Worldwide, street vending commerce has grown exponentially, representing in some countries, including Ecuador, a significant proportion of food consumed by the urban population. Pineapple is one of the common fruits sold as ready-to-eat slices by ambulant vendors in the street or on public transport at risk of contamination by various microorganisms. Previously, we selected Lactobacillus plantarum UTNCys5-4 and Lactococcus lactis subsp. lactis Gt28 strains producing peptides with high capacity to inhibit pathogen growth in vitro. In this study, the effect of different edited formulations containing a mixture of Cys5-4/Gt28 peptides was evaluated in vitro and ex vitro against a pathogenic cocktail containing E. coli (2), Salmonella (2) and Shigella (1). The growth of bacterial cocktail co-inoculated with cell-free supernatant containing peptides (formulation T1) and precipitated peptides (formulation T6), in a ratio of Cys5-4/Gt28:1:1 (v/v), results in a decrease of total cell viability with 1.85 and 1.2 log CFU/mL orders of magnitude at 6 h of incubation. About the same decrease (1.9 log CFU/g) was observed when pineapple slices artificially inoculated with the pathogenic cocktail were coated with T1 formulation, indicating the capacity to diminish simultaneous pathogens in situ, thus demonstrating its great biological control and protection. However, the E. coli cell counts reduced by 2.08 log CFU/g while Salmonella and Shigella cell counts reduced by 1.43 and 1.91 log CFU/g, respectively, at 5 days of refrigeration. In the untreated pineapple slices, the total cell density was maintained during storage, suggesting the adaptation of the pathogens to the fruit matrix. The peptide-based formulation exerted a bacteriolytic mode of action inducing pathogenic cell death. The results indicate that coating pineapple slices with peptide-based formulation is a promising approach to protect them from further contamination by microbial spoilage as well as an alternative to increase the food safety.

The occurrence of multidrug-resistant pathogens in the food chain causes health problems in humans, thus, research for novel antimicrobials to combat their growth is of interest. This study evaluates the antimicrobial potential of several combinations of peptide-protein extracts (PCs) consisting of peptide extracts from three native probiotic strains, Lactiplantibacillus plantarum UTNGt2, Lactococcus lactis UTNGt28, and L. plantarum UTNGt21A, alone or in combination with EDTA (ethylenediaminetetraacetic acid) against multidrug-resistant Staphylococcus aureus ATCC1026 and Citrobacter freundii UTNB3Sm1. Based on the antimicrobial assay, among the 19 tested PCs, two (PC11 and PC17) produced a greater zone of inhibition against both pathogens in vitro. Time-killing assays indicated the rapid death of S. aureus after exposure to PC11 and PC17, while C. freundii was rapidly inhibited by PC11 and PC1 (UTNGt2 only), suggesting that the inhibitory action is pathogen and dose-dependent of a particular molecule present in the extract. A marginal inhibitory effect was observed when the peptides were combined with EDTA. Transmission electron microscopy (TEM) revealed the structural membrane damage of both target strains upon interaction with individual peptide extracts. Different degrees of cell deformation, condensed cytoplasm, membrane blebbing, and ghost cell formation with visibly broken cell walls were observed in S. aureus. Likewise, the separation of the cytoplasmic membrane from the outer membrane, ghost cells, along with ovoid and deformed cells with undulated cell walls were observed for C. freundii. Furthermore, scanning electronic microscopy (SEM) analysis revealed different wrinkled and deformed cells covered by debris. A leakage of aromatic molecules was detected for both pathogens, indicating that PCs disrupted the cell wall integrity, inducing cell death. Given their inhibitory action and capacity to induce damage of the cytoplasmic membrane, the selected PCs may serve to slow bacterial growth in vitro; further research is required to prove their efficiency ex vitro to battle against food poisoning and subsequent human infection.

Consumption of ready-to-eat chopped fruits sold in the streets is a concern, as such activities are outside the regulation and protection in most developing countries. Ready-to-eat mangos are commonly sold as wedges in plastic cups at ambient temperature by mobile vendors in Ecuador, thus they are prone to contamination by bacteria, which poses a safety issue of concern. This work aimed to evaluate the effect of several antimicrobial cocktails consisting of previously designed specific peptide extract combinations from two probiotic bacteria Lactobacillus plantarum UTNCys5-4 and Lactococcus lactis subsp. lactis UTNGt28, along with nisin, a commercial food additive, on mango wedges artificially inoculated with a logarithmic phase culture of a five-strain bacterial mixture (FSBM). Preliminary bacteriological analysis of mango wedges purchased from mobile vendors showed the presence of multiple antibiotic-resistant isolates such E. coli spp., Enterobacter spp., Shigella spp., Salmonella spp., along with yeasts and molds, indicating non-compliance with the food safety standards. The results revealed that two antimicrobial cocktails, T2 and T5, containing cell-free supernatant based (CFS) and precipitated peptides (PP) based cocktails from UTNCys5-4 and UTNGt28 strains applied at dose 1:3 (v/v), were the most efficient combinations that inhibited the colonization of total bacterial counts with 56.03% and 55.61% in mango wedges stored with refrigeration. The reduction of total E. coli counts was 64.93%, while Salmonella and Shigella counts were reduced by 98.09% and 97.93%, respectively, when mango wedges were treated with T5-cocktail. The commercial nisin inhibited total Salmonella spp. counts by 40.13%, while E. coli spp. and Shigella spp. diminished by 28.20% and 37.22%, respectively. Moreover, we showed that T5 but not T7 (nisin) damaged the target cell integrity, thereby eventually inhibiting their growth and reproduction. The selected antimicrobial cocktails exerted a bacteriolytic effect by killing the FSBM simultaneously in a fruit matrix and preventing their accumulation in mango wedges. Furthermore, there is a possibility of using peptide combinatorial treatments to combat drug-resistant bacteria in ready-to-eat fruits.

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