Characterization and Genome Study of Novel Lytic Bacteriophages against Prevailing Saprophytic Bacterial Microflora of Minimally Processed Plant-Based Food Products

Abstract

The food industry is still searching for novel solutions to effectively ensure the microbiological safety of food, especially fresh and minimally processed food products. Nowadays, the use of bacteriophages as potential biological control agents in microbiological food safety and preservation is a promising strategy. The aim of the study was the isolation and comprehensive characterization of novel bacteriophages with lytic activity against saprophytic bacterial microflora of minimally processed plant-based food products, such as mixed leaf salads. From 43 phages isolated from municipal sewage, four phages, namely Enterobacter phage KKP 3263, Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 have lytic activity against Enterobacter ludwigii KKP 3083, Citrobacter freundii KKP 3655, Enterobacter cloacae KKP 3082, and Serratia fonticola KKP 3084 bacterial strains, respectively. Transmission electron microscopy (TEM) and whole-genome sequencing (WGS) identified Enterobacter phage KKP 3263 as an Autographiviridae, and Citrobacter phage KKP 3664, Enterobacter phage KKP 3262, and Serratia phage KKP 3264 as members of the Myoviridae family. Genome sequencing revealed that these phages have linear double-stranded DNA (dsDNA) with sizes of 39,418 bp (KKP 3263), 61,608 bp (KKP 3664), 84,075 bp (KKP 3262), and 148,182 bp (KKP 3264). No antibiotic resistance genes, virulence factors, integrase, recombinase, or repressors, which are the main markers of lysogenic viruses, were annotated in phage genomes. Serratia phage KKP 3264 showed the greatest growth inhibition of Serratia fonticola KKP 3084 strain. The use of MOI 1.0 caused an almost 5-fold decrease in the value of the specific growth rate coefficient. The phages retained their lytic activity in a wide range of temperatures (from −20 °C to 50 °C) and active acidity values (pH from 4 to 11). All phages retained at least 70% of lytic activity at 60 °C. At 80 °C, no lytic activity against tested bacterial strains was observed. Serratia phage KKP 3264 was the most resistant to chemical factors, by maintaining high lytic activity across a broader range of pH from 3 to 11. The results indicated that these phages could be a potential biological control agent against saprophytic bacterial microflora of minimally processed plant-based food products.

Two Newly Isolated Enterobacter-Specific Bacteriophages: Biological Properties and Stability Studies

“…Moreover, the observed decrease in titers as a result of a large deviation from physiological pH has been confirmed for most phages, including Enterobacter -specific [ 71 , 89 , 90 , 91 , 92 ] and other species (e.g., Escherichia coli -specific) [ 93 ]. Very similar tolerance to a wide pH range was also shown by the phages KKP 3262 and KKP 3263 described by Wójcicki et al (2021) [ 94 ]. Interestingly, two of five E. hormaechei -specific phages isolated by Chen and coworkers (2022) showed an almost complete resistance to a more alkaline environment (such as pH = 12) [ 95 ].…”

Application of Lytic Bacteriophages and Their Enzymes to Reduce Saprophytic Bacteria Isolated from Minimally Processed Plant-Based Food Products—In Vitro Studies

“…The statistics indicate that the number of illnesses is increasing year after year and a possible reason for this is the increasing consumer interest in minimally processed foods [ 8 , 9 ]. Reduced quality of MPF products resulting from the growth and development of saprophytic bacteria constitute a significant problem [ 10 , 11 , 12 ]. Vegetables are a source of nutrients for microorganisms because of their near-neutral pH, high content of vitamins, minerals, fiber [ 13 ], and high water activity [ 14 , 15 ].…”

“…Vegetables are a source of nutrients for microorganisms because of their near-neutral pH, high content of vitamins, minerals, fiber [ 13 ], and high water activity [ 14 , 15 ]. Plant-based food products have a short shelf life, even at low storage temperatures [ 16 ], due to the presence of saprophytic bacteria [ 12 ]. The search for methods to protect food has turned to the use of lytic bacteriophages for this purpose [ 17 , 18 ].…”

“…The search for methods to protect food has turned to the use of lytic bacteriophages for this purpose [ 17 , 18 ]. Scientific research shows that the use of phages and their enzymes does not affect the sensory properties of food (i.e., taste, appearance, color, and smell) [ 12 , 17 , 19 ]. Due to their high host specificity, bacteriophages and phage enzymes are becoming promising prospects in the detection and biocontrol of bacterial pathogens in agriculture, food production and processing, and medicine (phage therapy) [ 20 , 21 , 22 ].…”

“…The presence of bacteriophages in the environment is a natural mechanism to ensure bacterial balance [ 26 ]. Widespread occurrence is associated with the presence of phages in various environments, such as sewage, soil, and food products [ 12 ]. Their presence was confirmed in commercial vaccines and serums, in the mouth and gastrointestinal tract, skin and hair [ 18 , 27 , 28 , 29 ].…”