Synergistic effects of high hydrostatic pressure, mild heating, and amino acids on germination and inactivation of Clostridium sporogenes spores

Abstract

The synergistic effects of high hydrostatic pressure (HHP), mild heating, and amino acids on the germination of Clostridium sporogenes spores were examined by determining the number of surviving spores that returned to vegetative growth after pasteurization following these treatments. Pressurization at 200 MPa at a temperature higher than 40°C and treatment with some of the 19 l-amino acids at 10 mM or higher synergistically facilitated germination. When one of these factors was omitted, the level of germination was insignificant. Pressures of 100 and 400 MPa were less effective than 200 MPa. The spores were effectively inactivated by between 1.8 and 4.8 logs by pasteurization at 80°C after pressurization at 200 MPa at 45°C for 120 min with one of the amino acids with moderate hydrophobicity, such as Leu, Phe, Cys Met, Ala, Gly, or Ser. However, other amino acids showed poor inactivation effects of less than 0.9 logs. Spores in solutions containing 80 mM of either Leu, Phe, Cys, Met, Ala, Gly, or Ser were successfully inactivated by pasteurization by more than 5.4 logs after pressurization at 200 MPa at 70°C for 15 to 120 min. Ala and Met reduced the spore viability by 2.8 and 1.8 logs, respectively, by pasteurization at a concentration of 1 mM under 200 MPa at 70°C. These results indicate that germination of the spores is facilitated by a combination of high hydrostatic pressure, mild heating, and amino acids.

Fig 1

Effects of different amino acids on the inactivation of C. sporogenes spores by HHP-P. Spores contained in 80 mM amino acid solutions (pH 7.0) were subjected to pressure at 0.1 (atmospheric pressure), 100, 200, and 400 MPa at 45°C for 120 min and then heated at 80°C for 10 min to kill the germinated spores. The survival ratio of spores indicates the ratio of spores that survived and returned to vegetative growth. The values in parentheses beneath the amino acids are the hydropathy indexes. Error bars indicate 95% confidence limits. NA, no amino acids were added.

Fig 2

Effects of pressurizing temperatures on spore inactivation by HHP-P. The spores of C. sporogenes contained in 80 mM amino acid solutions were subjected to pressures at 0.1 MPa (A) and 200 MPa (B) at 30 to 70°C for 120 min and then heated at 80°C for 10 min. The survival ratio of spores indicates the ratio of spores that survived and returned to vegetative growth. Error bars indicate 95% confidence limits. Surviving spores were not detected under the treatment conditions (highlighted by asterisks) because the detection limit of the experiments had been reached. NA, no amino acids were added.

Fig 3

Effects of the pressurizing periods on spore inactivation by HHP-P. The spores of C. sporogenes placed in 80 mM amino acid solutions were subjected to 200 MPa of pressure at 45°C (A) and 70°C (B) for 10 to 120 min and pasteurized at 80°C for 10 min. The survival ratio of spores indicates the ratio of spores that survived and returned to vegetative growth. Error bars indicate 95% confidence limits. Surviving spores were not detected under the treatment conditions (highlighted by asterisks) because the detection limit of the experiments had been reached. NA, no amino acids were added.

Fig 4

Effects of amino acid concentrations on spore inactivation by HHP-P. The spores of C. sporogenes in solutions of 1 to 80 mM amino acids were pressurized at 200 MPa at 45°C for 120 min (A) or 70°C for 15 min (B) and then heated at 80°C for 10 min. The survival ratio of spores indicates the ratio of spores that survived and returned to vegetative growth. Error bars indicate 95% confidence limits.

Fig 5

Effects of high hydrostatic pressure, mild heating, and amino acids on spore germination. The spores of C. sporogenes in solutions of 80 mM amino acids were treated at 0.1 or 200 MPa at 30, 45, or 70°C for 15 or 120 min. Error bars indicate standard deviations (n = 2). NA, no amino acids were added.