Thermal-Stability and Reconstitution Ability of Listeria Phages P100 and A511

Abstract

The study evaluated the thermal-stability of Listeria phages P100 and A511 at temperatures simulating the preparation of ready-to-eat meats. The phage infectivity after heating to 71°C and holding for a minimum of 30 s, before eventually cooling to 4°C were examined. Higher temperatures of 75, 80, and 85°C were also tested to evaluate their effect on phages thermal-stability. This study found that despite minor differences in the amino acid sequences of their structural proteins, the two phages responded differently to high temperatures. P100 activity declined at least 10 log (PFU mL^-1) with exposure to 71°C (30 s) and falling below the limit of detection (1 log PFU mL^-1) while, A511 dropped from 10⁸ to 10⁵ PFU mL^-1. Cooling resulted in partial reconstitution of P100 phage particles to 10³ PFU mL^-1. Exposure to 75°C (30 s) abolished A511 activity (8 log PFU mL^-1) and both phages showed reconstitution during cooling phase after exposure to 75°C. P100 exhibited reconstitution after treatment at 80°C (30 s), conversely A511 showed no reconstitution activity. Heating P100 to 85°C abolished the reconstitution potential. Substantial differences were found in thermal-stability and reconstitution of the examined phages showing A511 to be more thermo-stable than P100, while P100 exhibited reconstitution during cooling after treatment at 80°C which was absent in A511. The differences in predicted melting temperatures of structural proteins of P100 and A511 were consistent with the observed differences in thermal stability and morphological changes observed with transmission electron microscopy.

Keywords: Bacteriophage; Listeria monocytogenes; food safety; ready-to eat meat; thermal-stability; transmission electron microscopy.

FIGURE 1

Changes in phage P100 infectivity (in log PFU mL^-1) following the heating-holding-cooling trial in SM buffer at peak heating temperatures of 71°C (A); 75°C (B); 80°C (C); 85°C (D). Thirty seconds on the x-axis refers to holding time of 30 s at pertaining temperatures. Different superscript letters associated with the numbers on the bars indicate statistical significance between two means with the level of significance of 0.05. Error bars denote standard deviation.

FIGURE 2

Changes in phage A511 infectivity (in log PFU mL^-1) following the heating-holding-cooling trial in SM buffer at peak heating temperatures of 71°C (A); 75°C (B); 80°C (C). Thirty seconds on the x-axis refers to holding time of 30 s at pertaining temperatures. Different superscript letters associated with the numbers on the bars indicate statistical significance between two means with the level of significance of 0.05. Error bars denote standard deviation.

FIGURE 3

Changes in phage P100 infectivity (in log PFU mL^-1) following the heating 71°C-holding-cooling trial in SM buffer with initial concentration of 10⁵ PFU mL^-1 (A); 10⁸ PFU mL^-1 (B); and phage A511 population following the heating 75°C-holding-cooling trial in SM buffer with initial concentration of 10⁵ PFU mL^-1 (C). Thirty seconds on the x-axis refers to holding time of 30 s at pertaining temperatures Different superscript letters associated with the numbers on the bars indicate statistical significance between two means with the level of significance of 0.05. Error bars denote standard deviation.

FIGURE 4

Transmission electron micrographs of Listeria phages P100 (A) and A511 (B) exposed to different treatments. Untreated control (1); heating to 71°C (2); heating to 75°C (3); heating to 80°C (4); heating to 85°C (5).