Determination of the survival levels of acid-adapted Escherichia coli O157:H7in sucuk (Turkish-type fermented sausage)

Abstract

This study aimed to determine the time taken for Escherichia coli O157:H7 to adapt to different types of acids and the effect of acid adaptation on the survival level of E. coli O157:H7 in sucuk (Turkish-type fermented sausage). E. coli O157:H7 cells were adapted to acids in a tryptic soy broth (TSB) set to pH 5.5 for 1-, 2-, 3-, and 4-h periods using hydrochloric, acetic, lactic, and citric acid. The acid tolerance of acid-adapted and nontreated cells was determined in TSB of pH 3.5 and then acid-adapted and nontreated E. coli O157:H7 (105 CFU/g) was added to sucuk. In the adaptation trial in which acetic acid was used, the highest acid resistance was seen in the 3-h adaptation samples, and in the lactic acid trials, the highest acid resistance was seen in the 3- and 4-h adaptation samples. In the adaptation trials with hydrochloric acid, it was observed that the duration of the adaptation period did not increase the resistance. It was determined that the survival rate of E. coli O157:H7 in sucuk was significantly increased through acid adaptation. As a result, it was understood that acid-adapted E. coli O157:H7 can maintain its viability in sucuk for at least 15 days.

Full text

485
http://journals.tubitak.gov.tr/veterinary/
Turkish Journal of
Veterinary and Animal Sciences Turk J Vet Anim Sci
(2015) 39: 485-492
© TÜBİTAK
doi:10.3906/vet-1401-96
Determination of the survival levels of acid-adapted Escherichia coli O157:H7
in sucuk (Turkish-type fermented sausage)
Fatma ÖZTÜRK1,*, Abdulkadir HALKMAN2
1Department of Fishing and Processing Technology, Faculty of Fisheries, İzmir Kâtip Çelebi University, Çiğli, İzmir, Turkey
2Department of Food Engineering, Faculty of Engineering, Ankara University, Dışkapı, Ankara, Turkey
* Correspondence: fatmayaman26@hotmail.com
1. Introduction
Escherichia coli O157:H7 is a foodborne pathogen that
causes hemorrhagic colitis and hemolytic uremic syndrome
(1,2). Inadequately cooked meat and unpasteurized milk
have led to a large number of infections resulting from
E. coli O157:H7 (3,4). ere have also been reports of
E. coli infections originating from acidic foods such as
apple cider, yogurt, and mayonnaise (5,6). e increase of
infections originating from these kinds of foods, which are
generally accepted as safe, has led to a rise in the number
of questions about the safety of acidic foods.
e acid tolerance response (ATR) occurs when
pathogen bacteria gain resistance to highly acidic media
by the synthesis of acid shock proteins, which is controlled
by specic genes. is process results from exposure
to moderately acidic (pH 5.0–6.0) media over a certain
period of time (7–12).
ATR allows E. coli O157:H7 to maintain its vitality in
both acidic and fermented foods by gaining resistance to
acids; thus, it can survive for an extended period of time
in acidic foods such as apple cider and mayonnaise (6),
fermented meat, and dairy products (7,13).
Sucuk (Turkish-type fermented sausage) is a traditional
meat product popular with Turkish people, and there are
other very similar fermented sausages such as saucisson,
chorizo, salami, summer sausage, pepperoni, teewurst,
and mettwurst widely consumed in many other countries
(14). Globally, dried fermented meat products such as
sucuk are considered to be a safe food group for humans.
Factors such as antimicrobial metabolites produced
by starter cultures, low pH, and low water activity in
fermented sausages made from raw ingredients inhibit
the growth of foodborne pathogens (15–17). However, in
recent years, it was determined that E. coli O157:H7 could
maintain its viability in these ‘safe’ foods for a long time
(18).
e purpose of the study was to determine the time
taken for E. coli O157:H7 to adapt to dierent types of acids
and the eect of acid adaptation on the survival level of E.
coli O157:H7 in sucuk (Turkish-type fermented sausage).
2. Materials and methods
2.1. Types and growth conditions of bacteria
e E. coli O157:H7 KUEN 1461 strain supplied by the
culture collection of the Food Engineering Department of
Ankara University was used in this study. Stock cultures of
E. coli O157:H7 were kept at –20 ± 2 °C in tryptic soy broth
(TSB, Merck) including glycerol (20%; v/v). For the trials,
Abstract: is study aimed to determine the time taken for Escherichia coli O157:H7 to adapt to dierent types of acids and the eect of
acid adaptation on the survival level of E. coli O157:H7 in sucuk (Turkish-type fermented sausage). E. coli O157:H7 cells were adapted
to acids in a tryptic soy broth (TSB) set to pH 5.5 for 1-, 2-, 3-, and 4-h periods using hydrochloric, acetic, lactic, and citric acid. e
acid tolerance of acid-adapted and nontreated cells was determined in TSB of pH 3.5 and then acid-adapted and nontreated E. coli
O157:H7 (105 CFU/g) was added to sucuk. In the adaptation trial in which acetic acid was used, the highest acid resistance was seen in
the 3-h adaptation samples, and in the lactic acid trials, the highest acid resistance was seen in the 3- and 4-h adaptation samples. In the
adaptation trials with hydrochloric acid, it was observed that the duration of the adaptation period did not increase the resistance. It
was determined that the survival rate of E. coli O157:H7 in sucuk was signicantly increased through acid adaptation. As a result, it was
understood that acid-adapted E. coli O157:H7 can maintain its viability in sucuk for at least 15 days.
Key words: Escherichia coli O157:H7, acid adaptation, acid tolerance, Turkish-type fermented sausage, sucuk
Received: 29.01.2014 Accepted/Published Online: 15.04.2015 Printed: 28.08.2015
Research Article

486
ÖZTÜRK and HALKMAN / Turk J Vet Anim Sci
100 µL of stock culture was added to 10 mL of TSB and
incubated for 24 h at 37 °C to activate the bacteria. is
activation process was repeated twice.
A lyophilized commercial starter culture (Lyocarni RBL-
73 from Sacco; http://www.saccosrl.it/pdfSchedeTecniche/
M92RBL730UK0.pdf) was used for the sucuk production.
e culture contained Staphylococcus carnosus,
Staphylococcus xylosus, and Lactobacillus curvatus strains
and was kept at –20 ± 2 °C.
2.2. Preparation of acid-adapted E. coli O157:H7 cells
e method dened by Cheng et al. (19) was slightly
modied and used in the preparation of the acid-adapted
cells. First, 5 µL of active E. coli O157:H7 was transferred
into 50 mL of TSB and incubated for 14 h at 37 °C. Aer
incubation, 9 mL of the activated culture was centrifuged
at 5000 rpm (Hettich EBA 12; Germany) for 10 min.
e suspended pellets were washed twice with 0.85%
NaCl solution. e pellets were transferred into 10 mL of
TSB that was set to pH 5.5 using 6 N HCl (37%; Merck),
5 M acetic acid (100%; Merck), 3 M lactic acid (85% DL-
lactic acid; Sigma), and 1 M citric acid (99.5%; Merck)
solutions and then incubated for 1, 2, 3, and 4 h at 37 °C.
For the preparation of the nontreated cells, 5 µL
of active culture was transferred to 50 mL of TSB and
incubated for 14 h at 37 °C. Aer incubation, the cultures
were centrifuged and the pellet was suspended in 10 mL of
TSB at pH 7.0.
2.3. Acid tolerance of E. coli O157:H7
To determine the resistance of E. coli O157:H7 cells to
strongly acidic conditions, samples of 50 mL of TSB (pH
3.5) were inoculated with acid-adapted and nontreated E.
coli O157:H7 cultures using HCl, acetic, lactic, and citric
acids and incubated at 37 °C.
For the HCl trials, aer 0, 1, 2, 3, 4, 5, 24, and 48 h
of incubation, 0.1 mL of culture was transferred to tryptic
soy agar (TSA, Merck) with the incubation periods being
0, 30, 60, 90, and 120 min for the organic acids. All results
were given as CFU/mL aer incubation for 24 h at 37 °C.
2.4. Preparation of bacteria cultures used in the
production of sucuk
First, 5 µL of active E. coli O157:H7 was transferred into
50 mL of TSB and incubated for 14 h at 37 °C. Aer
incubation, 9 mL of activated culture was centrifuged at
5000 rpm (Hettich EBA 12; Germany) for 10 min. e
suspended pellets were washed twice with 0.85% NaCl
solution. e pellets were transferred into 10 mL of TSB
that was set to pH 5.5 using 3 M lactic acid (85% DL-
lactic acid; Sigma) solution and then incubated for 3 h
at 37 °C. Aer incubation, the cultures were centrifuged
and the pellet was suspended in 9 mL of FTS. For the
preparation of the nontreated cells, 5 µL of active culture
was transferred to 50 mL of TSB and incubated for 14 h at
37 °C. Aer incubation, the cultures were centrifuged and
the pellet was suspended in 9 mL of FTS. e solution was
added to the sucuk batter so that the nal concentration
was 105 CFU/g.
2.5. Production of sucuk
Beef and sheep tail fat (25%) was cut into small pieces
and then minced through a 3-mm plate. e mixture was
irradiated at 25 kGy in the irradiation facility of the Turkish
Atomic Energy Authority, Sarayköy Nuclear Research and
Training Center, Ankara, and kept at –20 ± 2 °C until it
was to be used.
e mixture was defrosted overnight and then 1.6%
NaCl, 1.2% garlic, 0.5% sucrose, 0.5% chili pepper, 0.6%
sweet paprika, 0.6% black pepper, 0.8% cumin, 0.04%
NaNO3
, and 0.01% NaNO2 were added. A commercial
starter culture was added to the sucuk batter and
homogenized using a laboratory-type mixer. Aer this
stage, the sucuk batter was divided between the 2 trial
groups; acid-adapted E. coli O157:H7 (105 CFU/g) was
added to one group and the nontreated E. coli O157:H7 (105
CFU/g) was added to the other group as a control. ese
experiments were carried out in duplicate. e inoculated
sucuk batter was kept in a refrigerator at 4 °C overnight to
ensure adequate diusion throughout the meat.
A laboratory mixer was used to stu 50–60 g of the sucuk
batter into collagen casings 35 mm in diameter. e sucuk
batter samples were ripened in an automatically adjustable
air-conditioned room under conditions of 85%–90%
relative humidity at 22 °C, 80%–85% relative humidity at
22 °C, and 65%–70% relative humidity at 20 °C sequentially
for 3, 3, and 2 days, respectively (20). Aer this ripening
process, the sucuk samples were stored at 4 °C.
2.5.1. Bacteriological analysis of sucuk
Ripened sucuk samples (10 g) were transferred into
Stomacher bags containing 90 mL of Maximum Recovery
Diluent (Merck) and homogenized for 1 min at 235 rpm in
the Stomacher (Seward Stomacher®400 Circulator; UK).
Sorbitol MacConkey Agar (Merck) was used for the E. coli
O157:H7 and incubated at 37 °C for 24 h. Baird Parker
Agar (Merck) was used for the Staphylococcus spp. and
incubated at 37 °C for 48 h. For the lactic acid bacteria
(LAB), De Man Rogosa Sharp Agar (Merck) was used
and incubated at 30 °C for 72 h. Standard microbiological
methods were applied in the analyses (21).
2.5.2. Chemical and physical analyses of the sucuk
samples
e 10-g samples were homogenized in 100 mL of
distillated water and the pH was measured by pH meter
(WTW, Inolab Level 2; Germany). In order to determine
the dry matter, approximately 5 g of sucuk was weighed
and dried until a constant weight was obtained at 105 °C
(22).

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2.6. Statistical analysis
A variance analysis with repeated measurement (ANOVA)
was carried out on the experimental results. e Duncan
test was used to determine the dierences between the
groups. Statistical analyses were performed using SPSS
15 for the variance analyses and CMSTAT for the Duncan
tests.
3. Results
3.1. Acid adaptation
e survival rates of E. coli O157:H7 adapted to HCl at
pH 5.5 for 1, 2, 3, and 4 h and those of the nontreated
cells at pH 3.5 are shown in Table 1. In TSB (pH 3.5), no
noticeable decrease in the number of cells in the two groups
was observed in the rst 5 h of incubation. erefore, the
dierence among groups was not found to be signicant
(P > 0.01). In hours 24 and 48 of incubation, the number
of nontreated E. coli O157:H7 cells was lower than that of
the acid-adapted cells. In conclusion, it was determined
that E. coli O157:H7 gained resistance to the acid via acid
adaptation and the dierence between adaptation periods
was not signicant (P > 0.01).
e acid-adapted and nontreated cells in the acetic
acid were incubated for 120 min in TSB (pH 3.5) and
their survival levels were determined (Table 2). e acid
adaptation at pH 5.5 increased the resistance of E. coli
O157:H7 to acetic acid (P < 0.01) and the highest acid
resistance was detected in those cells exposed to a 3-h
adaptation period (P < 0.01).
e results regarding the survival level at pH 3.5 of
acid-adapted and nontreated E. coli O157:H7 cells for
1, 2, 3, and 4 h in TSB (pH set to 5.5 by lactic acid) are
Table 1. e survival level of HCl adapted and nontreated E. coli O157:H7 in pH 3.5 TSB (log CFU/mL).
Incubation (h) Acid-adapted period (h) Nontreated
1234
0 7.08Aa ± 0.04 7.03Aa ± 0.05 7.02Aa ± 0.02 7.04Aa ± 0.05 7.21Aa ± 0.10
1 6.93Aa ± 0.06 6.99Aa ± 0.06 6.97Aa ± 0.04 7.03Aa ± 0.05 7.20Aa ± 0.09
2 6.92Aa ± 0.05 6.93Aa ± 0.01 6.89Aa ± 0.11 6.95Aa ± 0.04 7.17Aa ± 0.09
3 6.92Aa ± 0.05 6.92Aa ± 0.02 6.87Aa ± 0.10 6.95Aa ± 0.05 7.14Aa ± 0.06
4 6.89Aa ± 0.04 6.83Aa ± 0.03 6.85Aa ± 0.10 6.92Aa ± 0.05 7.13Aa ± 0.05
5 6.83Aa ± 0.03 6.80Aa ± 0.01 6.79Aa ± 0.08 6.73Aa ± 0.13 7.08Aa ± 0.04
24 5.01Bb ± 0.47 5.46Bb ± 1.00 5.46Bb ± 1.00 6.41Aa ± 0.11 3.26Bc ± 0.11
48 2.24Cb ± 0.29 2.32Cb ± 0.72 2.33Cb ± 0.14 1.95Bb ± 0.10 <1.00Cc ± 0.00
Values are the average of 2 replicates.
*A, B, C (↓); a, b, c (→): e dierence between averages having the same letters is not statistically signicant (P > 0.01).
Table 2. e survival level of acetic acid-adapted and nontreated E. coli O157:H7 in pH 3.5 TSB (log CFU/mL).
Incubation (min) Acid-adapted period (h) Nontreated
1234
0 6.99Aa ± 0.01 6.95Aa ± 0.10 7.02Aa ± 0.02 7.15Aa ± 0.00 7.00Aa ± 0.00
30 6.46Bab ± 0.23 6.41Bb ± 0.03 6.66Ba ± 0.07 6.25Bb ± 0.02 3.91Bc ± 0.03
60 <1.00Ca ± 0.00 <1.00Ca ± 0.00 <1.00Ca ± 0.00 <1.00Ca ± 0.00 <1.00Ca ± 0.00
90 <1.00Ca ± 0.00 <1.00Ca ± 0.00 <1.00Ca ± 0.00 <1.00Ca ± 0.00 <1.00Ca ± 0.00
120 <1.00Ca ± 0.00 <1.00Ca ± 0.00 <1.00Ca ± 0.00 <1.00Ca ± 0.00 <1.00Ca ± 0.00
Values are the average of 2 replicates.
*A, B, C (↓); a, b, c (→): e dierence between averages having the same letters is not statistically signicant (P > 0.01).

488
ÖZTÜRK and HALKMAN / Turk J Vet Anim Sci
shown in Table 3. Aer 120 min of incubation, the highest
acid resistance was detected in the E. coli O157:H7 cells
exposed to adaptation for 3 and 4 h (P < 0.01).
Table 4 presents the results regarding the survival level
at pH 3.5 of acid-adapted and nontreated E. coli O157:H7
in TSB with pH set to 5.5 by citric acid. In the adaptation
trials performed using citric acid, it was seen that the
dierence between the average of the trial groups was
statistically insignicant and the application of citric acid
did not result in any increase in resistance. Interestingly,
the nontreated cells were not aected at a pH of 3.5 set
using citric acid.
In the adaptation trial using acetic acid, the highest
acid resistance was seen in the 3-h adaptation samples,
and in the lactic acid trials in the 3- and 4-h adaptation
samples. In the adaptation trials with HCl, it was detected
that the duration of the adaptation period did not increase
the resistance.
3.2. e numbers of acid-adapted and nontreated E. coli
O157:H7 in sucuk
e survival of E. coli O157:H7 in the sucuk samples and
the eects of acid adaptation were examined. For this
purpose, the samples were inoculated with acid-adapted E.
coli O157:H7 cells for 3 h in TSB of pH 5.5 with lactic acid
and nontreated cells. e survival levels of acid-adapted
and nontreated E. coli O157:H7 during the sucuk ripening
and storage period are shown in Table 5.
e numbers of acid-adapted E. coli O157:H7 decreased
to <2.00 log CFU/g on the 30th day and the nontreated E.
coli O157:H7 decreased to the same level on the 8th day.
e acid-adapted E. coli O157:H7 cells maintained their
survival for a longer time when compared to nontreated
E. coli O157:H7 cells (P < 0.01). ere was a signicant
increase in the survival rate of E. coli O157:H7 in sucuk
through acid adaptation (P < 0.01).
3.3. e number of LAB and Staphylococcus in sucuk
e dierence between the LAB numbers determined
in the sucuk samples inoculated with acid-adapted
and nontreated E. coli O157:H7 cells was found to be
statistically insignicant. e dierence in the number
of Staphylococcus spp. detected in the sucuk samples into
which acid-adapted and nontreated E. coli O157:H7 cells
were inoculated was found to be statistically insignicant
(Table 6).
Table 3. e survival level of lactic acid-adapted and nontreated E. coli O157:H7 in pH 3.5 TSB (log CFU/mL).
Incubation (min) Acid-adapted period (h) Nontreated
1234
0 7.01Aa ± 0.03 7.03Aa ± 0.05 7.04Aa ± 0.11 7.06Aa ± 0.06 7.08Aa ± 0.08
30 6.93Aa ± 0.07 6.97Aa ± 0.00 6.94Aa ± 0.03 6.94Aa ± 0.04 6.89Aa ± 0.10
60 5.39Bb ± 0.25 6.38Aa ± 0.12 6.68Aa ± 0.23 6.80Aa ± 0.06 5.31Bb ± 0.77
90 4.17Cb ± 0.25 5.30Ba ± 0.33 5.84Ba ± 0.39 5.81Ba ± 0.04 2.74Cc ± 0.26
120 <1.00Dc ± 0.00 3.19Cb ± 0.33 4.65Ca ± 0.54 4.34Ca ± 0.04 <1.00Dc ± 0.00
Values are the average of 2 replicates.
*A, B, C (↓); a, b, c (→): e dierence between averages having the same letters is not statistically signicant (P > 0.01).
Table 4. e survival level of citric acid-adapted and nontreated E. coli O157:H7 in pH 3.5 TSB (log CFU/mL).
Incubation (min) Acid-adapted period (h) Nontreated
1234
0 7.06Aa ± 0.02 7.08Aa ± 0.05 7.06Aa ± 0.02 7.08Aa ± 0.03 7.10Aa ± 0.05
30 7.00Aa ± 0.06 7.06Aa ± 0.02 7.04Aa ± 0.03 7.06Aa ± 0.02 7.04Aa ± 0.04
60 6.96Aa ± 0.04 7.02Aa ± 0.03 6.96Aa ± 0.08 7.00Aa ± 0.01 7.01Aa ± 0.03
90 6.94Aa ± 0.08 6.99Aa ± 0.02 6.95Aa ± 0.09 6.99Aa ± 0.02 6.97Aa ± 0.04
120 6.89Aa ± 0.03 6.96Aa ± 0.04 6.86Aa ± 0.04 6.91Aa ± 0.01 6.86Aa ± 0.06
Values are the average of 2 replicates.
*A, B, C (↓); a, b, c (→): e dierence between averages having the same letters is not statistically signicant (P > 0.01).

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3.4. Change in the pH and humidity levels during
ripening and storage
It was detected that as the number of LAB increased, the
pH decreased, and this interaction was found statistically
signicant (P < 0.01). Changes in the pH values of the
sucuk samples containing acid-adapted and nontreated E.
coli O157:H7 were observed during ripening and storage
(Table 7). It was seen that the acid-adapted E. coli O157:H7
proved resistant to pH changes occurring in fermentation
and that it could survive for at least 15 days.
e dierences between the humidity levels detected
in the sucuk samples with acid-adapted and nontreated
E. coli O157:H7 cells were found to be statistically
insignicant (Table 7). It was determined that there was
a positive interaction between the humidity level and the
pH value; as the former decreased, so did the latter (P <
0.05). Furthermore, as the humidity level decreased, it was
found that the survival level of the E. coli O157:H7 cells
also decreased (P < 0.01).
4. Discussion
Acid-tolerant pathogenic bacteria can survive for a long
time in acidic food. Furthermore, acid is one of the most
important defense mechanisms of the body. In particular,
the acidic medium of the stomach has a signicant role in
protecting the body against bacteria. Studies have shown
Table 5. e survival level of E. coli O157:H7 cells during ripening
and storage periods at 4 °C of the sucuk samples (log CFU/mL).
Days Acid-adapted Nontreated
H0* 5.80 ± 0.08 5.53 ± 0.05
Ripening
0 5.37 ± 0.07 4.64 ± 0.22
1 4.82 ± 0.06 4.37 ± 0.07
2 4.31 ± 0.05 3.89 ± 0.15
3 4.15 ± 0.27 3.68 ± 0.02
4 4.03 ± 0.33 3.51 ± 0.07
6 3.11 ± 0.21 2.30 ± 0.20
Storage
8 2.72 ± 0.12 <2.00 ± 0.00
15 2.50 ± 0.50 <2.00 ± 0.00
30 <2.00 ± 0.00 <2.00 ± 0.00
Acid-adapted cells were obtained by keeping them in TSB (pH
5.5) acidied with lactic acid for 3 h.
H0*: Keeping the sucuk batter at 4 °C overnight.
Values are the average of 2 replicates.
Table 6. e number of LAB and Staphylococcus spp. in the sucuk samples inoculated with E. coli O157:H7 cells
(log CFU/mL).
Days Acid-adapted Nontreated
LAB Staph. spp. LAB Staph. spp.
H0* 6.48 ± 0.05 6.43 ± 0.16 6.45 ± 0.02 6.33 ± 0.12
Ripening
0 6.65 ± 0.08 6.39 ± 0.05 6.52 ± 0.03 6.29 ± 0.01
1 9.25 ± 0.07 6.69 ± 0.07 9.14 ± 0.09 6.17 ± 0.43
2 9.42 ± 0.01 6.09 ± 0.05 9.31 ± 0.11 6.28 ± 0.20
3 9.35 ± 0.06 6.16 ± 0.01 9.36 ± 0.02 6.12 ± 0.12
4 9.19 ± 0.01 6.09 ± 0.05 9.20 ± 0.16 5.98 ± 0.13
6 9.21 ± 0.13 6.69 ± 1.09 9.32 ± 0.11 6.08 ± 0.04
Storage
8 9.22 ± 0.04 6.90 ± 0.09 9.12 ± 0.08 7.15 ± 0.07
15 8.75 ± 0.03 6.21 ± 0.21 8.79 ± 0.19 7.24 ± 0.36
30 8.83 ± 0.09 5.75 ± 0.40 8.57 ± 0.07 6.15 ± 1.33
Acid-adapted cells were obtained by keeping them in TSB (pH 5.5) acidied with lactic acid for 3 h.
H0*: Keeping the sucuk batter at 4 °C overnight.
Values are the average of 2 replicates.

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that E. coli O157:H7 adapts and gains resistance to acid
when subjected to a medium of pH 4.5–5.5. ATR increases
the acid resistance of E. coli O157:H7, allowing it to survive
both in acidic and fermented food (1,7).
Many researchers found that acid adaptation of E.
coli O157:H7 varied according to the duration of the
adaptation period. It was stated by Cheng et al. (19) that
aer being exposed to moderate acidic conditions (pH
5.0) the resistance of E. coli O157:H7 to strong acid stress
(pH 3.0) varied according to adaptation period, and the
highest acid tolerance was obtained aer the bacteria had
undergone a 4-h adaptation period. Tosun and Aktuğ
Gönül (23) reported an increase in the acid tolerance to
pH 3.0 of the acid-adapted E. coli O157:H7 and the highest
acid tolerance was obtained aer a 2-h adaptation period
at pH 4.5.
e current study has also shown that the HCl
adaptation of E. coli O157:H7 increased the acid tolerance;
however, the adaptation time did not result in a signicant
dierence (Table 1). e dissimilar results obtained from
the current study can be explained by the acid tolerance
of the bacteria. ere are studies showing that a decrease
in the pH increased the acid resistance of E. coli O157:H7.
Cheng et al. (19) reported that the acid adaptation of E.
coli O157:H7 subjected to mild acidic conditions (pH
5.0) varied depending on the adaptation time, and as
the pH increased (pH 4.0–5.0), the dierence between
the adaptation times was reduced. Öztürk (24) found
that the resistance of E. coli O157:H7 to pH 3.0 varied
with dierent adaptation times; however, there was no
signicant dierence between the adaptation times for pH
3.5.
e inhi bitory eect of hydrochloric, acetic, lactic, malic,
and citric acids on E. coli O157:H7 has been investigated.
It was determined that the highest and lowest inhibitory
eects were obtained from the lactic acid and HCl treatment,
respectively, with dierent sensitivity to acetic, malic, and
citric acid among strains (25). In the study carried out by
Ryu et al. (26) to detect the eect of dierent acids on E. coli
O157:H7, the bacteriostatic eect was determined to be in
the sequence of acetic > lactic > citric acid. In the current
study, it was found that acetic, lactic, and citric acids were
more eective than HCl and the bactericidal eect of the
organic acids was determined to be in the order of acetic >
lactic > citric acid.
Leyer et al. (7) detected that the resistance of acid-
adapted E. coli O157:H7 to lactic acid was increased by
culturing at pH 5.0 over a period of 1–2 generations. It
was determined by Goodson and Rowbury (27) that the
survival of 3 dierent strains of acid-adapted E. coli at
pH 5.0 increased in a culture medium of lactic, benzoic,
propionic, sorbic, and acetic acids with the pH set to 3.5.
Table 7. pH values and humidity levels (%) of the sucuk samples inoculated with E. coli O157:H7 cells.
Days Acid-adapted Nontreated
pH Moisture % pH Moisture %
H0* 5.78 ± 0.03 59.19 ± 0.57 5.75 ± 0.01 59.22 ± 1.47
Ripening
0 5.80 ± 0.07 57.15 ± 0.50 5.75 ± 0.03 58.62 ± 1.93
1 4.65 ± 0.01 56.11 ± 1.06 4.62 ± 0.02 58.51 ± 1.01
2 4.42 ± 0.06 54.35 ± 1.76 4.39 ± 0.04 54.00 ± 3.30
3 4.56 ± 0.04 50.21 ± 3.56 4.59 ± 0.11 50.47 ± 1.14
4 4.54 ± 0.05 48.20 ± 0.52 4.52 ± 0.02 49.19 ± 7.39
6 4.48 ± 0.01 43.01 ± 3.65 4.44 ± 0.07 38.49 ± 2.22
Storage
8 4.61 ± 0.01 39.82 ± 1.83 4.59 ± 0.11 35.88 ± 1.03
15 4.74 ± 0.03 35.37 ± 1.27 4.60 ± 0.03 31.04 ± 0.04
30 4.76 ± 0.06 34.39 ± 0.01 4.72 ± 0.13 30.06 ± 0.02
Acid-adapted cells were obtained by keeping them in TSB (pH 5.5) acidied with lactic acid for 3 h.
H0*: Keeping the sucuk batter at 4 °C overnight.
Values are the average of 2 replicates.

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e development characteristics of E. coli O157:H7 acid-
shocked, acid-adapted, and control cells in TSB acidied
with lactic acid and acetic acid were examined by Ryu and
Beuchat (6). In the acetic acid trials (pH 3.4), these three
cell types proved to have the same behavior. In the lactic
acid trials (pH 3.9), it was detected that the numbers of
acid-adapted and acid-shocked cells were higher than that
of the control cells, and the acid-adapted cells were more
tolerant of acidic conditions than acid-shocked cells.
As explained above, the ATR depends on the variety
of acid, pH, and cell type. In this current research, we
detected that the acid adaptation of E. coli O157:H7 varied
according to the variety of acid used and the adaptation
period. While the tolerance of E. coli O157:H7 to acetic
and lactic acid increased, there was no increase in its
tolerance to citric acid.
In this research, it was determined that the survival
rate of E. coli O157:H7 in sucuk increased through acid
adaptation. e same result was obtained in other studies
of various food systems and it has been detected that
acid adaptation increases acid resistance. Leyer et al.
(7) reported that E. coli O157:H7 has an acid adaptation
mechanism and the survival rate of the bacteria increases
in fermented sucuk and similar products, sliced dried
salami, and cider by this mechanism. Weagant et al. (5)
drew similar conclusions aer examining the survival rate
of acid-adapted and nontreated E. coli O157:H7 in ketchup
and mayonnaise.
Contrary to this research, in a study conducted by
Riordan et al. (28) it was detected that acid adaptation
did not result in any increase in the survival rate of E. coli
O157:H7 in pepperoni, another fermented meat product.
Dlamini and Buys (13) claimed that a signicant dierence
did not exist between survival levels of acid-adapted and
nontreated E. coli O157:H7 cells in amasi, also a traditional
fermented dairy product, but that in commercial amasi the
nontreated cells had a higher survival rate.
Dlamini and Buys (13) also reported that fermented
products inoculated with acid-adapted E. coli O157:H7
cells showed no signicant dierence in the number
of LAB. Similarly, the results of the current study have
shown no signicant dierence in the number of LAB.
As the number of LAB increased, the pH decreased, and
acid-adapted E. coli was resistant to changes in pH during
fermentation and could survive at a certain level.
In this research, it was determined that E. coli O157:H7
increases its acid toleration aer exposure to moderately
acidic (pH 5.5) conditions and it can survive for a long
time (at least 2 h) in highly acidic medium (pH 3.5),
which without the adaption process would have killed
the bacteria. In addition, it is understood that the type of
acid and the duration of the adaptation period have an
important eect on acid adaptation. In the sucuk samples
the survival level of E. coli O157:H7 with acid adaptation
in sucuk (pH of ~4.8) increased. e acid-adapted E. coli
O157:H7 cells maintained their survival in sucuk for 30
days. e existence of pathogenic bacteria in acidic foods
such as sucuk, which has traditionally been considered as
safe, is of great importance for both the food industry and
human health.
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