Food Microbiology 27 (2010) 266e273

Contents lists available at ScienceDirect

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

Effect of high pressure on the microbiological quality of cooked chicken

during storage at normal and abuse refrigeration temperatures
Margaret F. Patterson*, Alan M. McKay, Malachy Connolly, Mark Linton
Food Microbiology Branch, Agriculture, Food and Environmental Science Division, Agri-Food and Biosciences Institute, Newforge Lane, Belfast BT9 5PX, Northern Ireland, UK

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

Article history: Vacuum-packaged cooked poultry meat was treated at a range of pressures (400e600 MPa) and hold times
Received 21 August 2009 (1, 2 and 10 min), followed by storage at 4
, 8
or 12
C for up to 35 days. Weissella viridescens was found to
Received in revised form be the dominant microorganism in the pressure-treated meat, constituting 100% of the microflora iden-
8 October 2009
tified at 500 and 600 MPa. None of the pressure-treated samples had obvious signs of spoilage during the
Accepted 14 October 2009
35 day storage period, even when the Weissella count was >7 log10 cfu/g. Studies on a typical W. viridescens
Available online 21 October 2009
isolate showed it to be relatively pressure-resistant in poultry meat, with <1 log reduction in numbers
after a treatment of 2 min at 600 MPa. Agar diffusion assays showed that the isolate also caused the
Keywords:
High pressure inhibition of a number of Gram-positive and Gram-negative pathogens, including strains of Clostridium
Cooked meat botulinum, Listeria monocytogenes, Bacillus cereus and Escherichia coli. The selection of a pressure-resistant
Microbiological quality organism, such as this Weissella sp. could be advantageous in extending the shelf-life, and also microbi-
Storage temperature ological safety, of the cooked meat, as it could give protection in addition to the pressure treatment itself.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction
Cooked poultry meat is a highly perishable product with a shelf-
life of 14e15 days when stored in air at 4
C. (Patsias et al., 2006).
The cooking process should render the meat virtually sterile but this
lack of microbial competition means that some organisms that do
survive, or are introduced post-cooking, can readily grow and reach
high numbers (Borch et al., 1988). Post-cooking recontamination,
mainly from the resident factory microflora, and slicing procedures
are regarded as the main factors, along with storage temperature,
which affects the shelf-life of cooked meats. (Samelis et al., 1998).
The shelf-life can be extended through the use of appropriate
packaging, including modified atmospheres, where oxygen is
excluded and CO2 (>60%), balance N2 is used, or through vacuum
packaging (Patsias et al., 2006). The dominant microflora on cooked
chilled vacuum or modified atmosphere packaged meats consists of
psychrotrophic microorganisms, especially the lactic acid bacteria
(LAB) such as Leuconostoc spp., Lactobacillus spp. and Weissella spp.
(Samelis et al., 2000a,b). The composition, and spoilage potential, of
this LAB microflora can vary with product type and storage condi-
tions (Laursen et al., 2009). Typically, the initial numbers of LAB in
vacuum packaged ready-to-eat meats are low, but they increase
during storage under chill conditions and some types can cause
overt spoilage when numbers reach ca 7e8 log10 cfu/g (Santos et al.,
* Corresponding author. Tel.: þ44 2890 255316; fax: þ44 2890 255009.
E-mail address: margaret.patterson@afbini.gov.uk (M.F. Patterson).
2005; Vermeiren et al., 2005). Spoilage defects include sour odours
and tastes, green discolouration, slime formation and pack swelling
(Samelis et al., 2000a; Santos et al., 2005; Korkeala and Bjo €rkroth,
1997; Chenoll et al., 2007).
Post-cooking contamination may also result in pathogens being
introduced on to the cooked meats and these may not be inhibited by
subsequent vacuum e or modified atmosphere packaging followed
by chilled storage. Psychrotrophic organisms such as Listeria mono-
cytogenes are of particular concern and this pathogen has been
implicated in a number of foodborne outbreaks associated with
cooked meats (Gottlieb et al., 2006; Frye et al., 2002; Sim et al., 2002).
Growth of, and toxin production by, non-proteolytic Clostridium
botulinum is also potentially of concern in chilled foods, including
cooked meats, especially if the product is packaged in an atmosphere
with reduced oxygen. However, despite the large volume of cooked
meats, and other chilled foods being sold worldwide, they have not
normally been associated with foodborne botulism.
High hydrostatic pressure (HHP) can successfully be used as
a final decontamination step for pre-packed cooked meats (Slongo
et al., 2009). The technology is attractive as it can kill many
microorganisms, and so give improved product safety and shelf-life,
without significantly affecting the sensory or nutritional properties
of the food (Garriga et al., 2004) and it is being used commercially in
a number of countries in Europe and in the USA for this purpose.
Pathogens such as L. monocytogenes, Salmonella typhimurium
and Staphylococcus aureus are relatively sensitive to pressure and
a treatment of 400e600 MegaPascals (MPa) for a few minutes is

0740-0020/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2009.10.007
 M.F. Patterson et al. / Food Microbiology 27 (2010) 266e273
sufficient to give a significant reduction in numbers in ready-to-eat
foods (Hayman et al., 2004; Aymerich et al., 2005; Garriga et al.,
2004; Tassou et al., 2008). However, bacterial spores are extremely
pressure-resistant, just as they are resistant to many other physical
treatments such as heat and irradiation. Typically they can survive at
pressures >1000 MPa (Cheftel, 1995) and the use of extremely high
pressure alone is unlikely to be successful in reducing numbers.
However, there is increasing interest in using heat in combination
with high pressure to sterilize foods (Leadley et al., 2008).
Extending the shelf-life of cooked poultry meat to 4e5 weeks
has several advantages. Consumers would still regard the product
as fresh and minimally processed and the longer shelf-life would
allow more flexibility in the manufacturing and distribution supply
chain and, potentially, less waste. The use of pressure treatment
post packaging could improve the microbiological quality of the
cooked poultry meat to meet these requirements. There is already
evidence that the technology can improve food safety by reducing
numbers of pathogens, but there is relatively little published
information on the effect of HPP on the non-pathogenic microor-
ganisms present on cooked chicken and how any survivors may
subsequently multiply and influence the quality of the meat during
extended storage at ideal and abuse chill temperatures.
The purpose of the work reported here was to identify the microbial
interactions between high pressure processing conditions (level of
pressure and duration of application) and storage temperature (ideal
and abuse temperatures) on the cooked chicken and to identify those
organisms which dominate during storage for up to 35 days.
2. Materials and methods
2.1. Preparation of minced chicken
Chicken breast fillets, heated to a minimum internal temperature
of 80
C for 1 min were obtained from a local poultry producer
within 24 h of cooking The meat was maintained at 4
C during
transportation and on arrival at the laboratory, it was cut into strips
using a sterile knife and minced (to evenly distribute any surviving
microorganisms) using a sterile 0.4 cm mincing screen on a sterile
mincer attachment for a Kenwood Chef food mixer (Kenwood Ltd.,
Hampshire, England). Portions of the minced chicken (10  0.2 g)
were placed in polyethylene/polyamide vacuum pouches (Somer-
ville Packaging, Lisburn, Northern Ireland) and vacuum packaged as
described by Linton et al. (2004). The oxygen permeability of the
pouches was 50 cm3/m2/24 h at 1 bar, 23
C and 0% relative humidity.
2.2. High pressure treatment
Samples were treated in an Avure Quintus 35 L high pressure
press (Avure Technologies, Va €ster
as, Sweden), with a cylinder
18 cm in diameter and 1.2 m in length and a capacity of 35 L.
The pressure transmission fluid was potable water from the mains
water supply. The pressure come-up time was approximately
20e25 s per 100 MPa and pressure release time was between 10
and 15 s, depending on the pressure. The initial temperature of the
water was 18
C and the temperature increase due to adiabatic
heating was approximately 2
C per 100 MPa.
The samples were pressure treated in batches at 400, 500 and
600 MPa, each for 1, 2 and 10 min. Come-up and decompression
times were not included in the treatment time.
The entire experiment was replicated on two separate occasions.
2.3. Sample storage
After pressure treatment, one sample from each set of processing
conditions was analysed immediately (day 0) and the remaining
267
samples from each set of processing conditions were stored at 4
, 8

and 12
C and analysed on days 7, 14, 21 and 35 post-treatment.
2.4. Microbiological analyses
The packages were opened aseptically and the contents trans-
ferred to a sterile stomacher bag with a filter insert (Interscience,
St. Nom La Breteche, France). A 101 dilution of the minced chicken
was prepared in maximum recovery diluent (MRD) (Oxoid code
CM733, Oxoid, Basingstoke, U.K.) using a variable diluter (Baby
Gravimat, Interscience, St. Nom La Breteche, France). This was
homogenised for 2 min in a stomacher (Lab blender 400, Seward,
Bury St. Edmunds, U.K.). If necessary, appropriate further dilutions
were prepared in 9 ml MRD. Bacterial numbers were determined
using a WASP spiral plater (Don Whitley Scientific Ltd, Shipley, U.K.)
and colonies were enumerated using a Protocol automated colony
counter (Synoptics Ltd., Cambridge, U.K.).
Total aerobic psychrotrophic counts were obtained by plating
out on tryptone soya agar (Oxoid CM131) supplemented with 0.6%
yeast extract (Oxoid L21) (TSAYE). The plates were incubated
aerobically at 25
C for 72 h.
Anaerobic counts were obtained by plating out on TSAYE and
incubating the plates anaerobically at 30
C for 72 h. Anaerobic
incubation was carried out in an Electrotek anaerobic workstation
(Electrotek Scientific, Shipley, U.K.), using a gas mixture containing
5% CO2, 10% H2 and 85% N2.
Lactic acid bacteria (LAB) counts were obtained by plating out on
De Man, Rogosa and Sharpe agar (MRS) (Oxoid CM361). The plates
were incubated at 30
C for 72 h in a CO2 incubator (LEEC,
Nottingham, U.K.) with a 5% CO2 atmosphere.
Samples were also screened for the presence of galactose-fer-
menting LAB by plating onto MRS agar (de Man et al., 1960) which
had been modified by substituting galactose (1%) for glucose and
adding bromocresol purple (0.005%) as an indicator. These plates
(denoted as MRS-GAL) were incubated as for MRS.
2.5. Characterisation of microflora
When samples showed growth of surviving microorganisms,
isolates were randomly selected from MRS-GAL and TSAYE plates with
20e200 colonies, using the Harrison disc method (Harrigan,1998) and
characterised using standard biochemical tests as previously described
(Linton et al., 2004). A sub-selection of these isolates were identified by
16S RNA sequencing. DNA from the isolates was extracted and purified
(Pitcher et al., 1999). The 16S RNA gene was amplified by PCR using
primers UF (GAACGCTGGCGGCGTGCCT) and UR (ACGGGCGGTGTGTA)
with ABgene ReddyMix PCR Master Mix (1.5 mM MgCl2). The ampli-
fied fragments were purified using vacuum filtration (Millipore
MontageÔ kit) and sequenced with an Applied Biosystems BigDye
Terminator v3.1 cycle sequencing kit. The FASTA database (European
Bioinformatics Institute) was searched using sequences of at least
650 bp from both the 50 and 30 ends of the 16S RNA gene.
Selected galactose-negative isolates from MRS-GAL plates were
analysed by RAPD using ERIC1R and ERIC2 primers (Gillings and
Holley, 1997).
2.6. pH measurement of cooked poultry meat
The pH of the meat, mixed with deionised water (50:50) was
measured during the storage period using a Jenway pH Meter
Model 3150.
2.7. Pressure resistance of Weissella viridescens
Three of the W. viridescens strains isolated from the chicken storage
experiment were selected at random. Cultures were maintained on
268 M.F. Patterson et al. / Food Microbiology 27 (2010) 266e273
MRS agar slopes until required. A loopful of culture was inoculated
into 10 ml MRS broth (Oxoid CM359) and incubated for 18 h at 30
C.
A 103 dilution of this culture was prepared in MRD and 1 ml of the
dilution added to 100 ml MRS broth. The broth was incubated for 48 h
at 30
C. Cells from a 50 ml aliquot of the broth culture were harvested
by centrifugation (2400  g for 20 min). Pellets were washed twice in
phosphate-buffered saline (PBS) (Oxoid BR14a) before being resus-
pended into 50 ml PBS to give a final concentration of approximately
109 cfu/ml. The suspension was dispensed in 4.5 ml aliquots into
polyethylene/polyamide pouches (Somerville Packaging, Lisburn,
Northern Ireland) measuring approximately 10  6 cm. The pouches
were sealed using a heat sealer (R.S. Components, Corby, U.K.) taking
care to exclude as much air as possible. Each pouch was placed in
a larger pouch (13  8 cm) and vacuum packaged in a vacuum packer
(Henkelman BV, 's-Hertogenbosch, Netherlands). Pouches were
pressure treated within 1 h. An untreated suspension was held at
ambient temperature and pressure as a control.
Minced chicken meat was prepared and 10 g portions were
placed in 30  16 cm polyethylene/polyamide pouches as described
in Section 2.1. The meat was sterilised by irradiation to a dose of
25 kGy, using a 60Co Gamma-beam 650 facility (Nordion, Canada), as
described by Patterson (1989). A bacterial suspension was prepared
as described above. The pouch containing the sterile minced chicken
was opened aseptically and 1 ml of the suspension was added
to give a final concentration of approximately 8 log10 cfu/g. The
contents were mixed to distribute the suspension evenly and the
pouch was vacuum packaged. The resealed pouch was then placed
in a larger vacuum pouch and vacuum packaged again.
All samples were pressure treated within 1 h of inoculation. An
untreated, inoculated chicken sample was held at ambient pressure
as a control.
The samples were pressure treated using a Stansted Food
Lab 900 high-pressure isostat, which is capable of operating up
to 900 MPa (Stansted Fluid Power Ltd., Stansted, U.K.) and has
a cylinder 7 cm in diameter and 20.3 cm in length. The pressure
transmission fluid was 10% Cooledge B1 (Castrol, U.K.) in water.
The initial temperature was 18
C, the pressure come-up time was
approximately 200 MPa/min and the pressure release time was
approximately 2 min. In all the experiments the duration of the
pressure treatment did not include the come-up or pressure release
time. The temperature inside the cylinder was monitored by
a thermocouple that was immersed in the pressurisation fluid
during treatment. The temperature increase due to the adiabatic
heating effect during pressure treatment was approximately 2.5
C
per 100 MPa.
Initial inactivation studies were carried out in PBS for all three
strains at 200, 300, 400, 500, and 600 MPa for 1 min. After pressure
treatment, appropriate serial dilutions were prepared in MRD and
the samples plated onto MRS using a spiral plater as described in
Section 2.4. Plates were incubated at 30
C for 48 h.
As there was no significant difference between the strains (data
not presented), one strain was selected at random for further study.
A suspension of this strain in PBS and in minced chicken was
pressure treated at 400, 500 and 600 MPa with hold times in the
range 0e10 min. A hold time of 0 min was included to assess the
effect of pressure come-up time and decompression. After each
treatment enumeration was carried out as described above. Each
treatment was repeated on three separate occasions.
2.8. Inhibition of other bacteria by Weissella
The inhibition of a number of pathogenic bacteria by W. vir-
idescens (the isolate used in the detailed pressure resistance
studies, described above) was tested in vitro. The cultures came
from the designated culture collections or from the AFBI culture
collection, except for the C. botulinum cultures, which were kindly
provided by Dr P. McClure, Unilever, U.K.
W. viridescens was grown to stationary phase in MRS broth. A
1 ml subsample of the culture was transferred to a sterile Eppendorf
tube and centrifuged in a microfuge at 11 000  g for 5 min. The
supernatant was removed and filter-sterilised using a 0.2 mm filter.
The target microorganisms were grown to stationary phase in
TSBYE or cooked meat medium (for C. botulinum). This stationary
phase culture (200 ml) was added to an MRS agar plate and spread
using a sterile glass spreader. The plate was allowed to dry thor-
oughly and a drop of the W. viridescens culture was added to the plate
using a sterile pastette (drop diameter approx 1 cm). In addition,
a drop of the W. viridescens supernatant was added to a sterile filter
paper disc (diameter 6 mm) that had been placed on the surface
of the agar. The plate was incubated at 30
C for 24e48 h. If the
microorganism being tested against did not grow on MRS agar, the
microorganism was spread onto a suitable medium i.e. TSAYE or
Brain Heart Infusion agar (Oxoid CM375). When the plate was dry,
a plug of agar (6 mm in diameter) was aseptically removed and
replaced with a plug of MRS agar that had been aseptically removed
from a plate that had been spread with 200 ml of the W. viridescens
culture. After incubation the plates were examined for inhibition
around the spot or plug of W. viridescens and around the spot of
supernatant. A positive result was recorded if the zone of clearing
beyond the spot, disc or plug was 5 mm.
2.9. Statistical analyses
Factorial analysis of variance was used to determine the inter-
actions between the variables of pressure, hold time, storage
temperature and agars on the microbial counts obtained.
3. Results
3.1. Microbiological quality of pressure-treated cooked chicken
The initial microbial counts in the untreated and pressure-treated
cooked chicken were at, or below, the level of detection (<2.3 log10/g)
on TSAYE (aerobic), TSAYE (anaerobic) and MRS. For the purposes of
statistical analyses, these counts were taken to be 2.3 log10/g.
Overall, there were only minor differences, of no practical
significance, in the counts obtained aerobically, anaerobically or
on MRS agar at each of the sampling points, suggesting that the
surviving microorganisms could grow under all 3 conditions.
Therefore results are presented as averages of the 3 agars.
As expected, the 4 remaining factors (pressure, hold time,
storage temperature and storage time) were all significant when
considered individually (p < 0.001). As the pressure level increased
or the hold time increased, so the number of surviving microor-
ganisms decreased significantly (p < 0.001) (Tables 1 and 2). As the
storage temperature or the storage time increased, so the number
of surviving microorganisms increased significantly (p < 0.001)
(Tables 3 and 4). In all cases, the values presented in Tables 1e4 are
means over the other treatments.
There was no significant interaction between the 4 factors of
pressure, hold time, storage temperature and storage time (p > 0.1).
Table 1
Effect of pressure on the overall means of numbers of microorganisms in cooked
chicken. Values of means with superscripts of different letters are significantly
different. Least Significant Difference (5% level) ¼ 0.23 (n ¼ 270).
Log10 microbial numbers/g cooked chicken
400 MPa 500 MPa 600 MPa
6.06a 5.70b 4.81c
 M.F. Patterson et al. / Food Microbiology 27 (2010) 266e273 269

Table 2 Table 4
Effect of pressure-hold time on the overall means of numbers of microorganisms in Effect of storage time on the overall means of numbers of microorganisms in pres-
pressure-treated cooked chicken. Values of means bearing superscripts with different sure-treated cooked chicken. Values of means bearing superscripts with different
letters are significantly different. Least Significant Difference (5% level) ¼ 0.23 (n ¼ 270). letters are significantly different. Least Significant Difference (5% level) ¼ 0.30
(n ¼ 162).
Log10 microbial numbers/g cooked chicken
Log10 microbial numbers/g cooked chicken
1 min 2 min 10 min
6.09a 5.85b 4.63c 0 7 14 21 35
2.30a 4.14b 6.26c 7.31d 7.61d

However, there were significant interactions between pressure,

hold time and storage time (p < 0.001) and also between hold time, A more detailed microflora analysis was carried out on 65 isolates
storage temperature and storage time (p < 0.05). chosen at random from samples treated at 500 MPa for 10 min and
Fig. 1 shows the effects of pressure and hold time on the stored at 8
C for 10 days. These isolates were taken as representative
microbial counts during storage over 35 days. There was no of the homogenous microflora found in all the 500 and 600 MPa
significant difference (p > 0.05) in counts obtained with a hold time samples during the storage period. All isolates were Gram-positive,
of 1 or 2 min. However, counts tended to be significantly lower with
a hold time of 10 min. This effect was more pronounced as pressure
increased. At 400 MPa, the counts for a hold time of 10 min only
became significantly lower at day 14, while at 600 MPa, the counts
were significantly lower from day 7 onwards. Overall, a treatment
of 600 MPa for 10 min gave significantly lower counts throughout
storage, compared to the other treatments.
Fig. 2 shows the effects of hold time and storage temperature on
the microbial counts during 35 days storage. There was no signifi-
cant difference between counts obtained at either 8
or 12
C when
the hold time was 1 or 2 min. As expected, the counts were always
significantly lower when the storage temperature was 4
C at these
hold times. The only exception was with 1 min hold time after 35
days, when all the counts reached approximately 8.5 log10/g. All the
counts throughout the 35 day storage were lower when a hold time
of 10 min was used.
Overall, a treatment of 600 MPa for 10 min, followed by storage at
4
C gave the lowest microbial counts throughout the 35 day storage.

3.2. Characterisation of microflora

Twenty isolates from untreated samples stored at 8
C for 10 days

were characterised as Gram-negative and catalase positive. Ten of
these isolates were sequenced by 16S RNA and identified as Rahnella
aquatilis (6 isolates) and Serratia proteamaculans (4 isolates) by
FASTA search of the European Bioinformatics database. All isolates
had sequence homologies >98%. These would be typical of the
psychrotrophic Enterobacteriaceae recovered from vacuum-pack-
aged cooked meats (Gram et al., 1999; Barakat et al., 2000).
Visual inspection of plates for samples treated at 400 MPa for 1,
2, or 10 min and incubated at 8
or 12
C showed mixed microbial
populations of colonies. Twenty isolates were selected at random
from TSAYE plates of samples treated at 400 MPa for 1 min and
stored at 12
C. These were identified, from biochemical tests, as
Bacillus spp. (6 isolates), Enterobacter spp. (6 isolates), galactose-
negative lactobacilli (6 isolates) and lactococci (2 isolates). The
lactobacilli were later identified using RAPD and 16S sequencing as
W. viridescens. The agar plates from samples treated at 500 MPa or
600 MPa all showed one colony type, irrespective of storage
temperature and all the chosen isolates were galactose-negative
lactic acid bacteria when incubated at 4, 8 or 12
C.

Table 3
Effect of storage temperature on the overall means of numbers of microorganisms in
pressure-treated cooked chicken. Values of means bearing superscripts with different
letters are significantly different. Least Significant Difference (5% level) ¼ 0.23 (n ¼ 270).

Log10 microbial numbers/g cooked chicken

Fig. 1. Effect of high pressure and hold time on the microbiological quality of cooked
4
C 8
C 12
C poultry meat during storage. (a) Treatment at 400 MPa; (b) treatment at 500 MPa;
(c) treatment at 600 MPa. - 1 min hold time; C 2 min hold time; : 10 min hold time.
4.29a 5.83b 6.46c
Least Significant Difference (5% level) ¼ 0.90 (n ¼ 18).
270 M.F. Patterson et al. / Food Microbiology 27 (2010) 266e273
Fig. 2. Effect of storage temperature and pressure-hold time on the microbiological
quality of cooked poultry meat during storage. (a) Storage at 4
C; (b) storage at 8
C;
(c) storage at 12
C. - 1 min hold time; C 2 min hold time; : 10 min hold time. Least
Significant Difference (5% level) ¼ 0.90 (n ¼ 18).
catalase-negative rods which did not ferment galactose as tested on
MRS-galactose plates. Twenty of these isolates were selected at
random for RAPD analysis and all gave identical patterns; of these, 5
were identified by 16S RNA sequencing as W. viridescens.
3.3. pH of meat during storage
The pH of the chicken on Day 0 was 6.23 and it ranged from pH
5.98 to pH 6.45 during the 35 day period but there was no obvious
trend, based on treatment conditions, during storage.
3.4. Pressure resistance of W. viridescens
The results of the inactivation studies are given in Fig. 3. The
pressure come-up time (time 0) did not significantly reduce
numbers of the W. viridescens strain, when treated in chicken meat
or at 400 MPa in PBS. However, there was a 1.12 log10 and 2.55 log10
reduction in initial numbers when the organism was treated in PBS
at 500 and 600 MPa respectively. The W. viridescens isolate was
significantly more pressure-resistant, when treated in chicken meat
compared to buffer (p < 0.001). As expected, the level of inactivation
increased significantly as the pressure level increased (p < 0.001),
and after 600 MPa for 10 min there was a 3.26 log10 and 6.09 log10
reduction in the meat and buffer respectively.
3.5. Inhibition of other bacteria by Weissella
W. viridescens showed antimicrobial activity against a wide range
of microorganisms. (Table 5). Both Gram-negative and Gram-posi-
tive bacteria were inhibited, but inhibition was unpredictable as
some strains of species such as L. monocytogenes and Escherichia coli
were inhibited while others were not. In all cases, when microor-
ganisms were inhibited there was a large diffuse zone of clearing
around the spot of culture and no (or a very slight) zone around the
supernatant.
4. Discussion
In this study, the initial numbers of the cooked chicken, with or
without pressure treatment, were at or below the level of detection
(2.3 log10/g). This is similar to results reported by other workers,
where initial LAB counts on cooked meats were found to be
<1 log10/g. However, in many cases these products later became
spoiled during storage (Bjo € rkroth and Korkeala, 1996; Hamasaki
et al., 2003; Chenoll et al., 2007).
W. viridescens was found to be the most dominant microorganism
recovered from the pressure-treated poultry meat, irrespective of
Fig. 3. Pressure inactivation of Weissella viridescens in phosphate buffer and cooked
poultry meat. - 400 MPa treatment in buffer; C 500 MPa treatment in buffer; :
600 MPa treatment in buffer; , 400 MPa treatment in cooked chicken; B 500 MPa
treatment in cooked chicken; 6600 MPa treatment in cooked chicken. Log (N/No)
gives the level of inactivation. E.g. a value of 1 is equivalent to 1 log10 reduction in
numbers. Least Significant Difference (5% level) ¼ 2.04 (n ¼ 3).
 M.F. Patterson et al. / Food Microbiology 27 (2010) 266e273 271

Table 5 in numbers respectively in the poultry meat. This is in broad

Inhibition of pathogenic bacteria by Weissella viridescens. agreement with Park et al. (2001), who reported that a treatment
Species Strain Source Inhibition of 600 MPa for 5 min at 25
C gave a w4 log10 cfu/g reduction
Bacillus cereus NCTC 11145 Meat loaf þ numbers of Lactobacillus (now Weissella) viridescens in ham.
Bacillus cereus ATCC 14579 þ To date the majority of studies on the effect of pressure treatment
Bacillus cereus NCFB 578 þ on the microflora of ready-to-eat and cooked meats have concen-
Bacillus cereus NCFB 722 þ
trated on products other than poultry and most of the studies have
Bacillus cereus NCFB 1771 þ
Bacillus cereus LB1 NIZO þ been carried out under ideal storage conditions of w4
C, rather than
Bacillus cereus LB5 NIZO þ including abuse temperatures. In most cases, LAB have been found to
Bacillus cereus VC2 NIZO þ dominate the microflora of the products during storage.
Clostridium botulinum BL 77 þ Garriga et al. (2004) reported that pressure treatment of vacuum
Clostridium botulinum BL 34 
Clostridium botulinum BL 129 þ
packed cooked ham (600 MPa for 4 min at 16
C) resulted in aerobic
Escherichia coli ATCC 10536  total counts and LAB counts of 2.10 log10 and <2 log10/g respectively
Escherichia coli NCTC 9029 þ after 30 days storage at 4
C, compared to a total viable count (TVC)
Escherichia coli NCTC 9706 þ of 7.59 log10 and LAB count of 7.84 log10 cfu/g in the control samples.
Escherichia coli NCTC 9707 
By the end of the storage period (120 days), the LAB seemed to have
Escherichia coli NCTC 11601 Human faeces þ
Escherichia coli NCTC 11602 Human faeces þ recovered and the counts in control and pressure-treated samples
Escherichia coli NCTC 11603 Human faeces  were similar at 8.76  0.90 log10 and 7.62  0.97 log10 respectively.
Escherichia coli H03 þ This suggests that the microorganisms which survived the initial
Escherichia coli H117/86 þ pressure treatment may have been sub-lethally injured and were
Escherichia coli H461/84 þ
Listeria monocytogenes NCTC 4883 Bird þ
unable to grow rapidly in the ham which contained ingredients such
Listeria monocytogenes NCTC 4885 Clinical þ as sodium chloride (1.78%) which are known inhibitors of pressure-
(infantile meningitis) damaged cells (Patterson et al., 1995).
Listeria monocytogenes NCTC 5214 Sheep brain  Diez et al. (2009) reported that pressure-treated (600 MPa for
Listeria monocytogenes NCTC 5348 Mammal þ
10 min at 15
C) morcilla had TVC and LAB counts of 7.11 log10 and
Listeria monocytogenes NCTC 10357 Rabbit 
Listeria monocytogenes NCTC 10887 Dairy processing þ 7.41 log10 cfu/g respectively after 36 days storage at 4
C. These
environment samples were regarded as being spoiled as the LAB counts exceeded
Listeria monocytogenes NCTC 10888 Sheep þ 7 log10 cfu/g, although sensory evaluation scores did not decrease
Listeria monocytogenes NCTC 10890 Human faeces þ significantly during the 36 day storage and the product was found
Listeria monocytogenes NCTC 11994 
Listeria monocytogenes ATCC 19118 Chicken þ
suitable for consumption. Eleven of the 16 isolates identified at day
Listeria monocytogenes CIP 12502 þ 36 in pressure-treated samples were found to be W. viridescens. In
Listeria monocytogenes CIP 12503 þ contrast, the control, non-treated morcilla samples had TVC and LAB
Listeria monocytogenes AMB 114 þ counts of 8.07 log10 and 8.20 log10/g respectively after 36 days and 8
Listeria monocytogenes FMT 1750 Dairy processing 
of the 16 isolates identified were W. viridescens. Sensory evaluation
environment
Salmonella Chaco NCTC 6676  scores indicated that these samples had spoiled due to the presence
Salmonella Mbandaka NCTC 7892  of slime, off-odours, a sour taste and loss of vacuum in the packages
Salmonella Senftenberg NCTC 10384 Human faeces  (Diez et al., 2009). Overall, the results suggested there was no clear
Staphylococcus aureus NCTC 8325 Clinical þ selective effect, due to the pressure treatment, on the composition
Staphylococcus aureus 10562 þ
Staphylococcus aureus 10564 þ
of the microflora and the delay in spoilage may be due to lower
Staphylococcus aureus 10565 þ bacterial numbers being present in the pressure-treated samples.
Staphylococcus aureus 10566 þ The microbiological results from pressure-treated chicken ten-
Staphylococcus aureus 10567 þ ded to follow the same trend as morcilla, although the pressure
Staphylococcus aureus ATCC 27664 þ
treatment had a more pronounced selective effect on the microflora
of the chicken during storage, even at abuse temperatures. All of the
identified isolates from pressure-treated meat (500 and 600 MPa)
storage temperature. The dominance became more pronounced as were found to be W. viridescens and although numbers reached
the treatment conditions became more extreme and at 600 MPa it >8 log10 cfu/g at the end of storage, none of the samples showed
formed 100% of the identified surviving microflora. obvious signs of spoilage.
There are many reports of W. viridescens being recovered from The pH of pressure-treated cooked poultry meat did not change
ready-to-eat meats including cooked turkey (Samelis et al., 2000a), significantly during storage for 35 days, irrespective of storage
smoked pork loin and mortadella (Samelis et al., 2000b) and Morcilla temperature. This is similar to what was observed for morcilla samples
de Burgos, a cooked blood sausage from Spain (Koot et al., 2006). which had been pressure treated at 600 MPa for 10 min, where the
W. viridescens is a facultative heterofermentative psychrotroph initial pH of 6.40 did not change significantly during storage up to 36
which is relatively salt and pH tolerant (Mol et al., 1971) and some days at 4
C. However, after 50 days storage, the pH of the morcilla had
strains can cause spoilage (Borch et al., 1996; Santos et al., 2005). dropped to 5.68 (Diez et al., 2009).
However, none of the pressure-treated poultry meat samples in The W. viridescens strain chosen for the pressure resistance tests
the present study gave any sign of obvious spoilage, irrespective of also caused a number of pathogens to be inhibited during in vitro
pressure level, hold time or storage temperature even though, in studies. The effect was most pronounced against Gram-positive
some cases, microbial numbers were at 7e8 log10 cfu/g meat by day bacteria, including L. monocytogenes, S. aureus and spore formers
35 of storage. such as Bacillus cereus and C. botulinum (although not all strains of
W. viridescens is known to be relatively heat resistant and can C. botulinum were inhibited). Inhibition of Gram-negatives by
survive pasteurisation in cured meats (Milbourne, 1983; Borch W. viridescens was not so widespread; only a few of the E. coli strains
et al., 1988). Our results show it to also be relatively resistant to high tested were inhibited and none of the Salmonella serovars were
pressure. A pressure treatment of 600 MPa for 2 and 10 min at 18
C affected. It is not unusual for LAB to have antimicrobial activity
(initial temperature) resulted in <1 log10 and w3.5 log10 reduction against other microorganisms, especially Gram-positive bacteria.
272 M.F. Patterson et al. / Food Microbiology 27 (2010) 266e273
Barakat et al. (2000) found Carnobacterium piscicola isolated from
MAP cooked poultry produced an inhibitory substance which was
active against other LAB and several Listeria spp. and Lactobacillus
sakei CTC494 was found to inhibit the growth of L. monocytogenes in
raw chicken and cooked pork, especially when the meats were
packaged under vacuum (Hugas et al., 1998). Vermeiren et al. (2004)
also identified L. sakei strains which had antimicrobial activity
against L. monocytogenes present in cooked ham. The ham inocu-
lated with these isolates was acceptable in a sensory panel and the
authors concluded that they had potential to be used as protective
cultures in a cooked meat biopreservation system. This concept of
biopreservation has been proposed as a method for naturally
improving the shelf-life and safety of cooked meat products without
causing significant changes to the sensory properties (Lu € cke, 2000).
The antagonism between the LAB cultures and pathogens may result
from competition for nutrients and/or production of one or more
antimicrobial substances such as organic acids, antimicrobial
enzymes or bacteriocins (Holzapfel et al., 1995). Several studies have
reported synergism between HPP and bacteriocins, mainly nisin, for
the inactivation of pathogens in a range of foods, including cheese
(Rodriguez et al., 2005), sausage (Chung et al., 2005) and cooked ham
 et al., 2008). In these studies, the bacteriocin was added
(Jofre
directly to the food. This method can suffer the disadvantages of
limited diffusion in solid foods and inactivation through proteolytic
enzymes or binding to food ingredients (Holzapfel et al., 1995). An
alternative biopreservation method has been proposed. Here, non-
bacteriocinogenic, but very competitive cultures, could be added
successfully to meats where they would grow readily and inhibit the
growth of pathogens (Vermeiren et al., 2004).
Further work is required to determine the nature of the inhibition
shown by the W. viridescens strain in this study. Inhibition occurred
in the presence of the Weissella culture but not the supernatant. It is
possible that W. viridescens is producing small quantities of a small,
diffusible molecule and the microorganism needs to be growing
to keep producing enough antimicrobial to be inhibitory whereas
a small finite quantity of the substance, as found in the supernatant,
was not sufficient to be inhibitory. Another possibility is that the
putative inhibitor is not produced in planktonic growth in liquid
culture but is produced when grown on a solid medium. Weissella
paramesenteroides produces a small (w2.5 kDa) non-proteinaceous
antimicrobial that is active against both Gram-positive and Gram-
negative bacteria (Pal and Ramana, 2009).
Co-inoculation of a suitable poultry-based substrate with
W. viridescens and pathogens of potential concern in this type of
product would help to show whether the pathogens can survive and
compete with W. viridescens during pressure treatment and subse-
quent cold storage. Work is currently underway to determine the
survival of, and toxin production by, a range of strains of C. botulinum
during storage in pressure-treated cooked chicken. A comparative
study will be carried out in the presence of W. viridescens.
Treatments of 600 MPa were the most selective for the Weissella
spp. This pressure can be routinely achieved with modern equip-
ment but from a commercial viewpoint, as short as possible hold
time is desirable in order to maximise throughput of product and
minimise enzymatic changes (Patterson et al., 2006). Therefore,
a treatment regime of 600 MPa for 1e2 min rather than 10 min may
be more appropriate for the cooked poultry meat. This would allow
for greater survival of Weissella spp., but still give a significant
reduction in numbers of other, more pressure sensitive, microor-
ganisms. These results were obtained from one poultry processing
plant. Further work is required, using meat from other poultry
processing facilities, to determine if similar pressure-resistant, non-
spoilage Weissella spp. are commonly found in cooked poultry.
This study has also shown that high pressure treatment of the
cooked chicken selected for W. viridescens strains which did not
cause overt spoilage, even when present at high numbers, but
which did have inhibitory activity against pathogens. This could be
advantageous in extending the shelf-life and also microbiological
safety of the product, as it would give protection in addition to the
pressure treatment itself.
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