Kinetics and Thermodynamics of Thermal Inactivation of Novel Bacteriophages Specifically Targeting Non-O157 Shiga-toxigenic Escherichia coli

Introduction: Shiga toxin-producing Escherichia coli (STEC) are responsible for multiple foodborne illness outbreaks. It is therefore crucial to devise effective control strategies. Bacteriophages are advantageous over traditional antimicrobials by virtue of their high-specificity against target bacteria. The FDA-approval of bacteriophages as bio-preservatives in ready-to-eat meat and poultry products has paved way for development of phage-based antimicrobials. Understanding their thermal inactivation kinetics is essential to allow their use as bio-preservatives in food matrices. Kinetic modeling of thermal inactivation of STEC-specific bacteriophages could enable their predictive survival for successful application in food systems. 

Purpose: Evaluation of thermal inactivation models for selected bacteriophages and analysis of thermodynamic parameters to understand denaturation. 

Methods: Bacteriophages previously isolated from cattle farms in Oklahoma and those exhibiting inhibition towards non-O157 STEC (O26, O45, O103, O111, O121, and O145) were used. Thermal inactivation of bacteriophages (at 8-9 log PFU/ml), was determined at 40-90°C for 60 minutes with 10-minute-sampling intervals. Phages were heated in sealed tubes with host STEC-strain and enumerated using double-agar-layer technique. Kinetic degradation models from published literature: first-order, two-fraction, Weibull and n^th-order were used to understand degradation kinetics through non-linear regression module, using SigmaPlot13 (Systat Software, Inc., CA, USA). Activation enthalpy (ΔH), free energy of inactivation (ΔG) and activation entropy (ΔS) was calculated using rate-constant and activation energy (Ea) values.   

Results: The n^th-order-model provided the best description of thermal inactivation of bacteriophages at selected temperatures. The r² values ranged from 0.74-0.91 for all phages. High Ea values ranging from 195-619 kJ mol^-1 indicate that bacteriophages are relatively more thermostable than bacteriocins. D-values at reference temperature (t_ref) ranged from 22-346 minutes. High positive values of ΔH and ΔS was observed in our results. Thermodynamic parameters revealed that tested bacteriophages had structural thermo-stability.

Significance: Bacteriophages could serve as biopreservatives in food industry to provide synergistic effect with heat treatment.