Isolamento, caracterização e avaliação da atividade de bacteriófagos líticos para biocontrole de Pseudomonas fluorescens em leite cru

Abstract

The control of psychrotrophic growth in raw milk is crucial for maintaining its quality and its derivatives. Pseudomonas fluorescens stands out as the main contaminant, capable of producing thermoestable hydrolytic enzymes. Bacteriophages, viruses that exclusively infect bacteria, have been a promising alternative for biocontrol in food. However, its effectiveness in controlling the raw milk microbiota is still poorly studied. The objectives of this study were to isolate and characterize lytic bacteriophages, as well as to evaluate its effect on the bacterial count, proteolytic activity and proteolysis caused by P. fluorescens. Samples of water from streams, rivers and dairy effluents were used, as well as the enrichment method for isolating bacteriophages against P. fluorescens. Eighteen bacteriophages were isolated, of which seven were selected for preliminary characterization. The titration of these bacteriophages varied from 7 to 9 Log PFU/mL and the size of the lysis plates was 0.39 and 2.67 mm. They also showed infection capacity restricted to genus Pseudomonas. Two of these bacteriophages (Ph6 and Ph32) were more widely characterized. Ph6 and Ph32 showed genome sizes of 38.8 and 31.3 kb; latency period of 96 and 25 min; and burst size of 295 and 40 viral particles per infected bacterial cell, respectively. Neither bacteriophage was thermally inactivated after 30 min of treatment at 63 and 72 ° C, and maintained their titrations stable after exposure in pH ranges of 5 - 11 for 60 min. Both bacteriophages were inactivated after treatment with sodium hypochlorite at 200 mg/L for 30 min. The bacteriophages Ph6 and Ph32 reduced the count of P. fluorescens in a milk-based culture medium at 4 and 10 ° C during the 7 days of storage. Also, they delayed proteolytic activity and proteolysis by two days. In raw milk, they decreased the counts of Pseudomonas spp., psychrotrophic and aerobic mesophiles by 3.2, 2.5 and 2.9 Log CFU/mL, respectively, after 7 days of storage at 4 °C. The bacteriophages of the present study have potential for application as biological control agents for preserving raw milk.