JFS M: Food Microbiology and Safety

Evaluation of High Pressure Processing

as an Additional Hurdle to Control Listeria
monocytogenes and Salmonella enterica
in Low-Acid Fermented Sausages
BEGONYA MARCOS, TERESA AYMERICH, AND MARGARITA GARRIGA

ABSTRACT: Low-acid fermented sausages (fuet and chorizo) were manufactured to evaluate the combined effect of high
pressure processing (HPP) and ripening on foodborne pathogens. Raw sausages inoculated with a three-strain cocktail of
Salmonella ser. Derby, London, and Schwarzengrund, and a three-strain cocktail of L. monocytogenes ser. 1/2 c and 4b were
pressurized at 300 MPa for 10 min at 17 °C. Afterwards, sausages were ripened at 12 °C and 80% RH for 27 d. Salmonella
counts decreased in all studied sausages during ripening. However, the application of HPP as an additional hurdle to the
ripening process produced a greater decrease in the Salmonella population, showing lower counts (3 MPN/g) in ripened
sausages. By contrast, lower values of L. monocytogenes counts were obtained in non-treated (NT) than in pressurized
sausages due to the delay of pH drop caused by HPP inactivation of endogenous lactic acid bacteria. After pressurization of
raw sausages at 300 MPa, a discoloration of sausages was observed, coinciding with an increase in L* values.

Keywords: low-acid fermented sausages, ripening, high pressure processing, Salmonella, L. monocytogenes

Introduction L. monocytogenes is a ubiquitous microorganism with an impor-tant

F ermentation has traditionally been used to achieve a preserva-tion effect
in foodstuffs. During fermentation, meat products become more stable and
increase their safety as a consequence of different hurdles (Leistner 1995).
Acid fermented sausages can gen-erally be considered low-risk products as a
consequence of reduced water activity and pH (4.8 to 5.0) that inhibit
pathogenic bacteria even at ambient temperature (Barbuti and Parolari 2002).
Among Mediterranean countries, there is a preference for dry sausages with
a limited sour taste. Low-acid fermented sausage technology is based on
small diameter (# 30 to 40 mm) sausages, and low ripening tem-peratures
occurrence in meat and meat products (Farber and Peterkin 1991; Farber
and Daley 1994; EC 1999). Moreover, the prevalence of L.
monocytogenes has been detected in 10% of 60 surface sam-ples
investigated from Spanish meat industries after cleaning (Garriga and
others 2004). Although there is no epidemiological evidence of the
involvement of fermented sausages in recent outbreaks of listeriosis
(Talon and others 2004), L. monocytogenes may survive in meat
fermentations (Johnson and others 1988; Glass and Doyle 1989; Junttila
and others 1989). The Agence Française de Sécurité Sanitaire des
Aliments (2000) reported 12% to 16% Listeria-positive isolations in

M: Food Microbiology & Safety

(#10 to 12 °C) to prevent an intense and rapid acidification (Sanz and others
1998). The final pH values of this kind of product
are about 5.3 to 6.2 (Aymerich and others 2003a).
Over the last few years, there has been an important increase in
infections by foodborne pathogens, especially in cases of Salmonel-la
and L. monocytogenes food poisoning. In Spain, the cases of in-fection
by L. monocytogenes and Salmonella have increased since 1996 by
158% and 71%, respectively (CNE 1997, 2004).
The risk of Salmonella in fermented meats is generally consid-ered
low (EC 2003). However, the survival of Salmonella in this type of
product has been demonstrated (Smith and others 1975; Levine and
others 2001; Moore 2004). Several outbreaks due to Salmonella in
sausage-like products were reported (Van Netten and others 1986;
Cowden and others 1989; Pontello and others 1998). Recently an
outbreak linked to consumption of fermented sausages with a short
fermentation period was described (Bremer and others 2004).
French fermented sausages. Aymerich and others (2003a) also reported
the prevalence of low numbers of L. monocytogenes (4 MPN/g) in
29.4% of low-acid fer-mented sausages studied.
The prevalence of pathogens in fermented meats would require the
use of more hurdles to pathogen growth as suggested by Leist-ner
(2000). Research dealing with newly developing technologies, including
hydrostatic pressure, would provide potential benefits to the fermented
meat industry.
High pressure processing is a non-thermal food preservation method,
appropriate for foods whose nutritional and sensorial characteristics are
thermosensitive (Carlez and others 1994). High pressure disrupts
secondary and tertiary structures of macromol-ecules, such as proteins
and polysaccharides, and alters their struc-tural and functional integrity
in a pressure-dependent way. Micro-organisms are killed owing mainly
to membrane damage (Kalchayanand and others 1998).

In this study, high pressure processing was applied to raw sau-sages

MS 20040738 Submitted 11/9/04, Revised 5/16/05, Accepted 6/2/05. Authors are
with Inst. for Food Research and Technology (IRTA), Meat Technology Center, inoculated with S. enterica and L. monocytogenes. The objec-tive was to
Granja Camps i Armet, 17121 Monells, Spain. Direct inquiries to author Garriga evaluate the combined effect of pressurization and rip-ening on
(E-mail: margarita.garriga@irta.es). pathogen survival and to consider its commercial use.

© 2005 Institute of Food Technologists Vol. 70, Nr. 7, 2005—JOURNAL OF FOOD SCIENCE M339
Further reproduction without permission is prohibited Published on Web 8/19/2005
 High pressure in low-acid sausages . . .
Materials and Methods
Sausage manufacture
Two types of low-acid fermented sausages, fuet and chorizo, were
manufactured. Both products were made with 50% lean pork meat and
50% pork back fat. The ingredients of the fuet formulation were as
follows (g/kg): sodium chloride 20, black pepper 2.5, potassium nitrate
0.1, sodium nitrite 0.1, dextrose 1, and sodium ascorbate 0.5. The
ingredients of the chorizo formulation were (g/kg): sodium chlo-ride 20,
cayenne pepper 15, paprika 15, dextrose 1, and garlic 3.
2 monocytogenes was used (European Commission FOOD-PCR:
Batters were inoculated with 6 × 10 colony-forming units (CFU)/
g of a three-strain cocktail of Listeria monocytogenes (L. monocyto-
genes ser. 1/2 c CTC1010, L. monocytogenes ser. 1/2 c CTC1011, L.
2
monocytogenes ser. 4b CTC1034) and 6 × 10 CFU/g of a three-strain
cocktail of Salmonella enterica (S. enterica sp. enterica ser. Derby
CTC1022, S. enterica sp. enterica ser. London CTC1003, S. enterica sp.
enterica ser. Schwarzengrund CTC1015). All strains were isolated from
meat products.
Lean pork meat and pork back fat were minced in a meat mincer with
an adjustable plate set at a hole diameter of 6 mm, mixed with other
ingredients in a kneading machine and stuffed into collagen casings.
High pressure processing (HPP)
Raw sausages were vacuum packed in polyamide-polyethylene bags
(Sacoliva, Castellar del Vallè, Spain) and subjected to pres-sure, the day
after stuffing. Pressure was applied in an industrial hydrostatic
pressurization unit (Alstom, Nantes, France) with a chamber of 320 L
volume and 280 mm diameter. Sausages were treated at 300 MPa for 10
min at 17 °C. The come-up time was about 9 min and the pressure
release time was 1.5 min.
After pressure treatment, plastic bags were removed and both non-
treated (NT) and pressurized (HPP) sausages were ripened under the
same conditions.
Ripening conditions
Sausages (fuet and chorizo) were hung in a climate chamber Sanyo
MLR-350 H, and dried for 27 d at 12 °C and 80% RH. At select-ed
times—after stuffing (day 0), after pressurization (day 1), and during
ripening (days 6, 13, and 27)—3 different sausages from each treatment
(pressurized and non-treated) were analyzed.
M: Food Microbiology & Safety
Microbial analysis
At each selected time, 20 g sausage were 10-fold diluted in ster-ile
0.1% peptone water (PW) (Difco Laboratoriess, Detroit, Mich., U.S.A.)
and 0.85% Na Cl (Merck, Darmstadt, Germany). The solu-tion was
homogenized for 1 min in a Masticator (IUL Instruments, Barcelona,
Spain) and serially diluted in PW.
Enumeration was done in selective media: lactic acid bacteria (MRS
agar, Merck) incubated anaerobically at 30 °C for 72 h; L.
monocytogenes (PALCAM agar base and PALCAM Listeria Selective
Supplement according to Van Netten and others 1989, Merck) incu-
bated at 30 °C for 72 h; Salmonella (BGA agar, Difco) incubated at 37
°C for 24 to 48 h. Five presumed colonies of L. monocytogenes and
Salmonella from respective media were retained for polymerase chain
reaction (PCR) confirmation. The most probable number (MPN)
technique was used when counts under 10 CFU/g were ex-pected. Serial
dilutions of 3 or 5 PW tubes of 3 successive dilutions were incubated at
30 °C for 48 h. Peptone water was used as non-selective medium to
recover the sublethally injured bacteria with-out underestimating the
counts. The growth of L. monocytogenes and Salmonella in MPN tubes
was determined by validated species-specific PCR assays.
M340 JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 7, 2005
A PCR pretreatment was applied. Two milliliters of enriched cul-ture
were centrifuged at 4 °C for 5 min at 12500 × g. The pellet was
®
suspended in 300 mL 6% Chelex 100 (Bio-Rad Laboratories, Her-
cules, Calif., U.S.A.), incubated at 56 °C for 20 min, boiled for 10 min,
and cooled on ice. Mixing between each step was needed. Cell debris
were centrifuged at 4 °C for 5 min at 24600 × g. Twenty microliters of
the supernatant were used for PCR reaction.
PCR reactions
A validated PCR identification protocol of Salmonella and L.
D’Agostino and others 2004; Malorny and others 2003).
Twenty microliters of either colony suspended in distilled water or
MPN origin were mixed with 2 mL of buffer 10x (Invitrogen, Carls-
bad, Calif., U.S.A.), 1.5 mM MgCl2, 150 mM of each dNTP, and 0.3
mM of each primer (139 and 141 for Salmonella: Rahn and others
1992; Lip1 and Lip2 for L. monocytogenes: Simon and others 1996), 1
mg/ L of BSA, and 1 U of Platinum Taq DNA polymerase (Invitrogen).
Amplification was performed in a GeneAmp PCR System 2700 (Ap-
plied Biosystems, Foster City, Calif., U.S.A.) with the following pro-
gram: 30 s at 95 °C, 30 s at 64 °C, and 30 s a 72 °C during 38 cycles,
with a final extension of 4 min at 72 °C. The reaction was visualized on
an agarose gel (1.5% agarose and 0.5 mg/mL ethidium bromide) after
30 min of electrophoresis at 100 V.
pH and water activity measurements
pH determinations were taken using a Crison Basic 20 pH-meter,
equipped with a Crison penetration 52-32 electrode (Crison Instruments,
Alella, Spain). Water activity was measured at 25 °C using a Novasina
Thermoconstanter TH-500 (Novasina, Pfäffikon, Switzerland).
Instrumental color analysis
Color measurements were performed using a Minolta Chroma-meter
CR200 (Minolta, Tokyo, Japan). C illuminant and 0° standard observer
were chosen. L* (brightness), a* (redness), and b* (yellow-ness) color
values were determined in the 1976 CIELab system. The colorimeter
was calibrated before each series of measurements using a white
ceramic plate. Each measurement was repeated at least 6 times.
Statistical analysis
Data were subjected to analysis of variance of the General Linear
Model procedure of SAS software (SAS System for Windows NT, re-
lease 8.1, SAS Inst., Cary, N.C., U.S.A.) where product, treatment, time,
and interaction between these parameters were included as fixed effects.
Level significance was set for P < 0.05.
Results and Discussion
Water activity and pH
The water activity (aw) of sausages decreased throughout the drying
process from an initial value of 0.98 to values of 0.83 to 0.86 (data not
shown). The decrease of a w was as expected for this kind of product
(Baracco and others 1982; Lizaso and others 1999). No significant
differences (P > 0.05) were found between a w values of non-treated
(NT) and pressurized sausages during drying. There-fore, high pressure
treatment did not affect the normal evolution of a w during sausage
ripening.
From the beginning of the process lower pH values were obtained in
chorizo than in fuet, which can be related to the acidity of paprika and
cayenne pepper added to chorizo (data not shown). The pH of non-
treated sausages decreased from an initial range of 5.7 to 5.8 in
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High pressure in low-acid sausages . . .
the meat batter to about 5.3 in the final product (Figure 1). Minimum pH
values of 5.17 (chorizo) and 5.32 (fuet) were reached at day 6 of
ripening. High pressure processing had a significant effect (P < 0.05) on
the sausage pH. pH values increased 0.13 (fuet) and 0.15 (chorizo)
points as a consequence of pressurization at 300 MPa. Mandava and
others (1994) reported an increase of pH in raw sausages pressurized
above 200 MPa, as a consequence of protein denaturation. Macfar-lane
and others (1980) attributed the pH rise to a loss of protons as a
consequence of increased ionization by HPP and consequent redis-
tribution of ions. Interactions between treatment and time were sig-
nificant (P < 0.05), suggesting that HPP affected not only the pH values
but also the evolution of pH during ripening. Minimum pH values of
HPP sausages 5.32 (chorizo) and 5.47 (fuet) were obtained at d 13 and
stayed at this level until the end of ripening.
Instrumental color analysis
NT fuet showed the typical red color from raw meat, while the red
color of non-treated chorizo was provided by paprika and cay-enne
pepper. Pressurization (300 MPa) after stuffing induced visu-al changes.
A discoloration toward a pale pink color was observed in fuet, and
coloration of chorizo also became paler. Carlez and others (1995) also
reported the red color of minced beef meat turning into a much paler
pink color at pressures ranging between 200 to 350 MPa. The
“whitening” effect of pressure was attributed to globin denaturation
and/or to heme displacement or release.
The observed discoloration was numerically confirmed by an
increase in brightness (L*) (P < 0.001) as a consequence of pressur-
ization (Figure 2). Goutefongea and others (1995) related the in-crease
of L* values by pressure either to a loss of active pigment or to protein
coagulation, affecting structure of the sample and sur-face properties,
that, in turn, may have increased the share of re-flected light versus
absorbed light. An increase in L* values was also reported by Carlez and
others (1995) in minced beef meat at pres-sures ranging between 200
and 350 MPa. Shigehisa and others (1991) observed that L* values of
pork muscle homogenates started to increase between 100 and 200 MPa,
to reach a maximum L* val-ue between 300 and 400 MPa. No further
increase was observed even at 600 MPa.
a* (redness) values were not altered (P > 0.05) as a consequence of
HPP. b*(yellowness) values of fuet were reduced significantly (P <
0.05) by pressure, whereas no changes were observed in cho-rizo (P >
0.05). Variations of a* and b* values have been related to changes in the
chemical state of myoglobin. The presence of red col-orants in chorizo
could have hidden the possible alterations of myo-globin occurring in
the meat.
Figure 1—pH values of NT and HPP sausages during rip-
ening. Bars indicate standard deviation of triplicates.
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Carlez and others (1993) reported no variation in a* values at
pressures below 400 MPa, and no variation of b* values at any pres-sure
applied; however, Shigehisa and others (1991) observed a moderate
decrease of a* values between 100 and 200 MPa that progressed up to
600 MPa. Ananth and others (1998) did not detect changes of any of the
color parameters studied when pressurizing raw loin pork for 13 min at
414 MPa and 25 °C.
Microbial results
Figure 3 illustrates the evolution of lactic acid bacteria (LAB) pop-
ulation during ripening. At day 6 of drying, coinciding with mini-mum
9
pH values, LAB reached maximum growth levels (about 10 CFU/g)
that were maintained until the end of the process. High initial counts of
6
endogenous LAB (about 10 CFU/g), close to the level usually
inoculated as starter cultures, assured the rapid and intense pH decrease
in NT sausages during the first day of drying.
LAB population experienced an immediate reduction of 3 and 4 log
CFU/g in fuet and chorizo, respectively, after treatment at 300 MPa.
Carlez and others (1994) detected lower inactivation of lactic acid
bacteria (1 log CFU/g) when processing minced meat at 300 MPa (20
min, 20°C), while pressurization at 400 MPa (20 min, 20°C) showed a
4- to 5-log reduction.
After pressurization, a rapid recovery of LAB viability was ob-served
(Figure 3). At day 6, LAB counts of HPP sausages were about 7 log
CFU/g, and by day 13, they had already reached the same levels as NT
sausages. Similarly, Garriga and others (2002) reported the recovery of
LAB to levels close to initial ones during chilled stor-age of pressurized
meat homogenates (400 MPa for 10 min at 17 °C).
The growth of L. monocytogenes, inoculated at a level of 6 × 10
2
CFU/g, was prevented in all sausages (NT and HPP) during drying. The
growth limit of L. monocytogenes in foods has been fixed at values of
aw below 0.95 and pH below 5.5 (FSIS 2002). Both values were reached
during ripening, thus the physiochemical conditions during the process
prevented the growth of L. monocytogenes.
Ripening reduced L. monocytogenes counts in NT sausages by 2-log
units (Figure 4), a reduction mainly due to the rapid decrease of pH
values. After 27 d of drying (end product) L. monocytogenes lev-els
were below 3 MPN/g in fuet and absence in 25 g was reached in
chorizo. The absence of L. monocytogenes found in chorizo could be
explained by the presence of factors with antilisterial activity in such
products. In fact, 95% of enterococci isolated from non-treat-ed chorizo
showed activity against L. monocytogenes in vitro, while none of the
M: Food Microbiology & Safety
enterococci isolated from non-treated fuet showed this
Figure 2—Color parameters, L* (brightness), a* (redness),
and b* (yellowness), of NT and HPP sausages after treat-
ment at 300 MPa (day 1). Different letters indicate signifi-
cant differences (P < 0.05) between NT and HPP in fuet,
(a and b), and in chorizo (y and z).
Vol. 70, Nr. 7, 2005—JOURNAL OF FOOD SCIENCE M341
 High pressure in low-acid sausages . . .
effect. This finding is consistent with reports of several authors who
described the antilisterial activity of bacteriocins produced by en-
terococci (Aymerich and others 1999; Callewaert and others 2000).
HPP reduced L. monocytogenes counts by 1 log CFU/g (Figure 4). A
similar reduction (1.5 log CFU/g) was reported by Styles and others
(1991) in Ultra High Temperature (UHT) milk treated at 340 MPa (20
min, 23 °C), whereas much higher inactivation (7 log CFU/
g) was observed in phosphate buffer. A rapid recovery of L. mono-
cytogenes was observed after pressurization. From day 1 (after HPP) to
day 6 of ripening L. monocytogenes counts increased 0.9 log CFU/g in
fuet and 1.8 log CFU/g in chorizo. The recovery of L. monocytogenes
after pressurization has been reported in other meat products. Garriga
and others (2000, 2002) detected a recovery of L. monocytogenes, even
in chilled storage, after HPP of meat homogenates at 400 MPa.
There was a decrease in L. monocytogenes during ripening of
pressurized fuet from initial counts of 2.26 log CFU/g to final values of
1.42 CFU/g. By contrast, no changes in L. monocytogenes (P > 0.05)
were detected in pressurized chorizo after 27 d of ripening. This result
suggests that higher pressure levels should be applied to eliminate L.
monocytogenes cells in the end product. Carlez and others (1993)
reached complete inactivation of L. innocua in minced beef muscle at
400 MPa, and Yuste and others (1999) reported sig-nificant inactivation
of L. innocua in poultry sausages pressurized above 350 MPa.
M: Food Microbiology & Safety
Figure 3—Lactic acid bacteria counts of NT and HPP sau-sages
during ripening. Bars indicate standard deviation of triplicates.
Figure 4—L. monocytogenes counts of NT and HPP sau-
sages during ripening. Bars indicate standard deviation of
triplicates.
M342 JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 7, 2005
The reduction of L. monocytogenes in pressurized fuet reflects that
the addition of nitrate and nitrite influenced L. monocytogenes survival
during sausage fermentation, as observed by other au-thors (Junttila and
others 1989; Duffy and others 1994). In addi-tion, the nitrate and nitrite
effect would have been maximized by high pressure, which makes
sublethally injured cells more sensitive to the presence of antimicrobial
agents (Kalchayanand and others 1994).
In the ripened sausages, counts of L. monocytogenes (P < 0.05) were
lower in NT than in HPP sausages. This fact could be attributed to the
delay of pH drop in HPP sausages due to lactobacilli inacti-vation. HPP
also affected endogenous enterococci with reduction of antilisterial
activity from 95% (NT chorizo) to 27% (HPP chorizo). Thus, high
pressure processing of raw sausages in the studied con-ditions had a
counter-productive effect on L. monocytogenes con-trol as a
consequence of LAB inactivation. The addition of a bacte-riocin
producer starter culture resistant to pressure would prevent inactivation
of LAB by HPP and would improve the reduction of L. monocytogenes
counts.
The population of Salmonella decreased significantly during the
manufacture of both pressurized and non-treated sausages (Fig-ure 5).
Minimal growing conditions of Salmonella are pH of 4 to 4.5 and water
activity of 0.95 (ICMSF 1996; EC 2003). Thus, inhibition of Salmonella
in the manufacturing conditions (pH > 5.0) was main-ly obtained by
drying and not by acidification as usually happens in acid fermented
sausages (Talon and others 2004).
During ripening of NT sausages, Salmonella population de-creased 1
logarithm, from 2.45 log CFU/g to 1.35 CFU/g in fuet and from 2.67 log
CFU/g to 1.62 log CFU/g in chorizo. Until day 6, Sal-monella counts
were higher in chorizo, with no added nitrite and nitrate, compared with
fuet. From day 13 until the end of the pro-cess no differences in
Salmonella (P > 0.05) were found between both sausage types. Bard and
Townsend (1976) attributed a short life to the nitrite hurdle, because of
the microbial nitrite depletion during fermentation.
After pressurization, no immediate reduction of Salmonella counts
was observed. By contrast, inactivation of S. enteriditis (2-log reduction)
was reported by Aymerich and others (2003b) in cooked ham
pressurized at 400 MPa (10 min, 17 °C). A higher inactivation of S.
enteriditis (5-log reduction) was observed by Patterson and others
(1995) when pressurizing inoculated phosphate buffer at 450 MPa. This
suggests the baroprotective effect of food constitu-ents, which increase
the ability of bacteria to survive under high pressures (Hoover and
others 1989; Ponce and others 1998).
The pressure effect was observed later, when dehydration was
Figure 5—Salmonella counts of NT and HPP sausages dur-
ing ripening. Bars indicate standard deviation of triplicates.
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High pressure in low-acid sausages . . .
more pronounced. By day 6 of ripening, when a w was 0.96, the Sal-
monella population of HPP sausages started to decrease. Whereas counts
of NT sausages did not decrease until day 13, when a w was about 0.92.
These results are consistent with the affirmation that sublethally injured
cells become more sensitive to low water activ-ity (Smelt 1998). From
day 6 and until the end of ripening, Salmo-nella counts of HPP sausages
were lower (P < 0.05) than those of NT sausages (Figure 5). Pressurized
sausages showed a 2-log reduction of Salmonella levels during the
process, showing low counts (3 MPN/g) in the end product.
Conclusions
T he ripening process at 12 °C and 80% RH for 27 d was effective in preventing the
growth of Salmonella and L. monocytogenes.
Pressure treatment of raw sausages at 300 MPa (10 min, 17 °C) was an
additional hurdle to Salmonella control. However, HPP before ripening
had a negative effect on L. monocytogenes control as a con-sequence of
LAB inactivation.
Pressurization at 300 MPa before ripening is not commercially viable
because it alters the visual aspect of sausages significantly. Further
studies are in progress on pressurization after ripening at higher pressure
levels, to improve L. monocytogenes control and HPP commercial use.
Acknowledgments
This work was supported by the Natl. Inst. for Food and Agricultural
Research (INIA) project RTA01-084. B. Marcos is recipient of pre-
doctoral grant awarded by the Ministry of Science and Technology.
References
[AFSSA] Agence Française de Sécurité Sanitaire des Aliments. 2000. Rapport de la
Commission d’étude des risques liés à Listeria monocytogenes. Available from:
http://www.afssa.fr/dossiers/rapport.asp. Accessed May 1, 2003.
Ananth V, Murano EA, Dickson JS. 1998. Shelf life extension, safety and quality of fresh pork
loin treated with high hydrostatic pressure. J Food Prot 61:1649–56.
Aymerich MT, Artigas MG, Garriga M, Monfort JM, Hugas M. 1999. Effect of sau-sage
ingredients and additives on the production of enterocin A and B by Enterococcus faecium
CTC492. Optimization on in vitro production and anti-listerial effect in dry fermented
sausages. J Appl Microbiol 88:1–11.
Aymerich MT, Martín B, Garriga M, Hugas M. 2003a. Microbial quality and direct PCR
identification of lactic acid bacteria and nonpathogenic staphylococci from artisanal low-
acid sausages. Appl Environ Microbiol 69(8):4583–94.
Aymerich MT, Jofré A, Hugas M, Garriga M. 2003b. Inhibición de Listeria monocytogenes y
Salmonella en jamón cocido elaborado con antimicrobianos naturales sometidos a alta
presión hidrostática. Eurocarne 120:75–9.
Baracco P, Berger Y, Durand P, Frentz JC, Giron J, Guerin J, Jacquet B, Juillard A, Pinel M,
Poterre P, Sirami J. 1982. L’encyclopédie de la charcuterie. Vesoul: Soussana. 790 p.
Barbuti S, Parolari G. 2002. Validation of manufacturing process to control patho-genic
bacteria in typical dry fermented products. Meat Sci 62:323–9.
Bard J, Townsend WE. 1976. Carnes curadas. In: Price JF, Schweigert BS, editors.
Ciencia de la carne y los productos cárnicos. Zaragoza: Ed. Acribia. 582 p.
Bremer V, Leitmeyer K, Jensen E, Metzel U, Meczulat H, Weise E, Weber D, Tschaepe H,
Kreinbrock L, Glaser S, Ammon A. 2004. Outbreak of Salmonella Goldcoast infections
linked to consumption of fermented sausage, Germany 2001. Epi-demiol Infect 132:881–7.
Callewaert R, Hugas M, de Vuyst L. 2000. Competitiveness and bacteriocin pro-duction of
enterococci in the production of Spanish-style dry fermented sau-sages. Int J Food Microbiol
57:3–42.
Carlez A, Rosec JP, Richard N, Cheftel JC. 1993. High pressure inactivation of Citrobacter
freundii, Pseudomonas fluorescens, and Listeria innocua in inoc-ulated minced beef muscle.
Lebensm Wiss Technol 26:357–63.
Carlez A, Rosec JP, Richard N, Cheftel JC. 1994. Bacterial growth during chilled storage of
pressure-treated minced meat. Lebensm Wiss Technol 27:48–54.
Carlez A, Veciana-Nogues T, Cheftel JC. 1995. Changes in color an myoglobin of minced beef
meat due to high hydrostatic pressure processing. Lebensm Wiss Technol 28:528–38.
[CNE] Centro Nacional de Epidemiología. 1997. Resultados de la declaración al Sistema de
Información Microbiológica. Boletín Epidemiológico Semanal (se-rial online). 5(33):322–3.
Available from: http://193.146.50.130/htdocs/bes/ bes5397.pdf. Accessed Jan 1, 2005.
[CNE] Centro Nacional de Epidemiología. 2004. Resultados de la declaración al Sistema de
Información Microbiológica. Boletín Epidemiológico Semanal (se-rial online). 11(29):331–
4. Available from: http://193.146.50.130/htdocs/bes/ bes0353.pdf. Accessed Jan 1, 2005.
Cowden JM, O’Mahony M, Bartlett CL, Rana C, Smyth B, Lynch D, Tillett H, Ward L,
Roberts D, Gilbert RJ. 1989. A national outbreak of Salmonella typhimurium DT124 caused
by contaminated salami sticks. Epidemiol Infect 103:219–25.
URLs and E-mail addresses are active links at www.ift.org
D’Agostino M, Wagner M, Vazquez-Boland JA, Kuchtat T, Karpiskiva R, Hoorfar J, Novella
S, Cortti MS, Ellison J, Murray A, Fernandes I, Kuhn M, Pazlarova J, Heuvelinki A, Cook
N. 2004. A validated PCR-based method to detect Listeria monocytogenes using raw milk as
a food model—Toward an international standard. J Food Prot 67(8):1646–55.
Duffy LL, Vanderlinde PB, Grau FH. 1994. Growth of Listeria monocytogenes on vacuum-
packed cooked meats: effects of pH, aw, nitrite and ascorbate. Int J Food Micro 23:377–390.
[EC] European Commission. 1999. Opinion of the scientific committee on veter-inary
measures relating to public health on Listeria monocytogenes. Brussels: EC. Available from:
http://europa.eu.int/comm/food/fs/sc/scv/out25_en.pdf. Accessed Dec 1, 2004.
[EC] European Commission. 2003. Opinion of the scientific committee on veter-inary
measures relating to public health on Salmonellae in foodstuffs. Brus-sels: EC. Available
from: http://europa.eu.int/comm/food/fs/sc/scv/ out66_en.pdf. Accessed Dec 1, 2004.
Farber JM, Peterkin PI. 1991. Listeria monocytogenes: A food-borne pathogen.
Microbiol Rev 55:476–86.
Farber JM, Daley E. 1994. Presence and growth of Listeria monocytogenes in naturally-
contaminated meats. Int J Food Microbiol 22:33–42.
[FSIS] Food Safety and Inspection Service. 2002. Microbial sampling of ready-to-eat (RTE)
products for the FSIS verification testing program. Washington, D.C.: United States Dept. of
Agriculture.
Garriga M, Aymerich T, Costa S, Monfort JM, Hugas M. 2000. Las altas presiones en
combinación con bacteriocinas como nueva tecnología de conservación en productos
cárnicos. Eurocarne 87:59–63.
Garriga M, Aymerich T, Costa S, Monfort JM, Hugas M. 2002. Bacterial synergism through
bacteriocins and high pressure in a meat model system during stor-age. Food Microbiol
19(5):509–18.
Garriga M, Fadda S, Aymerich T, Hugas M. 2004. Evaluation of the hygienic status of
traditional dry sausage workshops in Catalonia. Paper presented at the 2nd Intl. Conference
on Food Factory of the Future. 2004 Oct 6-8; Laval, France: Laval Mayenne Technopole.
Glass KA, Doyle MP. 1989. Fate of Listeria monocytogenes in processed meat prod-ucts
during refrigerated storage. Appl Environ Microbiol 55(6):1565–9.
Goutefongea R, Rampon V, Nicolas J, Dumont JP. 1995. Meat color changes under high
pressure treatment. Paper presented at the 41st Intl. Congress of Meat Science and
Technology. San Antonio, Texas. 1995 Aug 19-21; Chicago, Ill.: National Live Stock and
Meat Board.
Hoover DG, Metrick C, Papineau AM, Farkas DF, Knorr D. 1989. Biological effects of high
hydrostatic pressure on food microorganisms. Food Technol 43:99–107 [ICMSF] Intl.
Commission on Microbiological Specifications for Foods. 1996. Microorganisms in foods. 5.
Microbiological specifications of food pathogens.
Suffolk: Blackie Academic & Professional. 513 p.
Johnson JL, Doyle MP, Cassens RG, Schoeni JL. 1988. Fate of Listeria monocyto-genes in
tissues of experimentally infected cattle and in hard salami. Appl Environ Microbiol
54(2):497–501.
Junttila J, Hirn J, Hill P, Nurmi E. 1989. Effect of different levels of nitrite and nitrate on the
survival of Listeria monocytogenes during the manufacture of fermented sausage. J Food
Prot 52(3):158–61.
Kalchayanand N, Sikes T, Dunne CP, Ray B. 1994. Hydrostatic pressure and elec-troporation
have increased bactericidal efficiency in combination with bac-teriocins. Appl Environ
Microbiol 60(11):4174–7.
Kalchayanand N, Sikes A, Dunne CP, Ray B. 1998. Factors influencing death and injury of
foodborne pathogens by hydrostatic pressure-pasteurization. Food Microbiol 15:207–214.
Leistner L. 1995. Stable and safe fermented sausages world-wide. In: Campbell-Platt G, Cook
PE, editors. Fermented meats. Glasgow: Black Academic & Pro-fessional. p 160–75.
Leistner L. 2000. Basic concepts of food preservation by hurdle technology. Int J Food
M: Food Microbiology & Safety
Microbiol 55:181–6.
Levine P, Rox B, Green S, Ransom G, Hill W. 2001. Pathogen testing of ready-to-eat meat and
poultry products collected at federally infected establishments in the United States, 1990–
1999. J Food Prot 64(8):1188–93.
Lizaso G, Chasco J, Beriain MJ. 1999. Microbial and biochemical changes during ripening of
salchichón, a Spanish dry cured sausage. Food Microbiol 16(3):219–28.
Malorny B, Tassios PT, Radstrom P, Cook N, Wagner M, Hoorfar J. 2003. Standard-ization of
diagnostic PCR for the detection of foodborne pathogens. Int J Food Microbiol 83:39–48.
Mandava R, Fernández I, Juillerat M. 1994. Effect of high hydrostatic pressure on sausage
batters. Paper presented at the 40th Intl. Congress of Meat Science and Technology. The
Hague, Netherlands. 1994 Aug 28-Sept 3; Chicago, Ill.: National Live Stock and Meat
Board.
Macfarlane JJ, McKenzie IJ, Turner RH. 1980. Pressure treatment of meat: effects on thermal
transitions and shear values. Meat Sci 5:307–17.
Moore JE. 2004. Gastrointestinal outbreaks associated with fermented meats.
Meat Sci 67(4):565–8.
Patterson MF, Quinn R, Simpson R, Gilmur A. 1995. Sensitivity of vegetative pathogens to
high hydrostatic pressure in phosphate-buffered saline and foods. J Food Prot 58:524–9.
Ponce E, Pla R, Mor-Mur M, Gervilla R, Guamis B. 1998. Inactivation of Listeria
monocytogenes inoculated in liquid whole egg by high hydrostatic pressure. J Food Prot
61(1):119–22.
Pontello M, Sodano L, Nastasi A, Mammina C, Astuti M, Domenichini M, Belluzzi G, Soccini
E, Silvestri MG, Gatti M, Gerosa E, Montagna A. 1998. A community-based outbreak of
Salmonella enterica serotype Typhimurium associated with salami consumption in Northern
Italy. Epidemiol Infect 120(3):209–14.
Rahn K, De Grandis SS, Clarke RC, McEwen SA, Galán JE, Ginocchio C, Curtiss R, Gyles
CL. 1992. Amplification of an invA gene sequence of Salmonella typh-imurium by
polymerase chain reaction as a specific method of detection of Salmonella. Mol Cell Probes
6:271–9.
Vol. 70, Nr. 7, 2005—JOURNAL OF FOOD SCIENCE M343
 High pressure in low-acid sausages . . .
Sanz Y, Vila R, Toldrá F, Flores J. 1998. Effect of nitrate and nitrite curing salts on microbial
changes and sensory quality of non-fermented sausages. Int J Food Microbiol 42:213–7.
Shigehisa T, Ohmori A, Saito S, Taji S, Hayashi R. 1991. Effects of high pressure on
characteristics of pork slurries and inactivation of microorganisms associat-ed with meat
and meat products. Int J Food Microbiol 12:207–16.
Simon MC, Gray DI, Cook N. 1996. DNA extraction and PCR methods for the detection of
Listeria monocytogenes in cold-smoked salmon. Appl Environ Microbiol 62(3):822–4.
Smelt JPPM. 1998. Recent advances in the microbiology of high pressure pro-cessing. Trends
Food Sci Technol 9:152–8.
Smith JL, Huhtanen CN, Kissinger JC, Palumbo SA. 1975. Survival of salmonellae during
pepperoni manufacture. Appl Microbiol 30(5):759–63.
Styles MF, Hoover DG, Farkas DF. 1991. Response of Listeria monocytogenes and
M: Food Microbiology & Safety
M344 JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 7, 2005
Vibrio parahaemolyticus to high hydrostatic pressure. J Food Sci 56(5):1404–7. Talon R,
Leroy-Sétrin S, Fadda S. 2004. Dry fermented sausages. In: Hui YH, Goddik LM, Hansen AS,
Josephsen J, Nip WK, Stanfield PS, Toldrá F, editors. Handbook of food and beverage
fermentation technology. New York: Marcel
Dekker. p 397–416.
Van Netten P, Leenaerts J, Heikant GM, Mossel DA. 1986. A small outbreak of salmonellosis
caused by Bologna sausage. Tijdschr Dieegeneeskd 24:1271–5.
Van Netten P, Perales J, van de Moosdijk A, Curtis GDW, Mossel DA. 1989. Liquid and solid
selective differential media for the detection and enumeration of Listeria monocytogenes.
Int Food Microbiol 8:299–316.
Yuste J, Mor-Mur M, Capellas M, Pla R. 1999. Listeria innocua and aerobic meso-philes
during chill storage of inoculated mechanically recovered poultry meat treated with HHP.
Meat Sci 53:251–7.
URLs and E-mail addresses are active links at www.ift.org