The inactivation of Listeria innocua BGA 3532 at subzero temperatures and pressures up to 400 MPa in buffer solution was studied to examine the impact of high-pressure treatments on bacteria in frozen matrices. The state of aggregation of water was taken into account. The inactivation was progressing rapidly during pressure holding under liquid conditions, whereas in the ice phases, extended pressure holding times had comparatively little effect. The transient phase change of ice I to other ice polymorphs (ice II or ice III) during pressure cycles above 200 MPa resulted in an inactivation of about 3 log cycles, probably due to the mechanical stress associated with the phase transition. This effect was independent of the applied pressure holding time. Flow cytometric analyses supported the assumption of different mechanisms of inactivation of L. innocua in the liquid phase and ice I (large fraction of sublethally damaged cells due to pressure inactivation) in contrast to cells subjected to ice I-to-ice III phase transitions (complete inactivation due to cell rupture). Possible applications of high-pressure-induced phase transitions include cell disintegration for the recovery of intracellular components and inactivation of microorganisms in frozen food.Water under high pressure. Under high pressure, water shows an unusual freezing point depression to Ϫ22°C at 210 MPa. In addition to the conventional ice I, various solid phases with a higher density than liquid water (ice II to ice V) exist in the pressure range between 210 and 500 MPa, as visualized in Fig. 1 (2). Since the early works of Tammann (33) and Bridgman (2), various other solid phases have been discovered under more extreme conditions (10), yet comparatively little is known about the kinetics of the phase transitions or metastable states.When pressurizing ice I to pressures above 210 MPa in the temperature range of Ϫ22 to Ϫ35°C, i.e., beyond the phase transition line of ice I to ice III, ice I does not transform instantly to ice III (2). Although Bridgman described this phase transition to be of explosive rapidity at temperatures above Ϫ30°C in cases where it occurs, it was also observed that ice I melts as soon as it approaches the prolongation of the ice I-to-liquid line (2, 18). Ice I melts, since a solid can never exist in the domain of the liquid, even though the liquid is relatively unstable with respect to ice III. The volume change during the phase transition is about 18% during the direct phase transition from ice I to ice III at Ϫ30°C and 212 MPa within a second and vice versa. When pressurizing ice I at temperatures below Ϫ35°C (triple point), it is possible to form ice II or ice III, since both phase transition lines were detected (2). Ice II and ice III have similar phase transition lines to ice I, and they do not vary much in specific volume (⌬vol Ͻ 3% at Ϫ34°C and 214 MPa) (2).High-pressure inactivation of microorganisms. It was shown frequently that high hydrostatic pressure is a powerful method for inactivating microorganisms in food m...