Inactivation of Pseudomonas Enveloped Bacteriophage Phi6 (H5N1 Surrogate) in Raw Bovine Milk using a Combination of...

Raw milk obtained from cows infected with H5N1 requires to be treated to inactivate the virus prior to disposal. In this context, a hurdle approach was evaluated to inactivate Phi6 bacteriophage (potential H5N1 surrogate) in raw milk as a risk management option for dairy farms. The treatment involved adding hydrogen peroxide (0.1, 1, or 10 mM) and sodium thiocyanate (0.2, 2.4, or 24 µM) to raw milk, which served as substrates for the lactoperoxidase system to produce hypothiocyanite. The substrates were also a source of free radicals via UV-C mediated photo-degradation in an Advanced Oxidation Process (AOP). The raw milk was inoculated with Phi6 bacteriophage (7-8 log PFU/mL) in the presence of thiocyanate and/or hydrogen peroxide, held for 10 min at 23 °C, before passing through a spiral UV-C reactor. The infective Phi6 phages were determined on a cell lawn of the Pseudomonas host cell. It was found that the lactoperoxidase system, in the absence of UV-C, supported a 3.01 ± 0.07 log PFU/mL reduction of phage when 10 mM hydrogen peroxide and 24 µM sodium thiocyanate were applied to raw milk. Hydrogen peroxide or thiocyanate alone supported <1.2 log reduction of Phi6. When UV-C (137 mJ/cm²) was applied in the presence of added hydrogen peroxide (10 mM) and thiocyanate (24 µM), the log reduction of phages increased to 4.49 ± 0.50 log PFU. The flow pattern of milk also influenced the efficacy of treatment, suggesting that homogenous mixing during passage through the reactor was an important factor. The Phi6 inactivation was partly supported by the direct action of UV photons, the generation of free radicals (hydroxyl-radical and thiocyanate), and lactoperoxidase. When the lactoperoxidase was thermally inactivated, the log reduction was increased to 5.63 ± 0.10 log PFU, suggesting competition for hydrogen peroxide and thiocyanate between the enzyme and AOP. The study demonstrated proof of principle for a nonthermal process for inactivating viruses in raw milk.