Study Of Using Bacteriophages As Antibacterial Agents For Pseudomonas Spp. From Raw Milk

Pseudomonas spp. as the predominant psychrotrophic bacterias are able to grow rapidly under4～7℃andmay cause spoilage of milk and milk products, such as rancidity, gelling of Ultra HighTemperature (UHT) treated milk, or the formation of bitter off-flavors, due to their production of heatstable extracellular enzymes. So exploring novel methods and technologies to control the spoilagePseudomonas spp. present in raw milk is significantly important for improving the safty and quality ofdairy products.The aim of this research is to isolate the bacteriophages combating Pseudomonas in raw milk tocontrol the predominant psychrophilic bacterias in raw milk. First, Pseudomonas strains producingextracellular proteases and lipases were screened and isolated from raw milk, and using the isolatedPseudomonas strains as hosts, lytic phages were isolated by double-layer plate method from the localsewage and identified initially by observing the morphology under transmission electron.Second,characterization of these phages, such as lysis spectrum, one step growth curve, host specificity,multiplicity of infection and phage infectivity and stability under different physical and chemicalconditions, were determined by double-layer plate method. Third, the exploitation of bacteriophages asbiocontrol tools in UHT milk containing Pseudomonas strains and raw milk were investigated byshelf-life study.The results showed that among the13Pseudomonas strains producing proteases or lipases (P1, P2, P5,P6, P9, P12, P17, P21, P22, P24, P25, P27, and P34), P1, P5and P34were Pseudomonas fragi; P2andP12were Pseudomonas libanensis; P6, P21and P22were Pseudomonas veronii，P24and P25werePseudomonas psychrophila; P9, P17and P27were Pseudomonas rhodesiae Pseudomonas koreensisand Pseudomonas gessardii, respectively.All of the13phages (PP1, PP2, PP5, PP6, PP9, PP12, PP17, PP21, PP22, PP24, PP27and PP34)against13Pseudomonas strains were lytic phages and PP1belonged to the family Pedoviridae, PP5belonged to the family Cystoviridae, and the other11phages belonged to the family Leviviridae. Eachphage had a broad host spectrum with the capacity of lysing at least10of the13Pseudomonas isolates.Most phages showed high infectivity under4oC or25oC, except for PP17that was the most infectiveunder37℃. The optimum pH and NaCl concentrations range for activity of phages were5～7and1.5%～5%, respectively. Thermal inactivation point (TIP) varied with different phages: TIP of PP1, PP6,PP12, PP27and PP34were below50℃, the TIP range of PP2, PP9, PP21, PP22, and PP24was50~60℃, and PP5, PP17, PP25were still able to survive even the temperature of treatment reached 60℃for10min. The range of latent period and the rise period for13phages were15~25min and10~30min, respectively, and13phages ranged in burst size from57±1to157±4PFU per infected cell.The Multiplicity of Infection (MOI) for PP1, PP2, PP12, PP22, PP27and PP34were100; the MOI ofPP5, PP6, PP9and PP24were10; and the MOI of PP17, PP21and PP25were1.The number of Pseudomonas in UHT milk containing phage cocktail A (107PFU/mL) under4℃wasdecreased to the lowest by2order of magnitude after3d, while that in UHT milk containing phagecocktail B (107PFU/mL) slumped to the bottom by2order of magnitude after8h.As to the investigation of the shelf life for raw milk containing phages, it was obvious that phagescocktail A (107PFU/mL) reduced the psychrophilic bacterias and total bacterias counts in raw milkunder4℃significantly by1order of magnitude after3d; and the number of the psychrophilic bacteriasand total bacterias counts in raw milk containing cocktail B (107PFU/mL) under25℃dropped by1order of magnitude after8h and4h, respectively.Based on the results showed in this study, the13phages with different morphologic and physiologiccharacteristics are able to control the Pseudomonas strains in milk effectively, which reveals their greatpotential for application in industry.