Food Research International 36 (2003) 703–712

www.elsevier.com/locate/foodres

Thermal inactivation of Escherichia coli O157:H7 and

Escherichia coli isolated from morcilla as affected by
composition of the product
Juan M. Oteiza, Leda Giannuzzi, Alicia N. Califano*
CIDCA, CONICET, Facultad Ciencias Exactas, Universidad Nacional de La Plata, 47 y 116, La Plata (1900), Argentina

Received 20 January 2002; accepted 6 March 2003

Abstract
Morcilla is a link sausage quite similar to black pudding, consisting of an inert casing stuffed with a mixture of beef blood, fat,
and seasonings. Thirty samples of morcilla showed total microbial counts (6.3
103–2.1
108 Cfu/g ), molds and yeasts (8.9
101–
6.3
104 Cfu/g), sulfite-reducing microorganism (2.0
101–2.1
102MPN/g); total coliforms (1.4
101–1.1
103 MPN/g); fecal coli-
forms (7.0–1.5
102MPN/g); Enterobactereaceae (1.6
102–5.0
105 Cfu/g). S. aureus and B. cereus were not detected. E. coli was
detected in 76.6% of the samples analyzed. The thermal resistance (D and z-values) of Escherichia coli O157:H7 and E. coli isolated
from morcilla were determined in nutrient broth and in a heating menstruun prepared with ground morcilla (discarding the casing)
and added fat or starch. Higher fat and starch levels resulted in higher D-values (min) at 54, 58 and 62  C for both strains. The
z-values ( C) for isolated E. coli in nutrient broth (M1), ground morcilla (M2), M2+10% fat (M3), M2+20% fat (M4), M2+10%
starch (M5), and M2+20% starch (M6) were 7.9, 7.8, 10.5, 10.4, 10.3, and 10.4, respectively, and for E. coli O157:H7 were 7.8, 7.4,
9.8, 10.2, 10.3, and 10.7. The composition of product affected heat lethality of the two strains of E. coli.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Escherichia coli O157:H7; Thermal resistance; Blood link sausage; Morcilla

1. Introduction hygienic practices and insufficient heat treatment at the

Morcilla is the name given in Spanish to a blood link
sausage quite similar to English black pudding. It con-
sists of a mixture of beef blood, pig fat, pig skins, salt,
onion and spices stuffed into natural or synthetic tripe,
tied manually and immersed in a hot water bath at
90  C. Actually Argentinean Food Law establishes nei-
ther any time-temperature standards for this treatment
nor detailed microbiological standards, it only prohibits
the presence of any pathogenic microorganism in meat
products. Morcilla is eaten in Argentina either cold,
without any further preparation, or warm, among other
sausages, tripe, sweet bread and meat cuts in a typical
barbecue called ‘‘asado’’. Morcilla is grilled for a few
minutes, just to warm it, to avoid bursting (thus the
center does not reach 60  C). A combination of poor
* Corresponding author. Tel.: +54-221-4254853; fax: +54-221-
4254853.
E-mail address: anc@quimica.unlp.edu.ar (A.N. Califano).
0963-9969/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0963-9969(03)00050-4
meat factory may result in a highly contaminated pro-
duct, posing a serious risk for the consumers.
Escherichia coli O157:H7 is recognized as a foodborne
pathogen of primary concern. The organism is an etio-
logical agent of hemorrhagic colitis, hemolytic uremic
syndrome (HUS), and thrombotic thrombocytopenic
purpura (Swerdlow et al., 1992). The etiology of these
diseases commonly involves foodborne transmission,
with ground beef being implicated often (Belongia et al.,
1991; Doyle, 1991; Riley, 1987). HUS is endemic in
Argentina, with the highest frequency in the world. The
Pediatric Argentine Society reported 4563 cases of
affected children, mostly from the second semester to 3
years of age, from 1973 to 1993 (Voyer, 1998). In 1998,
the rate of incidence was 75 cases for 10,000 children
lower than five years old (Miliwebsky et al., 1999). Most
of these cases have no known etiology. However, in a
recent study, Chinen et al., (2001) have detected E. coli
O157:H7 in a number of Argentine foodstuffs such as
ground beef, fresh sausages, dry sausages, and other
704 J.M. Oteiza et al. / Food Research International 36 (2003) 703–712
meat products. A preliminary study (Oteiza, Giannuzzi,
& Califano, 2001) showed a high incidence of E. coli in
morcilla, which makes the situation especially danger-
ous if one considers that many pediatricians recommend
to feed cooked morcilla to babies, because of its iron
and protein content, unaware of the frequent presence
of the microorganism.
Several authors have reported D and z-thermal in-
activation values of E. coli O157:H7 in ground beef,
turkey, lamb, pork, and poultry (Amhed, Conner, &
Huffman, 1995; Goodfellow & Brown, 1987; Juneja &
Marmer, 1999; Juneja, Snyder, & Marmer, 1997;
Kotrola & Conner, 1997; Line et al., 1991; Orta-
Ramirez, Price, Hsu, Veeramuth, Cherry-Merrit, &
Smith, 1997). Strains of E. coli vary considerably in heat
resistance and environmental conditions such as ther-
mal shock and growth atmosphere can increase the
D-values (Murano & Pierson, 1992). A key to opti-
mization of the heating step during manufacture of mor-
cillas is defining the target pathogen’s heat resistance in a
medium that closely resembles the morcilla stuffing.
The objectives of the present work were: (1) to study
the principal microorganisms present in morcilla sau-
sages purchased in local retail markets; (2) to determine
the thermal inactivation values (D and z) of a strain of
E. coli isolated from sausages and of E. coli O157:H7 in
both nutrient broth and heating menstruum prepared
with morcilla, and (3) to analyze the effect of the addi-
tion of fat or starch to the heating menstrua on heat
resistance for both strains.
2. Material and methods
2.1. Chemical composition of the product
Morcilla was obtained from local retail markets and
immediately analyzed. Water and fat contents were
determined by AOAC methods 24.002 and 24.005,
respectively (AOAC, 1984). Protein content was ana-
lyzed by Kjeldahl, AOAC method 24.027 (AOAC,
1984). Carbohydrates were determined by substracting
the water, fat and protein content from the total mass.
pH was measured with a combination pH electrode
(model 50215, Hach, Loveland, USA) on a pH meter
(model EC30, Hach, Loveland, USA).
2.2. Principal microorganisms present in the product
Thirty samples of morcilla sausages (approximately
100 g each), purchased in local retail markets, were
aseptically collected and placed into sterile containers.
They were immediately brought to the laboratory. Sub-
samples of 20 g were taken at random for each sample
and aseptically weighed into a sterile stomacher bag
with 180 ml of sterile 0.1% (w/v) peptone water (Merck
KGaA, Darmstadt, Germany) and homogenized for 1
min in a model 400 Stomacher (Seward Medical, Lon-
don, UK); appropriate serial dilutions were used to
determine different microorganisms according to the
following procedures.
1. Total microbial counts were enumerated follow-
ing pour plate technique in Plate count agar
(PCA; Merck KGaA, Darmstadt, Germany) and
incubated at 37  C for 48 h
2. Molds and yeasts counts were enumerated via
surface plating in YGC agar (Yeast extract, glu-
cose, chloranphenicol; Merck KGaA, Darmstadt,
Germany) and incubated at 25  C for 5 days.
3. Sulfite-reducing microorganism counts were
enumerated following MPN/g technique in SPS
agar (Sulfadiazine polymixin sulfite; Merck
KGaA, Darmstadt, Germany) and incubated at
37  C for 48 h in anaerobic jars (Gas Pack Plus
D-G100, Merck KGaA, Darmstadt, Germany).
4. Total coliforms, fecal coliforms, presence and
isolation of Escherichia coli in the products were
performed according to AOAC method 46016
(AOAC, 1984), including IMViC assay. Before
detection and isolation of E. coli by AOAC
method, Gene-Trak1 Escherichia coli assay
(Gene-Trak Systems Corp., Hopkinton, MA)
was performed as a screening procedure.
PCR assays were used to determine the pathogenicity
of the isolated strains, including stx1, stx2, and eae
genes (Heuvelink, van der Kar, Meis, Monnens, &
Melchers, 1995). Besides the isolated strains were sub-
mitted to the National Reference Laboratory (ANLIS
‘‘Carlos Malbrán’’, Buenos Aires, Argentina) to confirm
our results. One of the isolated strains (strain G335) was
chosen for the thermal inactivation studies. The follow-
ing biochemical characterization of E. coli G335 was
also performed: Indol test, Methyl Red test, Voges-
Proskauer test, Simmons citrate test, Gram, Catalase
test, Oxidase test, Motility test, Nitrate reduction test,
Urease test, b-Galactosidase test, Carbohydrate fer-
mentations (glucose, lactose, adonitol, sorbitol) (Mac
Faddin, 1979, Ørskov, 1984)
2.2.1. Staphylococcus aureus
Staphylococcus aureus were enumerated via surface
plating in Baird-Parker agar (Merck KGaA, Darm-
stadt, Germany) and incubated at 37  C for 48 h and
then examined by coagulase production (rabbit plasma
EDTA, Difco Laboratories, Detroit, MI). The detection
limit was 2
102 Cfu/g.
2.2.2. Bacillus cereus
Bacillus cereus counts were enumerated via surface
plating in phenol red egg yolk polymixin agar (Merck
 J.M. Oteiza et al. / Food Research International 36 (2003) 703–712
KGaA, Darmstadt, Germany) with incubation at 37  C
for 48 h, typical colonies were confirmed with starch,
citrate, Voges-Proskauer and motility-nitrate test. The
detection limit was 2
102 Cfu/g.
2.2.3. Enterobacteriaceae
Enterobacteriaceae counts were enumerated via sur-
face plating in violet red bile glucose agar (Merck
KGaA, Darmstadt, Germany) and incubating at 37  C
for 24–48 h.
2.3. Bacterial strains
Two strains of E. coli were used separately to study
thermal inactivation. E. coli G335 was isolated from
naturally contaminated sausages samples as described
earlier. The other strain used was E. coli O157:H7 strain
933 obtained from Servicio Fisiopatogenia, Instituto
Nacional de Enfermedades Infecciosas-ANLIS ‘‘Carlos
Malbrán’’, Buenos Aires, Argentina. Both strains used
for thermal inactivation studies were maintained in
nutrient broth (Merck KGaA, Darmstadt, Germany) at
4  C and periodically subcultured.
2.4. Inoculum preparation
To prepare cell suspensions, a 10 ml loop of stock
culture, either E. coli G335 or E. coli O157:H7, was
transferred to 10 ml of nutrient broth and aerobically
incubated at 37  C for 24 h. The population density in
each inoculum was enumerated by pour plate procedure
in PCA plates incubated at 37  C for 48 h.
2.5. Sample preparation and inoculation
2.5.1. Nutrient broth
Fifty microliters of inoculated nutrient broth con-
taining 108–109 Cfu/g (M1) were aseptically placed in
capillary tubes (external diameter=1.4 mm, length=75
mm, and volume=80 ml) using a Hamilton syringe. The
tubes were heat-sealed and immediately placed in a
temperature-controlled water bath as described later.
2.5.2. Sausage media
Several kilograms of ground sausage (without casing)
with a composition adjusted to resemble the average
composition of samples were kept frozen (18  C)
until use. At the time of each experiment, 100 g of
sausage were thawed, homogenized with 50 ml of water
and then heated at 100  C for 30 min twice to elim-
inate background microflora (M2). To analyze the
effect of fat and starch content, four additional men-
strua were prepared by adding known amounts of fat
or starch to M2. The heating menstrua were: M3
(100 g M2+10 g bovine fat); M4 (100 g M2+20 g
bovine fat); M5 (100g M2+10 g cornstarch); and M6
705
(100 g M2+20 g cornstarch). For the inoculation, 50 g
of M2, M3, M4, M5, and M6 media were transferred
aseptically to stomacher bags and 1 ml of each strain
of E. coli inoculum was added to each bag to give a
final concentration of approximately 108–109 Cfu/g. A
negative control containing sample M2 inoculated
with 1 ml of sterile 0.1% (w/v) peptone water was
included. The inoculated samples and negative control
were homogenized in a Stomacher for 1 min, and then
1 g was placed in sterile Pyrex1 tubes (1 cm diameter,
IVA S.A., Buenos Aires, Argentina). The tubes were
capped with bacteriological cups and immediately
placed in the temperature-controlled water bath as
described later.
2.6. Thermal inactivation
For experiments in nutrient broth and in the slurry
prepared with ground morcilla two extra tubes were
loaded, and type-T (copper-constantan) thermocouples
were inserted in the center of the media and sealed in to
serve as the temperature-monitoring tubes. Tubes were
completely immersed in a temperature-controlled bath
(Haake L, Haake Buchler Instruments, Karlsruhe, Ger-
many) during the heating cycle. Time/temperature data
were recorded (Servor 102 NGI, USA). Time and tem-
perature data points were as follows: 54  C: 0, 3, 5, 7,
10, 15, 20 min; 58  C: 0, 4, 6, 8, 10 min; 62  C: 0, 1, 2, 3,
4, 5, and 7 min.
Timing was started (time=0) when the tubes reached
the test temperature, two tubes were removed from the
bath to measure the actual initial count, and come up
times were recorded. The time necessary to reach the
desired temperature was always less than 15 sec. Dupli-
cate tubes were removed at each prescribed time inter-
val, then transferred immediately to an ice bath until
analyzed. Enumeration of the surviving microorganisms
was conducted as described later.
2.7. Enumeration of surviving microorganisms
2.7.1. Nutrient broth (M1)
The outside of the heat treated capillary tubes were
sterilized by rinsing first with sodium hypochlorite, sec-
ondly with 0.1% peptone water and then aseptically
dried and placed in sterile glass tubes containing 5 ml of
sterile 0.1% peptone water. Afterwards the capillary
tubes were aseptically broken with a glass rod and
appropriate serial dilutions (1:10) were made with sterile
0.1% peptone water and were plated in duplicate (1 ml
pour plate technique) on PCA (37  C, 48 h) and PCA
containing 1% sodium pyruvate, added to facilitate
repair damaged cells (Goodfellow & Brown, 1987)
(37  C, 48h) to culture E. coli. Colonies were counted
and the results were expressed as Cfu/g. The detection
limit was 2
102 Cfu/ml.
706 J.M. Oteiza et al. / Food Research International 36 (2003) 703–712
2.7.2. Slurry prepared with ground morcilla
Tubes containing the heat-treated samples were
opened; 9 ml of 0.1% peptone water were added and
homogenized in vortex. Appropriate serial dilutions
(1:10) of slurry were made with sterile 0.1% peptone
water and surface plated in duplicate (0.1 ml) on
CHROMagar1 ECC (CHROMagar, Paris, France)
(37  C, 24 h), eosine methylene blue agar (EMB) (Merck
KGaA, Darmstadt, Germany) (37  C, 24 h) and PCA
(37  C, 48 h) to culture E. coli. Typical colonies were
counted and the results were expressed as Cfu/g. The
detection limit was 2
102 Cfu/g. The three media were
used to assure that only E. coli was counted. For each
replicate experiment performed in duplicate, an average
of Cfu/g of 12 platings of each sampling time was used
to determine the D-values, four for each enumeration
media.
2.8. Experimental design
A factorial design was employed to assess the effects
of heating temperature (54, 58 and 62  C), two strains
(E. coli G335 or E. coli O157:H7), and six media (M1,
M2, M3, M4, M5, and M6). All 36 variable combina-
tions were replicated twice, each performed in duplicate.
2.9. Determination of D-values
For each temperature, media, and strain, D-values (in
minutes) were determined by regression from the
expression (Singh & Heldman, 1993):
logN ¼ logNo  t=D
where N is the experimental count value at time t and
No is the initial count value.
As z-value is the change in heating temperature nee-
ded to change the D-value by 90%, the relationship
between D and temperature can be expressed as (Singh
& Heldman, 1993):
Tref T
D ¼ Dref10 z ð2Þ
being Dref the D-value at reference temperature Tref. A
‘‘normal’’ procedure to determine z-value is to linearize
Eq. (2) by taking logarithms followed by linear regres-
sion of log D vs. T; but in that case the 95% confidence
intervals for the z-values are quite large due to the small
number of degrees of freedom (Himmelblaum, 1970;
Van Boekel, 1996), which in this case, considering three
temperatures and replicated experiments led to
n=62=4. A better way to estimate z-values is by
nonlinear regression of all experimental results at once,
since by combining Eqs. (1) and (2), it becomes:
t TTref
log ðNÞ ¼ log ðNoÞ  10 z
Dref
In this case, the dependent variable is the logarithm of
the survival count [log(N)], while time and temperature
are the independent variables. The parameters (con-
stants) to be fitted by a nonlinear procedure are No
(initial count), Dref and z. In the present work a
Tref=57  C was chosen since it is about the average of
the temperatures used in the experiments. Using this
procedure, the number of degrees of freedom for each
media and strain becomes n=38
23=73. Hence the
simultaneous analysis of all data, as opposed to the
determination of individual lethality constants, is clearly
preferable .
2.10. Statistical analysis
Linear regression analysis was performed on survival
curve data to calculate decimal reduction times
(D-values) according to Eq. (1). Nonlinear regression
analysis was performed on survival count data to evalu-
ate z-values [Eq. (3)]. Analysis of variance and post-hoc
multiple comparisons tests were conducted on D-values
and z-values. For simultaneous pairwise comparisons,
least significant difference (LSD) test was chosen. Dif-
ference in averages and F-tests (ANOVA) were con-
sidered significant when the computed probabilities were
< 0.05 (P < 0.05). All the statistical procedures were
carried out using SYSTAT (SYSTAT Inc, Everston,
Illinois).
3. Results and discusion
ð1Þ
3.1. Chemical characterization of the product
The chemical composition of the ground sausage,
without casing, was 15% protein, 32% fat, 1% carbo-
hydrates and 52% water. The pH of the product was
6.2. In the case of M2, M3, M4, M5, and M6 the
moisture contents were 68, 62, 57, 62, and 57%, respec-
tively. The composition of the product is suitable for
survival of pathogenic microorganisms.
3.2. Microorganisms present in the product
The range of values of the microorganisms analyzed
in 30 sausage samples were: total microbial counts
6.3
103–2.1
108 Cfu/g (40% of the isolated colonies
were Gram positive and 60% Gram negative), molds
and yeasts 8.9
101–6.3
104 Cfu/g, sulfite-reducing
microorganism 2.0
101–2.1
102 MPN/g, total coli-
forms 1.4x1011.1
103 MPN/g, fecal coliforms 7.0–
1.5
102 MPN/g, Enterobactereaceae 1.6
102–5.0
105
Cfu/g. S. aureus and B. cereus were not detected. The
presence of E. coli was detected in 76.6% of the samples
analyzed. Our PCR assays showed that none of the iso-
ð3Þ
lated strains were pathogenic, in accordance to the
 J.M. Oteiza et al. / Food Research International 36 (2003) 703–712
results given by the National Reference Laboratory.
The biochemical characterization of generic E. coli
G335 showed the following results: Indol test (+),
Methyl red test (+), Voges-Proskauer test (), Sim-
mons citrate test (), Gram (), Catalase test (+),
Oxidase test (), Motility test (+), Nitrate reduction
test (+), Urease test (), b-Galactosidase test (+),
D-glucose fermentations (+), Lactose fermentations (+),
Adonitol fermentations (), Sorbitol fermentations ().
No E. coli or E. coli-like microorganisms were detec-
ted on the ground sausage used to prepare the M2 to
M6 menstrua, thus, we concluded that the heating
treatment had been adequate.
3.3. Thermal inactivation studies
When M1 was used as heating menstuum no sig-
nificant differences (P < 0.05) in number between the
colony counts in PCA and PCA containing 1% sodium
pyruvate were observed. Since it has been previously
reported that when PCA supplemented with 1% sodium
pyruvate was used for recovery it gave the maximum
heat-injured E. coli O157:H7 recovery (Czechowicz,
Santos, & Zottola, 1996; Juneja, Marmer, & Eblen,
1999) the PCA procedure was considered as effective as
the PCA supplemented one. To check the adequacy of
the PCA procedure on menstrua containing ground
morcilla, ten tubes with inoculated M2 were heated for
5 min at 62  C, again, no significant differences were
found between the colony counts in PCA and PCA
containing 1% sodium pyruvate.
When the heating menstruum contained ground mor-
cilla, colony counts in PCA, CHROMagar1 ECC and
EMB did not differ for both strains tested, confirming
the absence of other contaminant microorganisms. For
this reason, the three sets of data were combined to
calculate D and z-values. An analysis of variance
showed that there were no significant differences
between D-values obtained in replicate experimental
runs.
The survivor curves for E. coli G335 and E. coli
O157:H7 in the different heating menstrua at three
temperatures assayed are shown in Figs. 1 and 2,
respectively, where average colony counts were plotted.
Tables 1 and 2 show the D-values, the corresponding
standard deviations and correlation coefficients (R) of
the regressions for the experiments using E. coli G335
and E. coli O157:H7, respectively. Correlation coeffi-
cients ranged from 0.926 to 0.998 for the E. coli G335
experiments and from 0.915 to 0.998 for E. coli
O157:H7.
It can be observed that D-values for M2 are always
higher than for M1 except for E. coli G335 at 62  C.
The heating menstruum prepared with sausage (M2)
offered protection compared with nutrient broth (M1),
indicated by higher recovery of heated cells that may be
707
attributed to the differences in fat content, salt and
other components present in the heating menstruum.
Juneja et al. (1999) showed that the addition of NaCl
was protective to E. coli O157:H7 against the lethal
effect of heat in pH 4 beef gravy while the trend was
opposite at pH 8; z-values increased with increasing salt
content regardless of gravy pH. In a study of Reichart
and Mohacsi-Farkas (1994), the heat destruction of
seven foodborne microorganisms increased with
increasing water activity. Analysis of variance also
showed that z-values for M1 and M2 were similar for
both strains ranging from 7.4 to 7.8 (Table 3).
An analysis of variance showed that the composition
of the heating menstrua prepared with sausage had a
signifficant effect on D and z-values for both strains.
The overall effect was that the addition of fat resulted in
longer decimal reduction times; higher fat content
implied higher average D-values. Analyzing the effect
produced on D-values at the different temperatures
Tables 1 and 2 showed that at 58 and 62  C, higher fat
levels in heating menstruum (M3 and M4) resulted in
higher D-values for E. coli G335, while at 54  C only
M4 was greater than M2 or M3. For E. coli O157:H7
M4 always produced longer decimal reduction times
than M2 and M3, while only at 62  C the addition of
10% fat resulted in higher D-values than M2. Fat in
heating menstrua typically enhances the survivability of
E. coli O157:H7 (Ahmed, Conner, & Huffman, 1995;
Doyle & Schoeni, 1987; Line et al., 1991). It has been
reported that vegetative cells and spores are more heat
resistant as the water activity of the heating menstruum
decreases (Lenovich, 1987). In this work increasing fat
content decreased water content, thus, M2, M3 and M4
have moisture content of 68, 62 and 57% respectively.
This likely accounted for differences in survival of both
strains in the different heating menstrua tested. These
results are consistent with other reports on this and
other foodborne bacterial pathogens (Doyle & Schoeni,
1987; Huang, Yousef, Marth, & Matthews, 1992; Line
et al., 1991). Ahmed et al. (1995) calculated D-values
using only linear regression analysis for the best fit line
of the survivor curve. These authors reported that the
D-values of E. coli O157:H7 in ground turkey and pork
sausage heated at 55  C in thermal death time tubes
ranged from 8.76 (chicken, 3% fat) to 9.74 (chicken,
11% fat) min and 6.37 (pork sausage, 7% fat) to 11.28
min (pork sausage, 30% fat), the values at 60  C ranged
from 0.38 (chicken, 3% fat) to 0.55 min (chicken, 11%
fat) and 0.37 (pork sausage, 7% fat) to 0.55 (pork sau-
sage, 30% fat). The same trend was also observed for
D-values estimated when the heating menstruum was
ground turkey or beef; D-values increased when fat
content decreased (Ahmed et al., 1995). However,
Kotrola and Conner (1997) reported that D-values for
E. coli O157:H7 ranged from 12.5 (turkey 3% fat) to 11
min (turkey, 11% fat) at 55  C, 2.8 min (turkey 3% fat)
708 J.M. Oteiza et al. / Food Research International 36 (2003) 703–712

Fig. 1. Survivor curves for Escherichia coli G335 in nutrient broth M1 (A), sausage stuffing, M2 (B), M2+10% bovine fat, (C), M2+20% bovine
fat (D), M2+10% cornstarch (E), and M2+20% cornstarch (F) at 54  C (*), 58  C (&), and 62  C (~).

to 2.4 min (turkey 11 fat) at 57  C, and the D-value at
60  C was 0.9 min regardless of the fat content in tur-
key. They attributed their results to the fine grinding of
the menstrua prior to heating, which could possibly
have affected dispersal of fat in the turkey meat allow-
ing emulsification. Kotrola and Conner (1997) also
showed that survivability of E. coli O157:H7 in turkey
was significantly enhanced (longer decimal reduction
times) by the use of additives in turkey meat formula-
tions such as NaCl, Na lactate and polyphosphate. In
all cases the percentage water content decreased, as well
as the mobility of water in the menstruum.
The effect of the starch concentration was evaluated
because it is common practice to add starch to the pro-
duct and in this way to extend the weight and product
yield. This practice is not approved by sanitary autho-
rities in Argentina but it is frequently used. Tables 1 and
2 indicated that the higher starch levels (M5 and M6)
resulted in D-values higher than M2. No previous
reports of D-values with added starch in meat products
 J.M. Oteiza et al. / Food Research International 36 (2003) 703–712 709

Fig. 2. Survivor curves for Escherichia coli O157:H7 in nutrient broth M1 (A), sausage stuffing, M2 (B), M2+10% bovine fat, (C), M2+20%
bovine fat (D), M2+10% cornstarch (E), and M2+20% cornstarch (F) at 54  C (*), 58  C (&), and 62  C (~).

exist. The increase in D-values implies a protective effect
to the cell in these heating menstrua (M5 and M6). The
moisture contents of M2, M5 and M6 were 68, 62 and
57%, respectively. Changes in the effect of heat on
microbial cells when starch was added could also be
explained by the fact that the products with higher car-
bohydrate composition contained lower moisture. Fig. 3
shows the effect of water content on calculated
D-values; regression coefficients ranged from 0.83 to
0.98 showing that there was a strong correlation
between both variables, with the exception of D-values
at 54  C for E. coli O157:H7 (R=0.32).
An analysis of variance of the z-values indicated that
the media used had a significant effect on the thermal
resistance constant (z). When the menstruum prepared
with morcilla was enriched either with fat or starch (M3,
M4, M5 and M6), z-values increased significantly with
respect to M2, although the sensitivity to temperature
change was independent of the amount of starch or fat
added. An analysis of variance also indicated there was
710 J.M. Oteiza et al. / Food Research International 36 (2003) 703–712

Table 1
D-values, z-values, and correlation coefficients for the regressions (R) obtained for survivor curves of E. coli G335 in the different media

Media

M1 (nutrient Sausage stuffing

broth)
M2 M3 (M2+ M4 (M2+ M5 (M2+ M6 (M2+
10%fat) 20%fat) 10%starch) 20% starch)

D* (min) 4.5a 4.9 b 4.9b 5.4c 5.2d 5.4c

T=54  C R=0.962 R=0.995 R=0.955 R=0.994 R=0.934 R=0.984
D* (min) 1.5a 1.7b 2.5c (0.1) 3.7d (0.1) 2.5c (0.1) 3.6d (0.1)
T=58  C R=0.926 R=0.968 R=0.966 R=0.975 R=0.962 R=0.968
D* (min) 0.5a(0.04) 0.7b(0.06) 1.1c (0.06) 1.2c (0.04) 0.9d (0.06) 1.2c (0.05)
T=62  C R=0.979 R=0.962 R=0.949 R=0.978 R=0.956 R=0.967
z** ( C) 7.90.6 7.80.6 10.50.8 10.40.8 10.3 0.8 10.4 0.8
R=0.996 R=0.998 R=0.998 R=0.998 R=0.997 R=0.998

Different letters (a–d) within the same row indicate that the values differ significantly (P<0.05).
* Standard deviation given between parentheses.
** Confidence limits calculated for P >0.95.

Table 2
D-values, z-values, and correlation coefficient for the regressions (R) obtained for survivor curves of E. coli O157:H7 in the different media

Media

M1 (nutrient Sausage stuffing

broth)
M2 M3 (M2+ M4 (M2+ M5 (M2+ M6 (M2+
10%fat) 20%fat) 10%starch) 20% starch)

D* (min) 4.9a 5.5b (0.2) 5.5b (0.3) 6.4c (0.3) 4.7d (0.2) 5.4b (0.2)
T=54  C R=0.922 R=0.943 R=0.971 R=0.961 R=0.964 R=0.983
D* (min) 1.6a (0.2) 2.1b (0.1) 2.1b (0.1) 3.2c (0.3) 2.5d (0.1) 3.6e (0.2)
T=58  C R=0.915 R=0.978 R=0.969 R=0.897 R=0.963 R=0.969
D* (min) 0.5a (0.04) 0.6a (0.03) 0.9b (0.09) 1.3c (0.02) 0.9b (0.05) 1.2c (0.05)
T=62  C R=0.973 R=0.987 R=0.924 R=0.993 R=0.963 R=0.977
z** ( C) 7.8 0.6 7.4 0.3 9.80.7 10.2 0.8 10.3 0.7 10.7 0.8
R=0.997 R=0.998 R=0.998 R=0.998 R=0.997 R=0.998

Different letters (a–d) within the same row indicate that the values differ significantly (P<0.05).
* Standard deviation given between parentheses.
** Confidence limits calculated for P >0.95.

Table 3
no significant difference between the z-values of the two
Comparison of z-values for E. coli G335 and E. coli O157:H7 in nutri-
ent broth, sausage stuffing, and sausage stuffing with 10 and 20% of fat strains (E. coli G335 and E. coli O157:H7). Line et al.
or starch added (1991) reported z-values of 4.3 and 4.6  C for E. coli in
ground beef containing 2% fat, depending on the
Media z-Value* ( C) z-Value* ( C)
E. coli G335 E. coli O157:H7
method of enumeration, and 4.7 for the same strain in
ground beef with 30.5% fat. Ahmed et al. (1995) repor-
Nutrient broth (M1) 7.9a 7.8a ted that the z-value for E. coli O157:H7 varied with fat
content of meat. In ground turkey with 3% or 11% fat
Sausage stuffing
M2 7.8a 7.4a content, the z-values were 4.74 or 4.35  C, respectively.
M3 (M2+10% fat) 10.5b 9.8b Similarly, the z-values determined in pork sausage con-
M4 (M2+20% fat) 10.4b 10.2b taining 7 or 30% fat, were 4.72 or 4.61  C, respectively.
M5 (M2+10% starch) 10.3b 10.3b However, the authors considered these values ‘‘similar’’
M6 (M2+20% starch) 10.4b 10.7b
but no statistical analysis was carried out to test for
Different letters (a–d) within the same column indicate that the values significant differences. We calculated, from the data
differ significantly (P<0.05). presented by Ahmed et al. (1995), the z-value standard
 J.M. Oteiza et al. / Food Research International 36 (2003) 703–712 711

Fig. 3. Decimal reduction times (D-values) as a function of water content at 54  C (*), 58  C (&), and 62  C (~): a—Escherichia coli G335;
b—Escherichia coli O157:H7.

deviation and applying a t-test, found that there were no
significant differences that might be attributed to fat
content.
The differences in D and z-values among studies may
be attributed to different E. coli O157:H7 strains or
isolates (assessed individually or as mixture), physi-
ological condition of the cells or the use of cultures in
different growth phases, fat content or pH of meat, or
methodology used for recovery of survivors and plating
procedures.
The findings in this work showed that when fat or
starch contents are raised to obtain better yields the risk
to consumers increases since both components have a
protective effect on the thermal inactivation of the
microorganisms. A combination of poor hygienic prac-
tices and insufficient heat treatment at the meat factory
may result in a product that does not meet the safety
standards established by law, posing a serious risk for
consumers. These results can be used to predict the time
required at a specific temperature to achieve a certain
number of log-cycle reduction of E. coli O157:H7 or
E. coli G335 in this type of sausage. Based on the thermal-
death-time values determined in this study, con-
taminated morcilla should be heated to an internal
temperature of 62  C and maintained for at least 2.9
min in the case of M2, 4.75 min (M3), 6.25 min (M4) to
provide a55D kill of E. coli O157:H7. When heating
media have 10 or 20% of cornstarch added contaminated
712 J.M. Oteiza et al. / Food Research International 36 (2003) 703–712
morcilla should be heated for a minimum of 4.6 min for
M5 and 6.0 min for M6 after an internal temperature of
62  C is reached. Thermal-death-times from this study
will assist meat factories to design heating regimes that
ensure safety of the product.
Acknowledgements
We thank Dr. Rivas for her PCR work on the isolated
strains. We also acknowledge the financial support of
the Consejo Nacional de Investigaciones Cientı́ficas y
Tecnológicas (CONICET), Agencia Nacional de Pro-
moción Cientı́fica y Tecnológica, Comisión de Investi-
gaciones Cientı́ficas de la Provincia de Buenos Aires
(CIC), and Universidad Nacional de La Plata.
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