acid after 2 days, or in samples treated with 1.4% citric acid
and 0.3% acetic acid after 6 days .
4.3. Antimicrobial activity of acetic or citric acid and their
combinations against S. aureus
Unlike Salmonella and E. coli, S. aureus was more resistant to the
acidic treatments used. In control samples without acids, S. aureus
numbers were reduced by 0.5, 2.5 and 1.3 log CFU/g when tab-
bouleh salad was stored at 21, 10 and 4
C, respectively after 7 days.
However, the treatments used showed similar inhibitory effects
(P > .05) by the end of storage, where the numbers of S. aureus were
reduced by 2.7e3.9 at 7 days at 21
C. However at 10
C, combina-
tion treatments (1.4% citric acid and 0.3% acetic acid or 1% citric acid

Table 1
Antimicrobial activity of acetic or citric acid and their combinations against E. coli O157:H7 in Tabbouleh salad stored at different temperatures.

Temperature Time Treatments

Control 1.4% Citric acid 1% Citric acid 0.4%Acetic acid 0.3%Acetic acid 1% Citric acid þ0.4% Acetic acid 1.4% Citric acid þ0.3% Acetic acid

21 0 7.17 ± 0.06a 7.03 ± 0.03a 7.31 ± 0.14a 7.16 ± 0.06a 7.01 ± 0.01a 7.20 ± 0.16a 7.27 ± 0.11a
0.33 7.43 ± 0.56a 6.97 ± 0.41a 6.58 ± 0.03a 7.11 ± 0.13a 7.41 ± 0.37a 6.70 ± 0.01a 6.85 ± 0.41a
1 7.46 ± 0.47a 6.36 ± 0.02a 6.50 ± 0.38a 7.13 ± 0.35a 6.92 ± 0.03a 6.30 ± 0.04a 6.36 ± 0.48a
2 7.43 ± 0.19a 5.42 ± 0.38b 6.50 ± 0.38ab 6.81 ± 0.27a 6.70 ± 0.03ab 7.10 ± 0.62a ND
3 6.72 ± 0.21a 4.58 ± 0.27c 4.55 ± 0.11c 6.49 ± 0.23a 5.78 ± 0.16b ND ND
4 5.79 ± 0.05a 4.53 ± 0.05b 4.40 ± 0.29b 5.19 ± 0.60ab 5.39 ± 0.36ab ND ND
5 5.39 ± 0.13a 5.40 ± 0.34a 5.30 ± 0.18a 4.66 ± 0.26a 3.40 ± 0.35b ND ND
6 4.78 ± 0.54a 4.77 ± 0.21a 5.54 ± 0.41a 3.40 ± 0.35b 3.19 ± 0.28b ND ND
7 4.56 ± 0.07a 5.31 ± 0.24a 4.98 ± 0.67a ND 4.81 ± 0.07a ND ND
10 0 7.19 ± 0.30a 7.01 ± 0.16a 7.30 ± 0.14a 7.05 ± 0.22a 7.00 ± 0.01a 7.39 ± 0.37a 7.27 ± 0.03a
0.33 7.09 ± 0.02a 6.98 ± 0.02a 6.92 ± 0.62a 7.41 ± 0.07a 7.11 ± 0.09a 6.96 ± 0.18a 7.11 ± 0.74a
1 7.14 ± 0.17ab 6.62 ± 0.18abc 7.08 ± 0.08ab 7.21 ± 0.20a 7.22 ± 0.18a 6.50 ± 0.10bc 6.42 ± 0.21c
2 6.95 ± 0.62a 6.57 ± 0.45a 6.76 ± 0.04a 6.61 ± 0.27a 7.15 ± 0.15a 6.49 ± 0.10a 6.36 ± 0.25a
3 5.99 ± 0.10ab 5.17 ± 0.06ab 5.54 ± 0.42ab 5.55 ± 0.53ab 6.37 ± 0.26a 4.93 ± 0.49b 6.08 ± 0.16ab
4 7.02 ± 0.27a 5.67 ± 0.42b 5.82 ± 0.11b 6.61 ± 0.40ab 7.03 ± 0.07a 6.13 ± 0.06ab 6.04 ± 0.21ab
5 6.77 ± 0.04ab 4.32 ± 0.12d 5.17 ± 0.19cd 6.25 ± 0.19abc 7.03 ± 0.67a 5.14 ± 0.23cd 5.60 ± 0.19bc
6 6.66 ± 0.15ab 4.80 ± 0.01d 5.02 ± 0.06cd 6.39 ± 0.10ab 7.04 ± 0.29a 5.73 ± 0.59bcd 6.01 ± 0.30abc
7 6.64 ± 0.11a 4.83 ± 0.30d 4.95 ± 0.09cd 6.17 ± 0.46ab 6.13 ± 0.07abc 4.74 ± 0.56d 5.36 ± 0.15bcd
4 0 7.08 ± 0.11a 6.82 ± 0.33a 7.14 ± 0.01a 7.18 ± 0.03a 7.00 ± 0.04a 7.12 ± 0.21a 7.78 ± 1.06a
0.33 7.29 ± 0.11a 6.40 ± 0.15c 7.07 ± 0.19ab 7.27 ± 0.01a 7.07 ± 0.08ab 6.78 ± 0.13bc 6.86 ± 0.05abc
1 6.91 ± 0.45a 6.45 ± 0.26a 6.95 ± 0.16a 7.00 ± 0.35a 7.06 ± 0.20a 6.23 ± 0.32a 6.51 ± 0.47a
2 7.30 ± 0.14a 7.09 ± 0.27a 7.27 ± 0.03a 6.28 ± 0.36a 7.32 ± 0.28a 6.28 ± 0.36a 6.75 ± 0.37a
3 7.26 ± 0.64a 6.03 ± 0.11b 6.28 ± 0.04ab 6.52 ± 0.31ab 6.63 ± 0.14a 5.75 ± 0.34b 6.14 ± 0.13ab
4 6.73 ± 0.02a 5.53 ± 0.32b 6.20 ± 0.15ab 6.80 ± 0.08a 6.72 ± 0.27a 5.47 ± 0.32b 5.94 ± 0.20ab
5 6.66 ± 0.34a 5.56 ± 0.15bc 6.13 ± 0.02ab 6.53 ± 0.01a 6.22 ± 0.02ab 5.17 ± 0.07c 5.46 ± 0.46bc
6 6.55 ± 0.16a 4.84 ± 0.23c 5.27 ± 0.02c 6.18 ± 0.01ab 6.43 ± 0.11ab 5.21 ± 0.16c 6.03 ± 0.06b
7 6.20 ± 0.08a 4.81 ± 0.16c 5.72 ± 0.14ab 5.49 ± 0.02b 6.20 ± 0.03a 4.77 ± 0.17c 5.58 ± 0.24b

Data are given as means ± SD. Different letters in the same row are significantly different (P < .05). ND: not detected. The detection limit was 2.0 Log CFU/g.
64 W.M. Al-Rousan et al. / Food Microbiology 73 (2018) 61e66

Table 2
Antimicrobial activity of acetic or citric acid and their combinations against S. Typhimurium in Tabbouleh salad stored at different temperatures.

Temperature Time Treatments

Control (T1) 1.4% Citric acid 1% Citric acid 0.4% Acetic acid 0.3% Acetic acid 1% Citric acid þ 0.4% Acetic acid 1.4% Citric acid þ 0.3% Acetic
(T2) (T3) (T4) (T5) (T6) acid (T7)

21 0 7.06 ± 0.09a 6.59 ± 0.47a 7.00 ± 0.01a 7.02 ± 0.04a 6.82 ± 0.10a 7.03 ± 0.11a 7.01 ± 0.17a
0.33 7.23 ± 0.30a 6.64 ± 0.04a 6.91 ± 0.03a 6.89 ± 0.17a 7.11 ± 0.20a 6.40 ± 0.04a 6.67 ± 0.43a
1 7.16 ± 0.03a 6.21 ± 0.14ab 6.06 ± 0.08ab 6.95 ± 0.14a 6.89 ± 0.31a 5.43 ± 0.12b 2.45 ± 0.64c
2 6.95 ± 0.25a 6.00 ± 0.01a 6.24 ± 0.34a 6.43 ± 0.32a 6.54 ± 0.07a 2.25 ± 0.35b ND
3 6.70 ± 0.43a 4.34 ± 0.19d 4.95 ± 0.07cd 5.48 ± 0.18bc 6.05 ± 0.07ab ND ND
4 5.30 ± 0.32a 4.13 ± 0.03b 4.22 ± 0.31b 4.18 ± 0.11b 4.48 ± 0.25b ND ND
5 4.41 ± 0.04a 3.92 ± 0.11b 4.29 ± 0.19a ND 3.74 ± 0.06b ND ND
6 3.82 ± 0.12a 3.00 ± 0.00b 3.88 ± 0.11a ND ND ND ND
7 3.00 ± 0.00a 2.74 ± 0.06b 3.00 ± 0.00a ND ND ND ND
10 0 6.94 ± 0.09a 7.00 ± 0.06a 6.99 ± 0.12a 6.98 ± 0.19a 7.12 ± 0.14a 6.98 ± 0.03a 6.75 ± 0.01a
0.33 7.26 ± 0.26a 7.09 ± 0.26a 7.19 ± 0.12a 7.17 ± 0.18a 7.19 ± 0.01a 7.22 ± 0.35a 7.11 ± 0.30a
1 6.97 ± 0.04a 7.12 ± 0.18a 7.01 ± 0.20a 6.96 ± 0.08a 7.23 ± 0.14a 5.78 ± 0.13b 4.00 ± 0.01c
2 7.09 ± 0.12a 5.58 ± 0.07c 6.48 ± 0.03b 6.34 ± 0.06b 6.66 ± 0.33ab 5.03 ± 0.01d 3.10 ± 0.00e
3 5.84 ± 0.08ab 3.85 ± 0.21d 5.05 ± 0.13c 5.61 ± 0.17b 6.16 ± 0.03a 3.42 ± 0.17d 2.70 ± 0.0e
4 6.87 ± 0.20a 4.82 ± 0.12b 5.01 ± 0.87b 5.43 ± 0.01b 6.87 ± 0.14a ND ND
5 6.57 ± 0.20a 4.64 ± 0.01c 5.10 ± 0.11bc 5.18 ± 0.22bc 5.52 ± 0.37b ND ND
6 6.57 ± 0.03a 3.79 ± 0.02e 4.69 ± 0.05d 5.85 ± 0.06c 6.13 ± 0.12b ND ND
7 6.50 ± 0.57a 3.12 ± 0.17d 4.20 ± 0.13cd 5.09 ± 0.36bc 5.42 ± 0.11b ND ND
4 0 7.02 ± 0.04a 7.00 ± 0.15a 6.91 ± 0.23a 7.03 ± 0.15a 6.98 ± 0.11a 6.90 ± 0.07a 6.63 ± 0.53a
0.33 7.20 ± 0.33a 6.95 ± 0.10a 7.51 ± 0.18a 7.06 ± 0.19a 7.20 ± 0.23a 6.32 ± 0.70a 6.65 ± 0.13a
1 6.99 ± 0.05a 5.19 ± 0.16b 6.91 ± 0.18a 6.93 ± 0.35a 6.94 ± 0.41a 4.96 ± 0.05b 4.98 ± 0.03b
2 7.14 ± 0.08a 5.00 ± 0.01b 7.13 ± 0.06a 6.97 ± 0.03a 7.19 ± 0.24a ND 4.70 ± 0.30b
3 6.58 ± 0.33a 4.24 ± 0.34b 6.22 ± 0.11a 6.48 ± 0.02a 6.85 ± 0.15a ND 3.40 ± 0.00c
4 6.82 ± 0.13a 3.98 ± 0.03c 5.77 ± 0.26b 6.44 ± 0.55ab 6.45 ± 0.16ab ND 2.57 ± 0.04d
5 6.76 ± 0.31a 4.26 ± 0.12c 5.42 ± 0.58b 6.28 ± 0.08ab 5.49 ± 0.22b ND 2.62 ± 0.11d
6 6.54 ± 0.03a 2.85 ± 0.21c 5.03 ± 0.16b 6.45 ± 0.03a 6.27 ± 0.05a ND ND
7 6.61 ± 0.39a 2.56 ± 0.54c 5.11 ± 0.09b 5.57 ± 0.03b 5.69 ± 0.14ab ND ND

Data are given as means ± SD. Different letters in the same row are significantly different (P < .05). ND: not detected. The detection limit was 2.0 Log CFU/g.

Table 3
Antimicrobial activity of acetic or citric acid and their combinations against S. aureus in Tabbouleh salad stored at different temperatures.

Temperature Time Treatments

Control (T1) 1.4% Citric acid 1% Citric acid 0.4% Acetic acid 0.3% Acetic acid 1% Citric acid þ 0.4% Acetic acid 1.4% Citric acid þ 0.3% Acetic
(T2) (T3) (T4) (T5) (T6) acid (T7)

21 0 7.17 ± 0.07a 6.98 ± 0.04ab 6.85 ± 0.01ab 6.60 ± 0.22b 6.70 ± 0.21ab 6.58 ± 0.10b 6.64 ± 0.14ab
0.33 6.11 ± 0.78a 5.74 ± 0.45a 6.17 ± 0.77a 6.23 ± 0.84a 5.61 ± 0.49a 5.85 ± 0.63a 5.49 ± 0.60a
1 6.65 ± 0.49a 6.07 ± 0.29ab 5.22 ± 0.54ab 6.31 ± 0.44ab 6.32 ± 0.45ab 4.89 ± 0.37b 6.18 ± 0.03ab
2 6.87 ± 0.12a 6.62 ± 0.66ab 5.69 ± 0.17ab 6.39 ± 0.18ab 6.87 ± 0.10a 5.43 ± 0.59b 3.84 ± 0.09c
3 6.78 ± 0.16a 4.93 ± 0.21b 4.85 ± 0.22bc 4.77 ± 0.07bc 5.32 ± 0.45b 4.02 ± 0.03cd 3.65 ± 0.18d
4 5.94 ± 0.35a 5.23 ± 0.07ab 5.57 ± 0.22ab 5.44 ± 0.16ab 5.71 ± 0.11a 4.95 ± 0.07b 3.38 ± 0.11c
5 7.18 ± 0.37a 5.13 ± 0.19c 4.84 ± 0.18c 6.20 ± 0.08b 6.30 ± 0.35ab 3.81 ± 0.25d 3.38 ± 0.11d
6 7.44 ± 0.46a 5.17 ± 0.13c 4.80 ± 0.29c 6.40 ± 0.11b 6.56 ± 0.27ab 3.80 ± 0.10d 2.36 ± 0.17e
7 6.63 ± 0.48a 3.67 ± 0.20b 3.97 ± 0.70b 3.78 ± 0.30b 3.97 ± 0.25b 3.15 ± 0.25b 2.78 ± 0.30b
10 0 6.96 ± 0.03a 6.98 ± 0.05a 6.83 ± 0.06a 6.93 ± 0.07a 6.69 ± 0.08a 6.82 ± 0.08a 6.89 ± 0.15a
0.33 6.58 ± 0.22a 6.60 ± 0.20a 7.17 ± 0.09a 6.68 ± 0.04a 6.43 ± 0.48a 6.40 ± 0.01a 6.69 ± 0.01a
1 6.85 ± 0.29a 6.73 ± 0.12a 6.97 ± 0.10a 6.52 ± 0.15a 6.85 ± 0.04a 6.62 ± 0.28a 7.04 ± 0.01a
2 6.59 ± 0.04a 6.18 ± 0.15a 6.28 ± 0.06a 6.66 ± 0.18a 6.48 ± 0.01a 6.27 ± 0.06a 5.13 ± 0.23b
3 6.27 ± 0.01a 4.57 ± 0.18c 5.36 ± 0.10b 5.42 ± 0.29b 5.32 ± 0.04b 5.00 ± 0.08bc 3.75 ± 0.14d
4 6.68 ± 0.22a 4.85 ± 0.81b 5.00 ± 0.01b 4.92 ± 0.12b 5.80 ± 0.15ab 4.70 ± 0.12b 3.06 ± 0.09c
5 5.44 ± 0.06a 3.15 ± 0.21b 4.69 ± 0.87a 5.64 ± 0.04a 5.80 ± 0.05a 3.01 ± 0.01b 3.24 ± 0.34b
6 5.59 ± 0.21ab 3.79 ± 0.02c 5.03 ± 0.35b 5.04 ± 0.06b 5.76 ± 0.03a 3.02 ± 0.01d 2.43 ± 0.06d
7 4.47 ± 0.28ab 3.41 ± 0.09bc 3.82 ± 0.07abc 3.93 ± 0.69abc 4.60 ± 0.18a 3.18 ± 0.08c 2.98 ± 0.07c
4 0 6.91 ± 0.31a 6.67 ± 0.39a 6.91 ± 0.35a 6.55 ± 0.27a 6.60 ± 0.03a 6.59 ± 0.13a 6.61 ± 0.18a
0.33 6.64 ± 0.53a 6.60 ± 0.29a 6.30 ± 0.17a 6.46 ± 0.16a 6.49 ± 0.32a 6.39 ± 0.05a 6.21 ± 0.13a
1 6.68 ± 0.47a 6.02 ± 0.08a 6.32 ± 0.01a 6.67 ± 0.13a 6.30 ± 0.06a 6.02 ± 0.08a 5.95 ± 0.11a
2 6.60 ± 0.17a 5.91 ± 0.34ab 6.48 ± 0.18ab 5.78 ± 0.16b 6.48 ± 0.17ab 6.18 ± 0.01ab 6.03 ± 0.01ab
3 6.59 ± 0.08a 4.87 ± 0.14c 5.43 ± 0.16bc 5.15 ± 0.32bc 5.65 ± 0.21bc 5.76 ± 0.26ab 5.25 ± 0.27bc
4 5.79 ± 0.16a 4.78 ± 0.06d 5.36 ± 0.10bc 5.74 ± 0.01a 5.66 ± 0.02ab 4.92 ± 0.12d 5.01 ± 0.02cd
5 6.02 ± 0.01a 4.05 ± 0.07e 5.49 ± 0.11b 4.94 ± 0.13c 5.74 ± 0.05ab 4.95 ± 0.07c 4.58 ± 0.08d
6 5.89 ± 0.33a 3.12 ± 0.05d 5.28 ± 0.07ab 4.77 ± 0.28b 5.63 ± 0.35a 4.45 ± 0.03bc 3.60 ± 0.03cd
7 5.60 ± 0.35a 2.38 ± 0.32c 3.74 ± 0.24b 2.72 ± 0.01bc 3.41 ± 0.35bc 3.24 ± 0.37bc 2.40 ± 0.14c

Data are given as means ± SD. Different letters in the same row are significantly different (P < .05). ND: not detected. The detection limit was 2.0 Log CFU/g.

and 0.4% acetic acid) were significantly more inhibitory than each citric acid or its combination with 0.3% acetic acid were the most
acid used separately, where numbers of S. aureus were reduced by inhibitory treatments against S. aureus which was reduced by 3.2
1.3e1.5 log CFU/g after 7 days compared to the control. At 4
C 1.4% log CFU/g by 7 days. Other treatments used reduced the S. aureus
 W.M. Al-Rousan et al. / Food Microbiology 73 (2018) 61e66
Table 4
Changes in pH of Tabbouleh salad treated with acetic and/or citric acids and stored at different temperatures.
Time Temperature Treatments
Control 1.4% Citric acid 1% Citric acid 0.4% Acetic acid 0.3% Acetic acid 1% Citric acid þ0.4% Acetic acid 1.4% Citric acid þ0.3% Acetic acid
a
Initial pH 4.97 ± 0.08 3.72 ± 0.02a 3.86 ± 0.06a 4.11 ± 0.35a
Final pH 4 4.72 ± 0.33a 3.15 ± 0.25a 3.49 ± 0.08a 4.12 ± 0.01a
10 4.30 ± 0.26a 3.18 ± 0.15a 3.39 ± 0.22a 4.10 ± 0.08a
21 3.69 ± 0.17b 3.62 ± 0.30a 3.60 ± 0.15a 3.51 ± 0.12b
Data are given as means ± SD. Different letters in the same column are significantly different (P < .05).
numbers by 1.9e2.9 log CFU/g by 7 days (Table 3).
5. Discussion
Salmonella and E. coli showed less ability to survive in tabbouleh
than S. aureus at 21
C and this may have been because S. aureusis
better able to tolerate salt stress (Garzoni and Kelley, 2009). In
general, acetic acid showed greater inhibitory activity than citric
acid at room temperature, while at refrigerator temperatures (4 and
10
C) citric acid showed the highest activity against S. Typhimu-
rium and E. coli O157:H7, which were not detected in tabbouleh
salad at 21
C, 5 and 7 days following the addition of 0.4% acetic
acid. Similar results were obtained by Al-Nabulsi et al. (2014) when
acetic and citric acids were used to reduce S. Typhimurium in
tahini. However, in another study citric acid at 0.4e0.8% reduced
the numbers of the same strains of S. Typhimurium, E. coli O157:H7
and S. aureus in eggplant dip by 1e1.5 log CFU/g after 7 days at 10 or
21
C. At 4
C, citric acid showed lower inhibitory effects against S.
Typhimurium and E. coli O157:H7, but it was more inhibitory
against S. aureus and the reduction reached to 3.2 to 3.6 log CFU/g
after 15 days (Osaili et al., 2015). It was clear that a variety of factors
including substrate (medium), temperature, organisms, type and
concentration of organic acids may affect the inhibitory activity.
Moreover, the antimicrobial activity of an organic acid depends on
its pKa value and thus, they are more active under acidic conditions
because of the presence of a higher proportion of the organic acid in
the undissociated form, which can pass through the bacterial
membrane and cause a reduction in internal cell pH following the
dissociation of hydrogen ions. However, other factors may be
involved in determining the antimicrobial activity of organic acids
such as pH reduction in the environment, disruption of membrane
transport and/or permeability, and anion accumulation (FDA, 2014;
Doores, 2005).
The combination of acetic and citric acids showed higher anti-
microbial activity than when either citric or acetic acid was used
individually. In some instances this may have been because of a
reduction in the pH of tabbouleh salads (Table 4). The initial pH of
tabbouleh salad samples treated with a mixture of citric acid plus
acetic acid was around 3.2, while the pH values of samples treated
with citric or acetic acids individually ranged from 3.7 to 4.2. In
contrast, Sengun and Karapinar (2005a, 2005b) found that the
addition of vinegar did not enhance the antimicrobial activity of a
washing solution containing lemon juice with 3.3% citric acid
against Yersinia enterocolitica on carrots or S. Typhimurium on
whole rocket salad leaves and shredded spring onion. However, the
dipping of carrots in a mixture of lemon juice and vinegar for
30 min was more effective in eliminating S. Typhimurium cells than
each agent alone (Sengun and Karapinar, 2005b). It should also be
noted that the initial pH of the control decreased from 4.97 to 3.69
after 7 days at 21
C which may have been due to the growth of
lactic acid bacteria and may have enhanced the reduction of S.
Typhimurium and E. coli O157:H7. Exposure of foodborne patho-
gens to severe stress or multiple-antimicrobials may rapidly lead to
65
4.18 ± 0.28a 3.15 ± 0.41a 3.24 ± 0.47a
4.15 ± 0.08a 3.14 ± 0.02a 3.32 ± 0.09a
4.33 ± 0.06a 3.28 ± 0.11a 3.22 ± 0.25a
3.61 ± 0.28b 3.28 ± 0.15a 3.10 ± 0.28a
exhaustion and death of organisms because of the additional en-
ergy required to overcome stress and maintain cell viability (Yousef
and Juneja, 2003). In the current study, pathogens were exposed to
several antimicrobials (citric acid, acetic acid, salt and olive oil)
simultaneously which caused sharp reductions in E. coli O157:H7
and Typhimirium viability, particularly when citric was combined
with acetic acid.
6. Conclusions
Acetic acid showed higher activity than citric acid against S.
Typhimurium, and E. coli O157:H7 in tabbouleh salad at room
temperature (21
C). Acetic acid at 0.4% reduced numbers of S.
Typhimurium and E. coli O157:H7 to undetectable levels in tab-
bouleh by 5 and 7 days, respectively, while citric acid was more
active at refrigerator temperatures (4 and 10
C). S. aureus was more
resistant to both acids compared to S. Typhimurium and E. coli
O157:H7. The combination of both acids was more effective than
each acid alone against S. Typhimurium and E. coli O157:H7, but not
against S. aureus. The combination of 0.4% citric acid with 0.3%
acetic acid or 1% citric acid plus 0.4% acetic acid reduced S. Typhi-
murium and E. coli O157:H7 to undetectable levels within 2 and 3
days at 21
C, respectively. These combinations also reduced S.
Typhimurium to undetectable levels by  6 days at 4 and 10
C.
Acknowledgments
The authors thank Al-Balqa’ Applied University for funding the
project (18/2014/2015).
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