Concerns regarding the role of antimicrobial resistance (AMR) in disease outbreaks are growing due to excessive antibiotic use. Moreover, consumers are demanding minimally processed and sustainably produced food products, without the use of chemical preservatives or antibiotics. Novel non-thermal technologies such as cold atmospheric plasma (CAP) and natural antimicrobials derived from by-products of the food industry such as grape seed extract (GSE) are attractive alternatives to conventional food decontamination methods as they meet the above demands. However, the level of microbial inactivation imposed by these methods varies depending on the type of microorganism, intensity of the treatment and substrate (solid or liquid). Therefore, further studies are required to investigate the potential of those novel approaches when applied individually or in combination.

Most studies exploring the antimicrobial efficacy of natural antimicrobials derived from food waste are not carrying out systematic quantitative monitoring of the microbial dynamics. Additionally, in most cases, the antimicrobial efficacy of novel processing control strategies is only studied in liquid environments or specific food products and their mechanism of inactivation is still not fully elucidated. In this thesis, a fundamental study on the antimicrobial efficacy of GSE and CAP in liquid and solid(like) in vitro models and their effect on the environmental stress response of pathogenic bacteria is presented for the first time.

This thesis demonstrates that GSE successfully inactivates L. monocytogenes and SigB appears to play an important role in the resistance of L. monocytogenes to the individual GSE and combined treatment with CAP. Additionally, it shows that the antimicrobial efficacy of the GSE in simplistic (monophasic) 3D models was comparable to liquid systems but not in more complex (biphasic and triphasic) 3D systems. The hurdle approach has promising microbial inactivation potential; however, the efficacy depends on the bacterial species, the structural and biochemical complexity of the substrate and the order of application.

This work contributes towards understanding the antimicrobial efficacy of mild processing control strategies in controlled in vitro models and mechanisms playing a role in tolerance/sensitivity of our model Gram-positive and Gram-negative bacteria to GSE, CAP and their combination. Therefore, our results contribute to the development of sustainable food safety strategies.