Determination of the combined effect of grape seed extract and cold atmospheric plasma on foodborne pathogens and their environmental stress knockout mutants.

The study aimed to explore the antimicrobial efficacy of grape seed extract (GSE) and cold atmospheric plasma (CAP) individually or in combination against L. monocytogenes and E. coli wild type (WT) and their isogenic mutants in environmental stress genes. More specifically, we examined the effects of 1% (wt/vol) GSE, 4 min of CAP treatment, and their combined effect on L. monocytogenes 10403S WT and its isogenic mutants ΔsigB, ΔgadD1, ΔgadD2, ΔgadD3, as well as E. coli K12 and its isogenic mutants ΔrpoS, ΔoxyR, and ΔdnaK. In addition, the sequence of the combined treatments was tested. A synergistic effect was achieved for all L. monocytogenes strains when exposure to GSE was followed by CAP treatment. However, the same effect was observed against E. coli strains, only for the reversed treatment sequence. Additionally, L. monocytogenes ΔsigB was more sensitive to the individual GSE and the combined GSE/CAP treatment, whereas ΔgadD2 was more sensitive to CAP, as compared to the rest of the mutants under study. Individual GSE exposure was unable to inhibit E. coli strains, and individual CAP treatment resulted in higher inactivation of E. coli in comparison to L. monocytogenes with the strain ΔrpoS appearing the most sensitive among all studied strains. Our findings provide a step toward a better understanding of the mechanisms playing a role in the tolerance/sensitivity of our model Gram-positive and Gram-negative bacteria toward GSE, CAP, and their combination. Therefore, our results contribute to the development of more effective and targeted antimicrobial strategies for sustainable decontamination.IMPORTANCEAlternative approaches to conventional sterilization are gaining interest from the food industry, driven by (i) the consumer demand for minimally processed products and (ii) the need for sustainable, environmentally friendly processing interventions. However, as such alternative approaches are milder than conventional heat sterilization, bacterial pathogens might not be entirely killed by them, which means that they could survive and grow, causing food contamination and health hazards. In this manuscript, we performed a systematic study of the impact of antimicrobials derived from fruit industry waste (grape seed extract) and cold atmospheric plasma on the inactivation/killing as well as the damage of bacterial pathogens and their genetically modified counterparts, for genes linked to the response to environmental stress. Our work provides insights into genes that could be responsible for the bacterial capability to resist/survive those novel treatments, therefore, contributing to the development of more effective and targeted antimicrobial strategies for sustainable decontamination.

A Systematic Quantitative Determination of the Antimicrobial Efficacy of Grape Seed Extract against Foodborne Bacterial Pathogens.

Concerns regarding the role of antimicrobial resistance (AMR) in disease outbreaks are growing due to the excessive use of antibiotics. Moreover, consumers are demanding food products that are minimally processed and produced in a sustainable way, without the use of chemical preservatives or antibiotics. Grape seed extract (GSE) is isolated from wine industry waste and is an interesting source of natural antimicrobials, especially when aiming to increase sustainable processing. The aim of this study was to obtain a systematic understanding of the microbial inactivation efficacy/potential of GSE against Listeria monocytogenes (Gram-positive), Escherichia coli and Salmonella Typhimurium (Gram-negative) in an in vitro model system. More specifically, for L. monocytogenes, the effects of the initial inoculum concentration, bacterial growth phase and absence of the environmental stress response regulon (SigB) on the GSE microbial inactivation potential were investigated. In general, GSE was found to be highly effective at inactivating L. monocytogenes, with higher inactivation achieved for higher GSE concentrations and lower initial inoculum levels. Generally, stationary phase cells were more resistant/tolerant to GSE as compared to exponential phase cells (for the same inoculum level). Additionally, SigB appears to play an important role in the resistance of L. monocytogenes to GSE. The Gram-negative bacteria under study (E. coli and S. Typhimurium) were less susceptible to GSE as compared to L. monocytogenes. Our findings provide a quantitative and mechanistic understanding of the impact of GSE on the microbial dynamics of foodborne pathogens, assisting in the more systematic design of natural antimicrobial-based strategies for sustainable food safety.

On the evaluation of the antimicrobial effect of grape seed extract and cold atmospheric plasma on the dynamics of Listeria monocytogenes in novel multiphase 3D viscoelastic models.

The demand for products that are minimally processed and produced in a sustainable way, without the use of chemical preservatives or antibiotics have increased over the last years. Novel non-thermal technologies such as cold atmospheric plasma (CAP) and natural antimicrobials such as grape seed extract (GSE) are attractive alternatives to conventional food decontamination methods as they can meet the above demands. The aim of this study was to investigate the microbial inactivation potential of GSE, CAP (in this case, a remote air plasma with an ozone-dominated RONS output) and their combination against L. monocytogenes on five different 3D in vitro models of varying rheological, structural, and biochemical composition. More specifically, we studied the microbial dynamics, as affected by 1 % (w/v) GSE, CAP or their combination, in three monophasic Xanthan Gum (XG) based 3D models of relatively low viscosity (1.5 %, 2.5 % and 5 % w/v XG) and in a biphasic XG/Whey Protein (WPI) and a triphasic XG/WPI/fat model. A significant microbial inactivation (comparable to liquid broth) was achieved in presence of GSE on the surface of all monophasic models regardless of their viscosity. In contrast, the GSE antimicrobial effect was diminished in the multiphasic systems, resulting to only a slight disturbance of the microbial growth. In contrast, CAP showed better antimicrobial potential on the surface of the complex multiphasic models as compared to the monophasic models. When combined, in a hurdle approach, GSE/CAP showed promising microbial inactivation potential in all our 3D models, but less microbial inactivation in the structurally and biochemically complex multiphasic models, with respect to the monophasic models. The level of inactivation also depended on the duration of the exposure to GSE. Our results contribute towards understanding the antimicrobial efficacy of GSE, CAP and their combination as affected by robustly controlled changes of rheological and structural properties and of the biochemical composition of the environment in which bacteria grow. Therefore, our results contribute to the development of sustainable food safety strategies.

Unravelling the impact of fat content on the microbial dynamics and spatial distribution of foodborne bacteria in tri-phasic viscoelastic 3D models.

The aim of the current study is to develop and characterise novel complex multi-phase in vitro 3D models, for advanced microbiological studies. More specifically, we enriched our previously developed bi-phasic polysaccharide (Xanthan Gum)/protein (Whey Protein) 3D model with a fat phase (Sunflower Oil) at various concentrations, i.e., 10%, 20%, 40% and 60% (v/v), for better mimicry of the structural and biochemical composition of real food products. Rheological, textural, and physicochemical analysis as well as advanced microscopy imaging (including spatial mapping of the fat droplet distribution) of the new tri-phasic 3D models revealed their similarity to industrial food products (especially cheese products). Furthermore, microbial growth experiments of foodborne bacteria, i.e., Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa and Lactococcus lactis on the surface of the 3D models revealed very interesting results, regarding the growth dynamics and distribution of cells at colony level. More specifically, the size of the colonies formed on the surface of the 3D models, increased substantially for increasing fat concentrations, especially in mid- and late-exponential growth phases. Furthermore, colonies formed in proximity to fat were substantially larger as compared to the ones that were located far from the fat phase of the models. In terms of growth location, the majority of colonies were located on the protein/polysaccharide phase of the 3D models. All those differences at microscopic level, that can directly affect the bacterial response to decontamination treatments, were not captured by the macroscopic kinetics (growth dynamics), which were unaffected from changes in fat concentration. Our findings demonstrate the importance of developing structurally and biochemically complex 3D in vitro models (for closer proximity to industrial products), as well as the necessity of conducting multi-level microbial analyses, to better understand and predict the bacterial behaviour in relation to their biochemical and structural environment. Such studies in advanced 3D environments can assist a better/more accurate design of industrial antimicrobial processes, ultimately, improving food safety.