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J Sci Food Agric 1998, 78, 486È490
Fate of Escherichia coli O157 : H7 in Chinese-Style
Sausage Subjected to Diþerent Packaging and
Storage Conditions
Chyang-Fuah Yu and Cheng-Chun Chou*
Graduate Institute of Food Science and Technology, National Taiwan University 59, Lane 144, Keelung
Rd, Sec 4, Taipei, Taiwan
(Received 21 July 1997 ; revised version received 16 February 1998 ; accepted 20 March 1998)
Abstract : In this study, Chinese-style sausages were subjected to air, vacuum or
nitrogen packaging and stored at either 5 or 25¡C. The survival characteristics of
Escherichia coli O157 : H7 during the storage period were determined. Results
revealed that, when stored at 5¡C, the number of viable E coli O157 : H7 in
sausages decreased slowly as the storage period extended, regardless of packaging methods. E coli O157 : H7 in sausages decreased from an initial population of ca 5É97 log CFU g~1 to ca 4É42È4É81 log CFU g~1 after 40 days of
storage at 5¡C. It was also found that viable cells of E coli O157 : H7 declined
more rapidly in sausage stored at 25¡C than at 5¡C. No viable E coli O157 : H7
was detected in either vacuum-packed or nitrogen-packed sausage after 40 days
of storage at 25¡C. On the other hand, the population of E coli O157 : H7
reduced to non-detectable levels in air-packed sausages after 20 days of storage.
Refrigerated storage and vacuum or nitrogen packaging provided conditions that
slowed down the death rate of E coli O157 : H7 in sausage. Furthermore, it was
noted that, among the curing agents tested, NaCl exerted the most signiÐcant
lethal e†ect on E coli O157 : H7 in sausage during the storage period. ( 1998
Society of Chemical Industry.
J Sci Food Agric 78, 486È490 (1998)
Key words : Escherichia coli O157 : H7 ; Chinese-style sausage ; packaging and
storage
pork, beef and poultry (Beery et al 1985 ; Borczyk et al
1987 ; Doyle and Schoeni 1987). Foods of animal origin
are regarded to be the primary sources of E coli
O157 : H7 infection.
Chinese-style sausage is made essentially from ground
pork with a larger particle size than is normal in other
types of sausage. It is cured and dried during the manufacturing process. The possible presence of E coli
O157 : H7 in the raw meat used for the manufacture of
Chinese-style sausage, mis-handling at processing facilities and the possibility of cross-contamination from
other principal vectors for transmission to pork may all
pose potential health risks. Previously, the authors
studied the fate of E coli O157 : H7 during the drying
steps of the manufacturing process and found that this
pathogen might still survive under some of the drying
INTRODUCTION
Escherichia coli O157 : H7 has emerged as a recognised
food-borne pathogen which causes haemorrhagic colitis
and haemolytic ureamic syndrome (Riley et al 1983 ;
Doyle and Schoeni 1987). Since the recognition of E coli
O157 : H7 in 1982, numerous incidents of food-borne
disease associated with this pathogen have occurred in
the US, Canada and the UK (Doyle 1991 ; Anonymous
1995). Furthermore, it was reported recently that foodborne outbreaks caused by E coli O157 : H7, the largest
in history, occurred in Japan, a†ecting more than 9000
individuals and resulting in seven deaths (Anonymous
1996). E coli O157 : H7 has been isolated from retail
* To whom correspondence should be addressed.
486
( 1998 Society of Chemical Industry. J Sci Food Agric 0022È5142/98/$17.50.
Printed in Great Britain
Fate of E coli in Chinese-style sausage
487
conditions currently employed in the sausage industry
in Taiwan (Yu and Chou 1997). In the present study,
they determined and compared the behavior of E coli
O157 : H7 in sausage subjected in air, vacuum and
nitrogen packaging and stored at 5 and 25¡C. In addition, the e†ect of curing agents on the survival characteristics of E coli O157 : H7 during the storage period
were also identiÐed.
MATERIALS AND METHODS
Organisms and preparation of inoculum
Four stains of E coli O157 : H7 including 933 (beef
isolate), A8993-C32(human isolate), MF6707(veal
isolate) and 18731A(veal isolate) were used as test
organisms. They were all obtained from the Food Safety
Inspection Service, Department of Agriculture, Washington, DC, USA. After two successive transfers of the
test organism in tryptic soy broth (TSB, Difco, Detroit,
MI, USA) at 37¡C for 24 h, the activated culture was
inoculated into TSB and incubated statically at 37¡C
for 18 h, when the population was ca 109 CFU ml~1.
Equal volumes of the cultures for each strain were then
combined to provide a four-strain mixture of E coli
O157 : H7 and were properly diluted with TSB to serve
as the inoculum.
Preparation and storage of the sausage
Detailed procedures for preparation of the sausage were
described in a previous paper (Yu and Chou 1997).
BrieÑy summarised, ground pork consisting of about
three parts lean to one part fat was mixed with the
TABLE 1
Curing agents for sausage
curing ingredient shown in Table 1, unless otherwise
speciÐed. After curing at refrigeration temperatures (5È
7¡C) for ca 12 h, the sausage batter was stu†ed into
edible collagen casing and sectioned by hand. Each
sausage was approximately 7 cm in length and 30 g in
weight. The sausages were then subjected to 50¡C airblast drying for 6 h. The prepared sausage was inoculated with E coli O157 : H7 (ca 5É0È6É0 log CFU g~1)
using a sterile syringe. They were inserted into either
ordinary oxygen permeable polyethylene or nylon/
polyethylene laminate bags (Sun A Enterprise Co, Taichiung, Taiwan). The latter bags, having a gas
transmission rate of 70 ml m~2 d~1 at 1 atm at 0% RH
and 30¡C, were further vacuum-packed or nitrogenpacked. All the packages were stored at either 5 or
25¡C.
Microbiological analysis
Samples were assayed for the presence of E coli according to the methods described by Okrend et al (1990) and
Abdul-Raouf et al (1993a). For enumeration of E coli
O157 : H7, a 25-g meat sample was Ðrst homogenised
with 225 ml sterile 0É1% peptone water. Serial dilutions were made with ButterÐeldÏs phosphate diluent.
One-tenth of a ml of each dilution was spread on MacConkey sorbitol agar (Difco) with 0É2 g litre~1
4-methylumbelliferyl-b-D-glucuronide (Oxoid, UK) and
incubated at 35¡C for 24 h. Typical colonies were
picked and conÐrmed as E coli O157 : H7 using biochemical tests of IMViC reaction and lysine decarboxylase production as well as a serological test with O157
and H7 antisera (Difco).
Aerobic plate counts (APC) were determined using
procedures described in the Bacteriological Analytical
Manual (FDA 1992).
Determinations of pH, temperature and moisture content
Ingredients
g kg~1 meat
Sucrose
Sodium chloride
Monosodium glutamate
Soybean protein isolate
Sodium tripolyphosphate
Potassium sorbate
Sodium ascorbate
Sodium nitrite
Rice wine
Spices
Wu-Shung powder
White pepper powder
Vanilla powder
Cinnamon powder
100É00
20É00
10É00
5É00
2É00
2É50
0É50
0É12
10É00
2É10
1É25
1É25
0É08
The pH of the samples was analysed by blending the
samples with equal amounts of distilled water and
determining the pH using a digital pH meter (Jenco
6200, Jenco Electronic, Taipei, Taiwan). The moisture
content of the sausage was determined by drying the
samples at 105¡C (AOAC 1984).
Statistical analysis
The mean values and standard deviation were calculated from data obtained from three separate experiments. These data were then compared by DuncanÏs
multiple range method (SAS 1989).
C-F Y u, C-C Chou
488
RESULTS AND DISCUSSION
Survival in sausage under di†erent packaging and storage
conditions
According to the Chinese National Standard, the wet
type of Chinese-style sausage should have a water
content of less than 40%. The sausages prepared in this
study had a water content of about 39É0%, thus meeting
the requirement. It is recommended that these sausages
be stored at refrigeration temperature (Anonymous
1984). However, in traditional retail markets, sausages
are often kept at room temperature for sale. Therefore,
the behaviour of E coli O157 : H7 at 25¡C, in addition
to that at 5¡C, was also examined in this study.
Table 2 shows the survival of E coli O157 : H7 in sausages subjected to vacumm, nitrogen or air packaging
and stored at 5 or 25¡C. Stored at 5¡C, regardless of the
packaging method, the viable cells of the inoculated E
coli O157 : H7 in all sausage showed a slight decrease
during the storage period. A population reduction of
only 1É16È1É55 log CFU g~1 was observed in sausage
after 40 days storage at 5¡C. No signiÐcant di†erence
(P [ 0É05) in the viable E coli O157 : H7 count was
observed in the sausage with the three di†erent packaging methods. This is consistent with the report of
Abul-Raouf et al (1993b), who indicated that no signiÐcant di†erence in the survival of E coli O157 : H7 in
salad vegetables subjected to ambient atmosphere packaging and those subjected to packaging under an atmosphere containing 3% oxygen and 97% nitrogen.
Regardless of packaging methods, a more rapid
decrease in the viable cells of E coli O157 : H7 was
noted in sausage kept at 25¡C than in those kept at 5¡C.
For example, a 1É69È2É29 log CFU g~1 reduction was
TABLE 2
Survival of Escherichia coli O157 : H7 in sausage with di†erent packaging methods and stored at 5¡C or 25¡Ca
Packaging
method
5¡C
Air
Vacuum
N
2
25¡C
Air
Vacuum
N
2
L og (CFU g~1) after days of storage
0
10
20
30
40
A5É97a
A5É97a
A5É97a
AB5É62a
B5É67a
AB5É70a
B5É20a
B5É51a
BC5É40a
B5É03a
C5É02a
CD5É13a
C4É42a
C4É75a
D4É81a
A5É83a
A5É83a
A5É83a
B3É54b
B4É14a
B4É00a
Èb
C3É67
C3É49
È
D3É33
D3É11
È
È
È
a Means in the same row with di†erent letters A, B, C, D or E and the
same column for each storage temperature with di†erent letters a or b
di†er signiÐcantly (P \ 0É05) according to DuncanÏs multiple range
test.
b No viable E coli O157 : H7 was detected when 0É1 ml of the lowest
dilution represent 0É1 g samples were surface plated on the media
used.
observed in sausage held at 25¡C for 10 days, compared
to a 1É16È1É55 log CFU g~1 in sausage held at 5¡C for
40 days. Additionally, a slightly higher number of viable
E coli O157 : H7 was noted in the vacuum-packed and
nitrogen-packed sausage than in the air-packed sausage.
When held at 25¡C for 20 days, E coli O157 : H7 in the
air-packed sausages decreased from an initial population of ca 5É83 log CFU g~1 to non-detectable level.
At the same time, a higher viable E coli O157 : H7
count of 3É67 and 3É49 log CFU g~1 with only 2É16È
2É34 log CFU g~1 reduction was observed in the
vacuum-packed and nitrogen-packed sausage, respectively.
Both refrigeration and vacuum packaging have been
generally regarded as beneÐcial for extending the shelflife of sausage (Anonymous 1984). Furthermore, a nitrogen gas atmosphere has been reported to be useful in
controlling the microÑora and extending the shelf-life of
lamb (Newton et al 1977), sandwiches (Kautter et al
1981 ; Bennett and Amos 1982), frankfurters (Simard et
al 1983a,b) and pork (Hu†man 1974 ; Enfors et al 1979).
However, results of the present study showed that
refrigeration, vacuum packaging and nitrogen packaging are actually less detrimental to E coli O157 : H7
than ambient atmosphere packaging (air packaging).
Yeh and Chou (1994), in a study of the fate of Campylobacter jejuni during the storage of Chinese-style sausage,
found that refrigerated storage and vacuum storage
provide conditions that slow down the death rate of C
jejuni in sausage. As shown in Table 3, the APC in
sausage held at 5¡C did not show any signiÐcant change
(P [ 0É05) during the 40-day storage period. Packaging
method caused no signiÐcant di†erence (P [ 0É05) in
the change of APC in the sausage either.
Held at 25¡C, the APC determined after 10 days
storage in vacuum-packed or nitrogen-packed sausage
showed a slight decrease. However, at the end of a
TABLE 3
Changes of aerobic plate count in sausage with di†erent packaging methods and stored at 5 or 25¡Ca
Packaging
method
5¡C
Air
Vacuum
N
2
25¡C
Air
Vacuum
N
2
L og (CFU g~1) after days of storage
0
10
20
30
40
A6É12a
A6É12a
A6É12a
AB5É85a
A5É93a
A6É05a
AB5É72a
A6É05a
A5É95a
AB5É90a
A6É00a
A5É92a
B5É47a
B5É52a
B5É71a
B6É10a
C6É10a
D6É10a
A7É16a
D5É27b
E5É12b
A7É42a
A7É44a
B7É69a
A7É04b
A7É70a
A8É00a
A7É29a
B6É63b
C6É84ab
a Means in the same row with di†erent letters A, B, C, D or E and the
same column for each storage temperature with di†erent letters a or b
di†er signiÐcantly (P \ 0É05) according to DuncanÏs multiple range
test.
Fate of E coli in Chinese-style sausage
489
Fig 1. E†ect of sodium chloride on the survival of E coli
O157 : H7 in sausage during storage at 25¡C in vacuum
package : (…) 0 g kg~1 ; (L) 10 g kg~1 ; (@) 20 g kg~1 ; (È)
30 g kg~1 ; (=) 40 g kg~1 ; standard deviations are represented by the error bars.
40-day storage period, a slight increase of APC (0É94È
1É90 log CFU g~1) was found in all the sausages,
regardless of the packaging method (Table 3).
Acidity was regarded to be one of the principle
factors which leads to the inactivation of E coli
O157 : H7 during the storage of fermented dry sausage
(Glass et al 1992). However, the pH of the sausages prepared, having an initial pH of 6É20È6É34, showed no signiÐcant change during the 40-day storage period,
regardless of the packaging method and storage temperature (Table 4). Therefore, under the test condition,
acidity should not exert any detrimental e†ect on the
survival of E coli O157 : H7 and other microÑora
during the storage of Chinese-style sausage.
Survival in sausage containing various amount of NaCl,
sorbate and nitrite during storage
In order to identify and compare the antimicrobial
activity of NaCl, sorbate and nitrite, sausages with
various formula were prepared. Ground meat was Ðrst
mixed with the curing ingredients listed in Table 1, with
the exception of NaCl, sorbate and nitrite. Various
amounts of either NaCl, sorbate or nitrite were then
Fig 2. E†ect of sodium nitrite on the survival of E coli
O157 : H7 in sausage during storage of 25¡C in vacuum
package : (…) 0 g kg~1 ; (L) 0É07 g kg~1 ; (@) 0É10 g kg~1 ; (È)
1É20 g kg~1 ; (=) 1É50 g kg~1 ; standard deviations are represented by the error bars.
added into the batter to prepare the sausage. Figures
1È3 show the survival of the inoculated E coli
O157 : H7 in the prepared sausages subjected to
vacuum packaging and held at 25¡C for a period of 15
days.
A signiÐcantly higher reduction (P \ 0É05) in the
viable E coli O157 : H7 was noted in sausages containing NaCl than that in sausage containing no NaCl (Fig
1). It was also found that, as the content of NaCl
increased or storage period extended, the reduction of
viable E coli O157 : H7 increased. After a 15-day
storage period, the viable E coli O157 : H7 in sausage
containing 20 g kg~1 NaCl, the level commonly
employed, decreased from an initial population of ca
5É89 to 5É16 log CFU g~1. On the other hand, sausage
with no added NaCl still had an E coli O157 : H7 population of ca 5É63 log CFU g~1, close to the initial population.
Although a slight change in the population of E coli
O157 : H7 was observed in sausages combined with the
amount of sodium nitrite (0É07È0É15 g kg~1) during the
storage period, the population in sausage containing
sodium nitrite displayed no signiÐcant di†erence
TABLE 4
Changes of pH in sausage with di†erent packaging methods
and stored at 5 or 25¡C
Packaging
method
5¡C
Air
Vacuum
N
2
25¡C
Air
Vacuum
N
2
pH value after days of storage
0
10
20
30
40
6É20
6É20
6É20
6É16
6É21
6É16
6É22
6É20
6É17
6É18
6É24
6É21
6É19
6É19
6É24
6É34
6É34
6É34
6É36
6É41
6É38
6É43
6É41
6É41
6É35
6É36
6É45
6É39
6É37
6É41
Fig 3. E†ect of potassium sorbate on the survival of E coli
O157 : H7 in sausage during storage at 25¡C in vacuum
package ; (…) 0 g kg~1 ; (L) 1É0 g kg~1 ; (@) 1É5 g kg~1 ; (È)
2É0 g kg~1 ; (=) 2É5 g kg~1 ; standard deviations are represented by the error bars.
C-F Y u, C-C Chou
490
(P [ 0É05) from that in sausage without sodium nitrite
(Fig 2). Meanwhile, the Ðnal E coli O157 : H7 population found in sausage containing 2É0 g kg~1 or less
sorbate was not signiÐcantly di†erent from that without
sorbate (Fig 3). However, a signiÐcantly less E coli
O157 : H7 population (P \ 0É05) was detected in
sausage containing 2É5 g kg~1 sorbate.
CONCLUSIONS
Based on the data obtained in this study, it is concluded
that, among the curing agents tested, NaCl exhibited
the most profound lethal e†ect on E coli O157 : H7 in
sausage during the storage period. If E coli O157 : H7
in the raw material was not completely inactivated
during the sausage processing, as noted previously (Yu
and Chou 1997), or if post-processing contamination
occurs, the general practice of vacuum packaging or
nitrogen packaging and refrigerated storage, while
extending the shelf-life of the sausage, would also enable
the contaminating E coli O157 : H7 to survive longer.
ACKNOWLEDGEMENT
This research was supported by the Department of
Health, Executive Yuan (DOH 84-FS006), Taiwan,
ROC.
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