Isolation of virulent phages infecting dominant mesophilic aerobic bacteria in cucumber pickle fermentation

Pickle is a type of mildly lactic acid fermented vegetable and is a traditional dish favored in China, Japan, and Korea. Corruption of spoilage bacteria and accumulation of nitrite during vegetable fermentation are common problems that affect the pickle industry and consumer health. In this work, cucumber juice was used as a vegetable model to study the dominant mesophilic aerobic bacteria (MAB) producing nitrite during pickle fermentation. Virulent phages infecting the dominant MABs combined with Lactobacillus plantarum M6 were used to control these bacteria. Enterobacter cloacae and Pseudomonas fluorescens are the dominant MABs in the fermentation of cucumber juice containing 4% or 8% NaCl, with isolation percentages reaching 30.6% and 23.1%, respectively. Virulent phages PspYZU5415 and EcpYZU01 were isolated using P. fluorescens J5415 and E. cloacae J01 as the host bacteria, respectively. These two phages show a broad host range and strong lytic activity, and their genomes contain no toxins and antibiotic resistance genes. PspYZU5415 and EcpYZU01 were combined into a cocktail (designated as Phage MIX) that effectively inhibits the growth of E. cloacae and P. fluorescens in cucumber juice with different salt concentrations. PhageMIX combined with L. plantarum M6 decreased the counts of P. mendocina and E. cloacae to undetectable levels at 48 h during the fermentation of cucumber juice artificially contaminated with P. mendocina and E. cloacae. In addition, nitrite content increased to 11.3 mg/L at 20 h and then degraded completely at 36 h. By contrast, P. mendocina and E. cloacae remained in the groups without PhageMIX during fermentation (0-48 h). Nitrite content rapidly increased to 65.7 mg/L at 12 h and then decreased to 21.6 mg/L at 48 h in the control group. This study suggests that PhageMIX combined with lactic acid bacterial strains can be used as an ecological starter for controlling the dominant MABs P. mendocina and E. cloacae and for reducing nitrate production during the early stage of pickle fermentation.