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

Michał Wójcicki,Paulina Emanowicz,Barbara Sokołowska,Stanisław Błażejak,Olga Świder,Edyta Juszczuk-Kubiak,Iwona Gientka,Monika Kowalczyk,Paulina Średnicka

doi:10.3390/ijms222212460

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.

Highlights

Interest in minimally processed food is still growing, which is resulting in an extending assortment of food products of this type and in their increased availability to consumers
Pure bacterial cultures were isolated during the evaluation of the microbiological quality of the tested products: RUC, mixed leaf salad with carrot (MLSC), and mixed leaf salad with beetroot (MLSB), respectively
Our results showed that Serratia phage KKP 3264 against Serratia fonticola KKP 3084 was the most resistant to chemical factors; it retained lytic activity in the pH range from 3 to 11 (Figure 15D)

Summary

Introduction

Interest in minimally processed food is still growing, which is resulting in an extending assortment of food products of this type and in their increased availability to consumers. The production of minimally processed food involves only basic treatments that enable manufacturing ready-to-eat (RTE) food products, without compromising their natural properties [1]. Owing to the use of mild methods of heat treatment and preservation, often combined with physicochemical and biological methods, minimally processed food products should retain the sensory traits of freshness, including turgor, aroma, taste, and color [2,3,4,5]. Their production process is expected to preserve the thermolabile nutrients (vitamins and provitamins, minerals, or phytocompounds). The packaging process of food usually makes use of conditions of modified atmosphere and special (intelligent) packages tailored to individual food products [6,7]

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Conclusion