Journal of Applied Microbiology 1998, 85, 849–854 Inactivation of Bacillus subtilis spores by combining ultrasonic waves under pressure and mild heat treatment J. Raso, A. Palop, R. Paga ´ n and S. Condo ´n Tecnologı ´ a de los Alimentos, Universidad de Zaragoza, Spain 6629/03/98: received 13 March 1998, revised 5 June 1998 and accepted 9 June 1998 J. RASO, A. PALOP, R. PAGA ´ N AND S. CONDO ´ N. 1998. The inactivation of Bacillus subtilis spores by ultrasonic treatments under static pressure (Mano-Sonication, MS) and a combined MS/heat treatment (Mano-Thermo-Sonication) was investigated. The sporicidal effect of MS treatments depended on static pressure, amplitude of ultrasonic waves and treatment temperature. At 70 °C, pressure increments up to 500 kPa caused progressively more inactivation. An MS treatment at 500 kPa and 117 mm of amplitude for 12 min inactivated approximately 99% of the B. subtilis spore population. Over 500 kPa, further increments in pressure did not increase the percentage of inactivation. In the range 90–150 mm, an exponential relationship was observed between the amplitude of ultrasonic waves under pressure and the number of survivors. While an MS treatment (20 kHz, 300 kPa, 70 °C, 12 min) at 90 mm inactivated 75% of the B. subtilis spore population, the same treatment at 150 mm inactivated 99·9% of this population. The MS treatments at temperatures higher than 70 °C (MTS) led to more spore inactivation. In the range 70–90 °C, the combination of heat with an MS treatment (20 kHz, 300 kPa, 117 mm, 6 min) had a synergistic effect on spore inactivation. The inactivating effect of ultrasound was due neither to titanium particles eroded from the sonication tip, nor to free radicals released during ultrasonic treatment. The MS treatments sensitized spores of B. subtilis to lysozyme. INTRODUCTION Heat treatment of liquid foods is predominantly used as a means of inactivation of spoilage or pathogen micro-organ- isms that might grow under conditions normally encountered in storage. However, heat treatment can adversely affect the flavour, taste and nutritive value of foods. Currently, efforts are being made to find new methods of food preservation to avoid the unwanted effects of heat. These methods are based on non-thermal processes of inactivation (Mertens and Knorr 1992; Knorr 1994; Qin et al. 1996) or a combination of pres- ervation methods already known (Gould and Jones 1989; Ama et al. 1994). The inactivation of vegetative forms of micro-organisms by ultrasound in liquid media was observed in the late 1920s (Harvey and Loomis 1929). The lethal effect of ultrasound has been attributed to cavitation. Cavitation is due to growth Correspondence to: Santiago Condo ´n, Tecnologı ´a de los Alimentos, Facultad de Veterinaria, C/Miguel Servet, 177 50013 Zaragoza, Spain (e-mail:scondon@posta.unizar.es). © 1998 The Society for Applied Microbiology and subsequent collapse of microscopic bubbles when ultra- sonic waves travel through a liquid. Cavitation can affect a biological system by virtue of the localized temperature rise and mechanical stress (Scherba et al. 1991). Moreover, the dissociation of water molecules into H- and OH- free radicals, as a consequence of the very high temperature and pressures produced by cavitation, may induce adverse chemical changes such as DNA or protein denaturation (Riesz and Kondo 1992). However, the ultimate reason for the lethality of ultra- sound on micro-organisms is still unknown. The use of ultrasound has been suggested for disinfection and food preservation but its limited lethal effect on bacterial spores (Berger and Marr 1960) has discouraged any attempt at using it as a sterilization procedure. The combination of ultrasound with, for example, chemical treatment (Sierra and Boucher 1971; Ahmed and Russell 1975; Lillard 1993), heat treatment (Burgos et al. 1972; Garcı ´a et al. 1989) or ionizing radiation (Dharkar 1964), increased the lethal effect of ultra- sound on vegetative cells, but the inactivation of bacterial spores was still low.