International Journal of Food Microbiology 55 (2000) 115–119

www.elsevier.nl / locate / ijfoodmicro

Effects of high hydrostatic pressure on membrane proteins of

Salmonella typhimurium
a b b a,
M. Ritz , M. Freulet , N. Orange , M. Federighi *
a
Unite´ associee
´ INRA d’ Hygiene
` Alimentaire, Ecole Nationale Veterinaire
´´ de Nantes, Route de Gachet, BP40706,
F-44307 Nantes, France
b
Laboratoire de Microbiologie du Froid, Universite´ de Rouen, 43 rue Saint-Germain, F-27000 Evreux, France

Abstract

Salmonella typhimurium is a leading cause of foodborne diseases. Today high hydrostatic pressure treatments are
considered as alternative methods of preservation. To select optimal conditions of treatment, we have to characterize the cell
targets of pressure. In this study the action of pressure on the bacterial membrane proteins is analysed. The total membrane
extract is obtained by lysis of cells separated by equilibrium density gradient centrifugation. Protein content is analysed by
electrophoresis SDS–PAGE and visualised by silver stain. Electrophoretic profiles reveal the presence of three major outer
membrane proteins and 12 minor proteins in control bacteria outer membranes. Outer membrane protein content is
drastically modified after treatments. In some cases, except for the major proteins OmpA and LamB, other outer membrane
proteins seem to totally disappear. LamB is more resistant to hyperbaric exposure when the pH of the media is acidic. This
behaviour could be explained by a different conformation adopted by the LamB protein depending on the extracellular pH.
This work allows us to define membrane proteins as a target of high hydrostatic pressure treatments. Knowledge of the
behaviour of these bacterial membrane proteins subjected to pressure under different conditions (pH, temperature, a w . . . )
could allow an increase in the efficiency of treatments.  2000 Elsevier Science B.V. All rights reserved.

Keywords: High pressure; Salmonella; Membrane protein

1. Introduction preserving food (Knorr, 1993). One of the primary

High hydrostatic pressure treatment is currently
considered as an attractive non-thermal process for
*Corresponding author. Tel.: 133-2-4068-7681.
E-mail address: federighi@vet-nantes.fr (M. Federighi)
considerations in evaluating the effectiveness of any
preservation treatment is its ability to eradicate
pathogenic micro-organisms and thus ensure product
safety. Previous studies showed that a pressure
treatment of 500 to 700 MPa readily kills vegetative
cells of bacteria, yeast and mould, while bacterial
spores are more resistant (Hayakawa et al., 1994;
Mackey et al., 1994; Wuytack et al., 1998). Industrial
equipment used to preserve foods is routinely de-

0168-1605 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0168-1605( 00 )00165-3
116 M. Ritz et al. / International Journal of Food Microbiology 55 (2000) 115 – 119
signed to generate at least 400 MPa of pressure every
cycle (Zimmerman and Bergman, 1993). Resistance
by micro-organisms to high pressure is variable, and
reduction of microbial loads is directly related to the
amount of hydrostatic pressure applied (Arroyo et
al., 1997). High pressure brings about a number of
changes in the morphology, cell membrane, cell
wall, biochemical reactions and genetic mechanisms
of micro-organisms (Hoover et al., 1989; Cheftel,
1995; Simpson and Gilmour, 1997; Wouters et al.,
1998). The cell membrane represents probably one
of the major targets for pressure-induced inactivation
of micro-organisms. Previous studies demonstrated
modifications of the fatty acid composition of mem-
branes in relation to pressure treatment of lipid
bilayers and pressure growth of micro-organisms
(Macdonald, 1992; Fujii et al., 1997). Other reports
demonstrated some changes in cell morphology and
structure (Mackey et al., 1994), and changes in
intracellular and membrane-bound enzyme activity
(Wouters et al., 1998). Elucidation of the mechanis-
tic aspects of high-pressure inactivation of micro-
organisms will help in food preservation.
The cell envelope of Salmonella and other gram-
negative enteric bacteria is a complex structure
composed of three morphologically distinct layers:
cytoplasmic membrane, a rigid peptidoglycan layer
external to the cytoplasmic membrane, and a second
membrane structure, the outer membrane or L-layer
at the surface of the cell. The outer membrane
contains substantial amounts of protein and phos-
pholipid and in addition most or all of the
lipopolysaccharide of the cell envelope. The aim of
this work was to characterize the cell targets of
pressure treatment and know if the membrane pro-
teins of Salmonella typhimurium pressure could be
this potential target. For this purpose a technique for
separation of cytoplasmic membrane from outer
membrane was necessary. We followed the method
described by Osborn et al. (1972) with few modi-
fications and used a technique involving a sucrose
density gradient centrifugation of membranes ob-
tained by spheroplast lysis.
This communication presents general characteriza-
tion of the membrane fractions obtained from Sal-
monella typhimurium by this technique and provides
evidence that the proteins of membranes are really a
target of high hydrostatic pressure treatment.
2. Materials and methods
2.1. Bacteria culture preparation
S. typhimurium (ATCC 13311) was obtained from
Collection de l’Institut Pasteur (Institut Pasteur,
Paris, France) and stored at 2 308C in cryobeads
(AES, Combourg, France). The first bacterial culture
was obtained by inoculating a cryobead into brain-
heart infusion (BHI; Biokar, Beauvais, France) incu-
bated at 378C for 24 h. This first culture was used to
prepare a subculture by inoculating 1 ppt. (v / v) into
fresh BHI to incubate for 18 h at 378C. In these
conditions, the count for the initial cell population
was 8 log 10 , with a standard error of 0.1.
2.2. Inoculation of the suspending medium
The pressurized samples were composed of 150
ml of subculture diluted in 600 ml of buffer. Two
buffers were used: a phosphate buffer (pH 7.00)
composed of Na 2 HPO 4 (0.2 mol l 21 ) and NaH 2 PO 4
(0.2 mol l 21 ), and a citrate buffer (pH 5.6) com-
posed of Na 2 HPO 4 (0.2 mol l 21 ) and C 6 H 8 O 7 (0.1
mol l 21 ) (Merck, Darmstadt, Germany). The sam-
ples were placed in a sterile bag.
2.3. Enumeration of surviving cells
The number of cells was determined before and
after treatment, and the efficiency of treatment was
calculated by comparing these two counts in log 10 .
Cells were enumerated by plating 0.1-ml volumes
twice on plate count agar (PCA) (Biokar), which
were then incubated for 48 h at 378C. For low
numbers of viable cells, 10 ml of cell suspension
were divided between two plates and poured with
PCA. Thus, the pitch limit was 0.1 c / ml, corre-
sponding to the result 0.
2.4. High-pressure treatment
The sterile bags were sealed after eliminating air
inside, placed in a hydrostatic pressure vessel (3 l) in
a high-pressure apparatus (Alstom, Nantes, France),
and treated at 208C for 10 min. High-pressure levels
(350 to 600 MPa) were generated using water
pressurized by a hydrostatic pump. Each treatment
 M. Ritz et al. / International Journal of Food Microbiology 55 (2000) 115 – 119
Table 1
Cell enumeration of Salmonella typhimurium populations before and after treatment
Phosphate buffer pH 7.0
Non-treated sample 8.04 log 10
400 MPa, 10 min ,1 cell / 10 ml
600 MPa, 10 min ,1 cell / 10 ml
was a combination of a pressure level, duration, and
pH as shown in Table 1.
2.5. Isolation of cytoplasmic and outer membrane
fractions
The procedure was based on an equilibrium
density gradient centrifugation of the total membrane
extract obtained by lysis of lysozyme–EDTA sphero-
plasts. Cells were harvested by centrifugation at
8000 rev. / min for 15 min at 48C (Sorvall RC5B,
rotor GSA). The cell pellet was rapidly resuspended
in cold 0.75 M sucrose–10 mM Tris–HCl buffer, pH
7.8. Lysozyme was immediately added to a final
concentration of 100 mg / ml, 2 volumes of cold 1.5
mM EDTA (Na 1 ), pH 7.5 were slowly added and
the bacterial suspension was incubated at 308C for 90
min. Conversion to spheroplasts was monitored by
optical microscopy. When spheroplast formation
attempted about 95%, cell suspension was immersed
in an ice-cold bath and sonicated four times for 30 s
with Vibracell (Bioblock Scientific). Intact cells were
removed by centrifugation at 10,000 rev. / min for 15
min (Sorvall RC5B, rotor SS34). Membrane extracts
were collected by centrifugation at 37,000 rev. / min
for 2 h (Beckman, rotor 70 Ti). Step gradients of
sucrose density were prepared by layering 2 ml each
of 50%, 45%, 40%, 35% and 30% sucrose solutions
(w / w) containing 5 mM EDTA, pH 7.5. The mem-
brane pellet was resuspended in 0.5 ml of cold 25%
sucrose, 5 mM EDTA, pH 7.5 and applied on the top
of the gradient. Centrifugation was carried out for 12
h at 37,000 rev. / min and 48C (Beckman, rotor SW
41 Ti). Fractions of 0.5 ml were collected from the
bottom of the tube with a peristaltic pump. Protein
content was assayed by ESL Boehringer kit and
membrane extracts were analysed by electrophoresis
SDS–PAGE (7% and 15% gel system). Proteins
were visualised by silver stain.
117
Citrate phosphate buffer pH 5.6
Non-treated sample 7.99 log 10
350 MPa, 10 min ,10 cell / 10 ml
600 MPa, 10 min ,1 cell / 10 ml
3. Results
3.1. Outer membrane fractions analysis
After hyperbaric exposure, cells were harvested,
bacterial membranes extracted and analysed by
electrophoresis SDS–PAGE.
Efficiency of spheroplast formation was lower
(50%) for treated cells than for control bacterial
suspension (95%). This suggests that the outer
membranes (OM) seem to be more resistant to
EDTA–lysozyme treatment after hyperbaric shock.
Electrophoretic profiles revealed the presence of
three major outer membrane proteins and about 12
minor proteins in the control bacteria outer mem-
branes (Figs. 1 and 2). Major outer membrane
proteins are identified as LamB (44 kDa), OmpC (37
kDa) and OmpA (35 kDa) (Nikaido and Vaara,
1985). The quantities of these proteins were found to
be quite similar whatever the extra-cellular pH is, 7.0
or 5.6.
After treatment at 350 MPa or 400 MPa, and more
so at 600 MPa, the Salmonella typhimurium popula-
tion was totally inactivated, outer membrane protein
content is drastically modified whatever the extra-
Fig. 1. Outer membrane profile. Cell suspension in phosphate
buffer pH 7.0. Protein content is analysed by electrophoresis
SDS–PAGE and visualised by silver stain.
118 M. Ritz et al. / International Journal of Food Microbiology 55 (2000) 115 – 119
Fig. 2. Outer membrane profile. Cell suspension in citrate phos-
phate buffer pH 5.6. Protein content is analysed by electrophoresis
SDS–PAGE and visualised by silver stain.
cellular pH; 350 MPa, pH 5.6 and 400 MPa, pH 7.0
treated cells presented a different outer membrane
protein pattern whereas the effect on enumeration is
similar. Outer membrane extracts from treated cells
in phosphate buffer at 400 MPa (Fig. 1), showed
each minor and major protein except the 44 kDa
protein which almost entirely disappeared.
On the contrary, outer membranes obtained from
cells suspended in citrate buffer at an acidic pH of
5.6 (Fig. 2), revealed only two proteins; the major
outer membrane protein of 35 kDa appearing in a
similar quantity as in controls, and a 44 kDa outer
membrane protein less concentrated. Each other
major or minor outer membrane protein seemed to
disappear.
Only the 35 kDa protein remained after the 600
MPa treatment whatever the pH of the suspending
medium. After heating for 10 min at 1008C, the
major outer membrane protein of 35 kDa is modified
and migrates as a band corresponding to a relative
molecular weight of 37 kDa. This heat modified
behaviour is characteristic of porins (Nikaido et al.,
1991). The heat modification property is conserved
after hyperbaric exposure for cells in phosphate
buffer but seems to be partially altered for cells
suspended in citrate buffer.
3.2. Inner membrane analysis
Sonication of spheroplasts from hyperbaric ex-
posed cells needs less time than for spheroplasts
from control cells. This suggests that the inner
membrane seems to be made fragile by hyperbaric
Fig. 3. Internal membrane profile. Cell suspension in citrate
phosphate buffer pH 5.6. Protein content is analysed by electro-
phoresis SDS–PAGE and visualised by silver stain.
treatment. Before treatment the inner membrane
protein content is similar whatever the pH of the
suspending medium. This content seems to reduce
with the increase of pressure and after 600 MPa
exposure (Fig. 3). The protein content in inner
membrane protein is drastically diminished, but no
significant difference of sensitivity was observed
depending on the pH of the suspending medium.
4. Discussion
Previous studies demonstrated some changes in
cell structures (Mackey et al., 1994) and membrane-
bound enzymes activity (Wouters et al., 1998). This
work permits us to confirm the role of the cell
membrane in the inactivation and characterizes a
physical target of high hydrostatic pressure.
The presence of the major outer membrane pro-
teins identified as LamB, OmpC and OmpA for the
control bacterial suspension confirm the capacity of
this technique employed to characterize the content
of membrane proteins.
We observed lower formation of spheroplasts for
treated cells than for control bacterial suspensions.
This behaviour could be explained by a reorganisa-
tion of the outer membrane. The disappearance of
the porins used for the macromolecule transport
could explain the difficulties of lysozyme permea-
tion.
The results show that hyperbaric exposure alters
drastically outer membrane proteins. However, the
 M. Ritz et al. / International Journal of Food Microbiology 55 (2000) 115 – 119
major outer membrane protein OmpA seems to be
more resistant to this treatment, probably due to its
conformation and surrounding. This protein is well
known to be involved in cell shape and integrity,
which could explain its very resistant conformation
to many stresses (Nikaido and Vaara, 1985).
Moreover, the protein LamB displays a particular
behaviour. Indeed, the protein resistance to hyper-
baric shock depends on the extra-cellular pH. LamB
is more resistant to hyperbaric exposure when the pH
of the media is acidic. When the extra-cellular pH is
close to neutrality, the porin is less resistant and 350
MPa are sufficient to cleave it. This behaviour could
be explained by a different conformation adopted by
the LamB depending on the extra-cellular pH.
5. Conclusion
This study demonstrates the action of pressure on
the membrane proteins. Even if the pressure is
isostatic, the external membrane seems to be more
damaged than the cytoplasmic membrane. In fact,
except for the 35 kDa protein, each other major or
minor outer membrane protein seems to disappear.
The different destruction of membrane proteins
between acidic and neutral pH could explain the
greater inactivation recorded when the cells are
treated in acidic suspension or food. These observa-
tions combined with the knowledge of pH of food
could be integrated for the bacterial destruction
models.
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