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Third, treatment at elevated temperature (>40��C) greatly enhances process lethality and eliminates even pressure-resistant strains (Table ?(Table1).1). The combination of pressure treatment with elevated temperature and/or low pH, however, is not suitable for all food products and preservation of low-acid and temperature sensitive food thus required the identification of additional antimicrobial hurdles that act synergistically with pressure. Pressure sensitive targets in cells of E. coli and the possible exploitation of these targets for development of hurdle technologies are discussed in the subsequent sections. TABLE 1 Pressure-inactivation of different strains of E. coli in food. Pressure Mediated Disruption of the Outer Membrane The barrier properties of the RRAD Gram-negative outer membrane mediate resistance against antimicrobial peptides including lysozyme, lactoferrin, and bacteriocins from lactic acid bacteria, and hydrophobic inhibitors including bile acids, which are ingredients of most selective media for E. coli or coliform bacteria (Vaara, 1992; G?nzle et al., 1999; Nikaido, 2003). The observation that pressure permeabilizes the outer membrane of Gram-negative bacteria was initially based on the synergistic activity of pressure and pediocin or nisin (Kalchayanand et al., 1992). Pressure application also sensitizes E. coli to lactoferrin and lysozyme, lactoferrin, and the lactoperoxidase system (Hauben et al., 1996; Garcia-Graells et al., 2000; Masschalck et al., 2001a,b). In situ determination of the permeabilization of the outer membrane suggested that the outer membrane is reversibly permeabilized concomitant with compression, followed by the irreversible loss of lipid A and outer membrane proteins (Figure ?(Figure1;1; Ritz et al., 2000; G?nzle and Vogel, 2001). The outer membrane is stabilized by electrostatic interactions of Ca2+ and lipid A (Vaara, 1992); electrostatic interactions are pressure sensitive. Outer membrane porins are over-expressed during growth at elevated pressure (Nakashima et al., 1995) and the pressure-resistant strain E. coli AW1.7 is distinguished by most other strains of E. coli by expression of the porin NmpC (Ruan et al., 2011; Liu et al., 2012). The outer membrane lipoproteins NlpI, YbaY, and OsmE increase pressure resistance of E. coli, presumably through stabilization of the outer membrane (Charoenwong et al., 2011). FIGURE 1 Pressure effects on the outer membrane of E. coli. The outer leaflet of the outer membrane is composed of a lipopolysaccharide layer which prevents penetration of large or hydrophobic molecules to the periplasm. Lipid A molecules are cross-linked by divalent ... Pressure-mediated disruption of the outer membrane does not compromise the viability of E.