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.

Combined Antimicrobial Effect of Bio-Waste Olive Leaf Extract and Remote Cold Atmospheric Plasma Effluent.

A novel strategy involving Olive Leaf Extract (OLE) and Cold Atmospheric Plasma (CAP) was developed as a green antimicrobial treatment. Specifically, we reported a preliminary investigation on the combined use of OLE + CAP against three pathogens, chosen to represent medical and food industries (i.e., E. coli, S. aureus and L. innocua). The results indicated that a concentration of 100 mg/mL (total polyphenols) in OLE can exert an antimicrobial activity, but still insufficient for a total bacterial inactivation. By using plain OLE, we significantly reduced the growth of Gram positive S. aureus and L. innocua, but not Gram-negative E. coli. Instead, we demonstrated a remarkable decontamination effect of OLE + CAP in E. coli, S. aureus and L. innocua samples after 6 h. This effect was optimally maintained up to 24 h in S. aureus strain. E. coli and L. innocua grew again in 24 h. In the latter strain, OLE alone was most effective to significantly reduce bacterial growth. By further adjusting the parameters of OLE + CAP technology, e.g., OLE amount and CAP exposure, it could be possible to prolong the initial powerful decontamination over a longer time. Since OLE derives from a bio-waste and CAP is a non-thermal technology based on ionized air, we propose OLE + CAP as a potential green platform for bacterial decontamination. As a combination, OLE and CAP can lead to better antimicrobial activity than individually and may replace or complement conventional thermal procedures in food and biomedical industries.

The antimicrobial efficacy of remote cold atmospheric plasma effluent against single and mixed bacterial biofilms of varying age.

Cold atmospheric plasma (CAP) is a minimal food processing technology of increasing interest in the food industry, as it is mild in nature compared to traditional methods (e.g. pasteurisation) and thus can maintain the food's desirable qualities. However, due to this mild nature, the potential exists for post-treatment microbial survival and/or stress adaptation. Furthermore, biofilm inactivation by CAP is underexplored and mostly studied on specific foods or on plastic/polymer surfaces. Co-culture effects, biofilm age, and innate biofilm-associated resistance could all impact CAP efficacy, while studies on real foods are limited to the food product investigated without accounting for structural complexity. The effect of a Remote and Enclosed CAP device (Fourth State Medicine Ltd) was investigated on Escherichia coli and Listeria innocua grown as planktonic cells and as single or mixed bacterial biofilms of variable age, on a biphasic viscoelastic food model of controlled rheological and structural complexity. Post-CAP viability was assessed by plate counts, cell sublethal injury was quantified using flow cytometry, and biofilms were characterised and assessed using total protein content and microscopy techniques. A greater impact of CAP on planktonic cells was observed at higher air flow rates, where the ReCAP device operates in a mode more favourable to reactive oxygen species than reactive nitrogen species. Although planktonic E. coli was more susceptible to CAP than planktonic L. innocua, the opposite was observed in biofilm form. The efficacy of CAP was reduced with increasing biofilm age. Furthermore, E. coli produced much higher protein content in both single and mixed biofilms than L. innocua. Consequently, greater survival of L. innocua in mixed biofilms was attributed to a protective effect from E. coli. These results show that biofilm susceptibility to CAP is age and bacteria dependent, and that in mixed biofilms bacteria may become less susceptible to CAP. These findings are of significance to the food industry for the development of effective food decontamination methods using CAP.