Avaliação da eficácia de bacteriófagos na redução de Pseudomonas fluorescens e seu efeito na vida de prateleira de queijo Minas Frescal

Abstract

Minas Frescal cheese (QMF) is one of Brazil's most produced and consumed cheeses. However, this type of cheese has a short shelf life due to the ease of microbial contamination during production. Microbial deterioration of QMF occurs mainly through the growth of psychrotrophic bacteria, which can grow and produce proteolytic and lipolytic enzymes even at temperatures of 4°C. In this sense, the genus Pseudomonas deserves attention due to its isolation frequency and problems related to its growth in milk and dairy products. This study aimed to evaluate the activity of lytic bacteriophages in controlling Pseudomonas fluorescens in Minas Frescal cheese to reduce spoilage and extend the shelf life of this dairy product. The experiments used two lytic bacteriophages (UFJF_PfDIW6 and UFJF_PfSW6) and a strain of P. fluorescens (UFV 041). Initially, the phages were evaluated for brine stability (23% NaCl and pH 5.3) for 28 days. The titer of phages UFJF_PfDIW6 and UFJF_PfSW6 remained constant throughout storage, at 7,0 and 4,0 Log PFU/mL, respectively. Next, the influence of adding phages to cheese on the reduction of bacteria was evaluated. Microcheeses were prepared and inoculated with 104 CFU/mL of bacteria, and the bacteriophages UFJF_PfSW6 (106 PFU/mL) and UFJF_PfDIW6 (109 PFU/mL) via milk, dough, or brine. Statistically significant differences (p<0.05) were observed between the bacterial count in micro cheeses with or without bacteriophages added only after three days of refrigerated storage. Phage treatment reduced around 1,1 to 1,5 Log CFU/g in micro cheeses after this storage period, regardless of how the viruses were added. However, the efficiency of phages in bacterial reduction decreased throughout storage, reaching values of 0,1 and 0,2 Log CFU/g at 28 days. In this way, the effect of phages on the count of P. fluorescens added via brine to cheeses manufactured on was evaluated considering different microbial loads and storage temperatures (103 CFU/g and 10 ºC; 103 CFU/g and 4 ºC; 107 CFU /g and 10 ºC). It was observed that P. fluorescens grew rapidly in cheeses even at low microbial loads and low storage temperatures, reaching counts above 108 CFU/g between 7 and 14 days. Phage treatment showed low efficacy in controlling P. fluorescens in QMF produced on a macro scale, except in the experimental condition of inoculum of 103 CFU/g and 4 ºC in which the phages caused reductions > 1,0 Log CFU/g at 3 days of storage. However, over the storage period, this effect was reduced. Therefore, adding phages could not effectively reduce the P. fluorescens count in cheeses and, consequently, did not interfere with the deterioration process. The extension and depth indices, which measure protein degradation, reached values above 20% after 28 days, regardless of the storage temperature and whether or not phages were added to the cheeses. Lipid degradation was also observed through increased titratable acidity, with over 2 mg KOH/g expenditure to neutralize fat at the end of storage. Another important result of this study was the survival capacity of P. fluorescens in brine. Despite the reduction in the initially inoculated count over time, counts of 2,4 Log CFU/mL were obtained after 40 days of storage at 10 ºC. Phages were also unable to reduce bacterial counts in brine. Therefore, the bacteriophages studied were ineffective in controlling the deterioration of QMF by P. fluorescens and in reducing these bacteria in brine, an important source of cheese contamination. New studies must be carried out to elucidate the influences of the matrix/food on the bacteriophage-bacteria interaction, the influences of the physiological state of the bacteria on the infection process, and the influence of NaCl on the infectivity of the phages used in this study. It is also recommended to analyze the application of bacteriophages in other dairy products.