Low Temperature Inactivation Kinetics to Determine Bacteriophage Shelf-life Stability

Introduction:  Bacteriophages, clinically useful and safe in both human and animals have garnered much attention as antimicrobials in food applications. However, efficacy of antimicrobials in the cold distribution chain for foods largely depends on stability at low temperature range. Retaining activity at low temperatures, for longer periods of time could render bacteriophages as cold-stable biocontrol agents. It is therefore important to understand their cold temperature inactivation kinetics at low and ultra-low temperature.

Purpose:  Determine cold temperature inactivation kinetics of bacteriophages specific for non-O157 Shiga-toxigenic Escherichia coli (STEC).

Methods:  Previously isolated bacteriophages, specific for non-O157 STEC (O26, O45, O103, O111, O121, O145), were used. Phages were exposed to low temperatures of 4, -20, and -80°C for 90 days in sealed-tubes. Phage population was enumerated on days 0, 1, 30, 60, and 90, using double-agar-layer technique. First-order model, commonly used to understand temperature-degradation kinetics, was fitted through regression module, using SigmaPlot13^TM (Systat Software, US). The model was evaluated using regression coefficient (r²), mean square error (MSE), and standard error of means (SEM). Several kinetic parameters: order of reaction (n), half-life (t_1/2), and inactivation-time (D-value), for low-temperature inactivation were calculated.

Results:  The r² values obtained from first-order model ranged from 0.82-0.98 for all phages. Irrespective of the host, all phages remained stable at storage temperatures for 90 days, except O103-phage which lost activity on day-90 at -20 and -80°C. The inactivation rate constant (k) for all the phages at 4°C ranged from 5.0×10^-4 to 1.10×10^-3 per day. However, it increased slightly at -20°C (1.80x10^-3-3.03×10^-2) and -80°C (1.47×10^-3-3.37×10^-2). Predicted D-values for all phages at 4, -20 and -80°C, ranged from 2.03-12.61, 0.20-3.50, and 0.18-2.52 years, respectively. The high predicted D-values indicate that bacteriophages are stable at low and ultra-low temperatures.

Significance:  Tested bacteriophages exhibited cold-storage stability, enabling them to be used as biopreservatives in cold chains.