Building of Pressure-Assisted Ultra-High Temperature System and Its Inactivation of Bacterial Spores

Abstract

The pressure-assisted ultra-high temperature (PAUHT) system was built by using soybean oil as pressure-transmitting medium, and the multiple regression equation of soybean oil temperature change (ΔT_P ) during pressurization as a function of initial temperature (T_i ) and set pressure (P) was developed: ΔT_P = -13.45 + 0.46 T_i + 0.0799 P - 0.0037 $T_i^2$ - 2.83 × 10^-5 P². The fitted model indicated that the temperature of the system would achieve ≥121°C at 600 MPa when the initial temperature of soybean oil was ≥84°C. The PAUHT system could effectively inactivate spores of Bacillus subtilis 168 and Clostridium sporogenes PA3679 (less than 1 min). Treatment of 600 MPa and 121°C with no holding time resulted in a 6.75 log reductions of B. subtilis 168 spores, while treatment of 700 MPa and 121°C with pressure holding time of 20 s achieved more than 5 log reductions of C. sporogenes PA3679 spores. By comparing the PAUHT treatment with high pressure or thermal treatment alone, and also studying the effect of compression on spore inactivation during PAUHT treatment, the inactivation mechanism was further discussed and could be concluded as follows: both B. subtilis 168 and C. sporogenes PA3679 spores were triggered to germinate firstly by high pressure, which was enhanced by increased temperature, then the germinated spores were inactivated by heat.

Keywords: bacterial spores; inactivation mechanism; mathematical model; pressure-assisted ultra-high temperature; sterilization.

FIGURE 1

Schematic diagram of the high-pressure experimental system: T₁ and T₂ are thermocouples in soybean oil insulator medium and high hydrostatic pressure vessel medium, respectively.

FIGURE 2

Typical pressure–temperature profiles of soybean oil during PAUHT treatments: (A) 600 MPa and 121°C (1 min), and (B) 700 MPa and 130°C (1 min).

FIGURE 3

The phase contrast and fluorescence microscope of B. subtilis168 (A–C) and C. sporogenes PA3679 (D–F) spores: (A,D) untreated spores, (C,F) 600 MPa at 121°C for 1 min, (B) 600 MPa at ambient temperature for 10 min, (E) Incubation in 20 mM Tris buffer pH 7.4, L-alanine (100 mM), L-lactate (50 mM), and NaHCO₃ at 30°C for 1 h.

FIGURE 4

The inactivation of B. subtilis 168 (•) and C. sporogenes PA3679 spores () by thermal treatment at 121°C for different times, and its correlating amount of released DPA for B. subtilis 168 spores and () for C. sporogenes PA3679 spores (), respectively.

FIGURE 5

The phase contrast and fluorescence microscopy of B. subtilis 168 (A–C) and C. sporogenes PA3679 (D–F) spores: (A,D) untreated spores, (B,E) 121°C for 1 min at ambient pressure. (C) 600 MPa at ambient temperature for 10 min used as control, (F) Incubation in 20 mM Tris buffer pH 7.4, L-alanine (100 mM), L-lactate (50 mM), and NaHCO₃ at 30°C for 1 h used as control.

FIGURE 6

The DPA release or inactivation of unactivated and heat activated (75°C, 15 min) spores after treatment of 600 MPa at ambient temperature for 1 min (A) or PAUHT treatment of 600 MPa at 121°C without pressure holding time (B).