Dr Katie Costello

Minimal processing for microbial decontamination, such as the use of natural antimicrobials, is gaining interest in the food industry as these methods are generally milder than conventional processing, therefore better maintaining the nutritional content and sensory characteristics of food products. The aim of this study was to quantify the impact of (i) structural composition and complexity, (ii) growth location and morphology, and (iii) the natural antimicrobial nisin, on the microbial dynamics of . More specifically, viscoelastic food model systems of various compositions and internal structure were developed and characterised, i.e. monophasic Xanthan gum-based and biphasic Xanthan gum/Whey protein-based viscoelastic systems. The microbial dynamics of at 10oC, 30oC and 37oC were monitored and compared for planktonic growth in liquid, or in/on (immersed or surface colony growth) the developed viscoelastic systems, with or without a sublethal concentration of nisin. Microscopy imaging was used to determine the bacterial colony size and spatial organisation in/on the viscoelastic systems. Selective growth of on the protein phase of the developed biphasic system was observed for the first time. Additionally, significant differences were observed in the colony size and distribution in the monophasic Xanthan gum-based systems depending on (i) the type of growth (surface/immersed) and (ii) the Xanthan gum concentration. Furthermore, the system viscosity in monophasic Xanthan gum-based systems had a protective role against the effects of nisin for immersed growth, and a further inhibitory effect for surface growth at a suboptimal temperature (10oC). These findings give a systematic quantitative insight on the impact of nisin as an environmental challenge on the growth and spatial organisation of , in viscoelastic food model systems of various structural compositions/complexities. This study highlights the importance of accounting for system structural composition/complexity when designing minimal food processing methods with natural antimicrobials.

Novel processing methods such as cold atmospheric plasma (CAP) and natural antimicrobials like nisin, are of interest to replace traditional food decontamination approaches as, due to their mild nature, they can maintain desirable food characteristics, i.e., taste, texture, and nutritional content. However, the microbial growth characteristics (planktonic growth/surface colonies) and/or the food structure itself (liquid/solid surface) can impact the inactivation efficacy of these novel processing methods. More specifically, cells grown as colonies on a solid(like) surface experience a completely different growth environment to cells grown planktonically in liquid, and thus could display a different response to novel processing treatments through stress adaptation and/or cross protection mechanisms. The order in which combined treatments are applied could also impact their efficacy, especially if the mechanisms of action are complementary. This work presents a fundamental study on the efficacy of CAP and nisin, alone and combined, as affected by food system structure. More specifically,  was grown planktonically (liquid broth) or on a viscoelastic Xanthan gum gel system (1.5% w/v) and treated with CAP, nisin, or a combination of the two. Both the inactivation system, i.e., liquid versus solid(like) surface and the growth characteristics, i.e., planktonic versus colony growth, were shown to impact the treatment efficacy. The combination of nisin and CAP was more effective than individual treatments, but only when nisin was applied before the CAP treatment. This study provides insight into the environmental stress response/adaptation of  grown on structured systems in response to natural antimicrobials and novel processing technologies, and is a step towards the faster delivery of these food decontamination methods from the bench to the food industry.

Ultrasound, alone or in combination with natural antimicrobials, is a novel food processing technology of interest to replace traditional food decontamination methods, as it is milder than classical sterilisation (heat treatment) and maintains desirable sensory characteristics. However, ultrasound efficacy can be affected by food structure/composition, as well as the order in which combined treatments are applied. More specifically, treatments which target different cell components could result in enhanced inactivation if applied in the appropriate order. The microbial properties i.e. Gram positive/Gram negative can also impact the treatment efficacy. This work presents a systematic study of the combined effect of ultrasound and nisin on the inactivation of the bacteria  (Gram positive) and  (Gram negative), at a range of  conditions (44, 500, 1000 kHz). The order of treatment application was varied, and the impact of system structure was also investigated by varying the concentration of  used to create the food model systems (0 – 0.5% w/v). Microbial inactivation kinetics were monitored, and advanced microscopy and flow  techniques were utilised to quantify the impact of treatment on a cellular level. Ultrasound was shown to be effective against  at 500 kHz only, with  demonstrating resistance to all frequencies studied. Enhanced inactivation of  was observed for the combination of nisin and ultrasound at 500 kHz, but only when nisin was applied before ultrasound treatment. The system structure negatively impacted the inactivation efficacy. The combined effect of ultrasound and nisin on  was attributed to short-lived destabilisation of the outer membrane as a result of , allowing nisin to penetrate the cytoplasmic membrane and facilitate cell inactivation.

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  was more susceptible to CAP than planktonic , the opposite was observed in biofilm form. The efficacy of CAP was reduced with increasing biofilm age. Furthermore,  produced much higher protein content in both single and mixed biofilms than . Consequently, greater survival of  in mixed biofilms was attributed to a protective effect from . 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.

Natural antimicrobials are of interest to replace traditional food decontamination methods: they are milder and maintain desirable sensory characteristics. However, efficacy can be affected by food structure/composition, thus structural effects in a co-culture pathogen/microflora system are investigated.  was grown planktonically (liquid broth) or on a biphasic viscoelastic system, in monoculture with/without artificial nisin, or in co-culture with  (nisin/non-nisin producing). Microbial growth kinetics were monitored and advanced microscopy techniques were utilized to quantify cellular interactions and spatial organization. Microstructural effects are observed on the kinetics, with differences in monoculture/co-culture. Significant microscopic differences are observed in spatial organization and colony size. We are the first to observe changing growth location for all species in monoculture/co-culture, with differences in colony size/organization through stationary phase. This study provides insight into the environmental stress response/adaptation of  grown on structured systems in response to  and natural antimicrobials.