(347g) Keynote: Combined Effects of Microstructure and Minimal Processing on the Response and Spatial Organization of Listeria in Viscoelastic Models

Introduction and Aim:

Foods retaining their taste, texture, natural colouring and nutritional content are in increasing demand¹. To maintain these characteristics, there is great interest in minimal food processing techniques for microbial decontamination, e.g. natural antimicrobials, ultrasound, and cold atmospheric plasma (CAP)^2–4. Such treatments can be combined synergistically to act as a hurdle for microbial growth thereby increasing processing efficiency^5–7. However, the efficacy and mechanisms of such treatments, both individually and combined, are still unclear, especially as most studies have been done in actual food products; making the results very case specific. Furthermore, as these treatments are milder than traditional processing methods (pasteurization, heat sterilization), there is the potential for conditions to present a mild, sublethal stress which may induce an adaptive response in bacteria, leading to the risk of post-treatment survival and/or potential development of antimicrobial resistance (AMR).

The chemical composition and rheological/structural properties of foods can vary significantly, and can affect the growth kinetics, stress response, and potential AMR development. Natural antimicrobials may be added artificially or produced in situ by natural microflora (e.g. nisin-producing Lactococcus lactis), thus the production rate, quantity and efficacy of natural antimicrobials, and the efficacy of ultrasound and CAP treatments, may also be affected. More specifically, cells on a solid or solid(like) surface are immobilised and grow as 2-dimensional (2D) colonies. These cells will experience a significantly different biochemical and structural environment to cells grown planktonically in a liquid system: diffusional limitations of oxygen and nutrients to the colony will exist, while (acidic) metabolic by-products can accumulate around the colony causing a self-induced (acid) stress which can affect both the microbial kinetics and the environmental stress response^8–10. Overall, microorganisms could display different levels of AMR as a result^11–13.

As previously stated, most available studies on the inactivation of pathogens by natural antimicrobials such as nisin, applied individually or in combination with ultrasound or CAP, have been conducted in/on real foods (solid(like) and liquid) or in laboratory broths. However, studies in real food products are useful only for the system studied, and liquid broth models do not account for structural effects. Furthermore, a fundamental study of microbial inactivation by nisin, alone or combined with ultrasound/CAP, on systems of controlled rheological and structural properties is currently lacking.

Our previous work presents (to the best of our knowledge), the only systematic study thus far on the combined effects of artificially added nisin and system structure on the growth kinetics and spatial organization of Listeria. Surface growth of Listeria on a complex biphasic protein-polysaccharide model system was observed, for the first time, to be selective for the protein phase. Furthermore, colony size/distribution was different depending on the system viscosity and growth type (surface/immersed), suggesting structural effects on a microscopic scale¹⁴.

The present work is a fundamental study on the combined effects of the natural antimicrobial nisin with ultrasound or CAP treatments in/on structured food model systems. A range of monophasic and biphasic complex model systems that mimic a variety of real food products are created using Xanthan gum (XG) and/or Whey protein isolate (WPI) and/or fat, which are all components used in the food industry and are stable at a range of temperatures.

Materials and Methods:

Food model systems were prepared using Tryptic Soy Broth supplemented with 0.6% Yeast Extract (TSBYE), with (1) no added gelling agent (planktonic growth), (2) 0.5 - 1.5% XG (monophasic systems), or (3) a combination of 5% XG and 10% WPI in 1:1 ratio (biphasic complex system) or (3) a triphasic system (XG/WPI/Fat). For nisin-containing systems, nisin was added during food model system preparation for a final concentration of 140 IU/mL (a sublethal concentration which allows the observation of combined effects with ultrasound or CAP treatments).

Ultrasound inactivation of L. innocua stationary phase immersed colonies, with/without added nisin, was investigated in the monophasic XG systems (500 kHz, treatment time up to 30 min). CAP inactivation of L. innocua stationary phase surface colonies, with/without added nisin, was investigated on the surface of all systems (dielectric barrier discharge reactor, helium-oxygen plasma, treatment time up to 15 minutes).

Inactivation kinetics were monitored, and the inactivation model of Geeraerd et al. was fitted to the experimental data¹⁵.

Morphological changes of colonies following inactivation treatments were studied at a cellular level, using scanning electron microscopy (SEM). Samples were fixed in a 3% formaldehyde solution and dehydrated in an ethanol/water series before imaging.

Results and Discussion:

Effects of microstructure on L. innocua inactivation by ultrasound and CAP are observed, with additional differences in systems containing nisin. SEM imaging after inactivation treatments shows a change in cell morphology with differences in systems of different structure. Furthermore, SEM images show topographical differences between monophasic and biphasic food model systems on a cellular scale, which are observed to influence colony size, outward growth, and density. These have a significant impact on the environmental stress response within a colony, i.e., oxidative stress, self-induced acid stress, metabolic stress. Thus, different responses to microbial decontamination processes arise for systems of varying physicochemical and rheological/structural properties.

Significance and Impact:

Our findings give a systematic quantitative insight on the impact of natural antimicrobials, alone or combined with ultrasound/CAP treatments, on the inactivation of Listeria in food model systems of different physiochemical and structural complexity. They highlight the importance of accounting for bacterial stress adaptation in solid(like) systems when designing novel decontamination processes. Furthermore, significant differences can exist on a microscopic scale, thus it is important to account for microscopic differences when designing decontamination processes, as different survival rates of Listeria may be observed in different systems.

Acknowledgements:

This work was supported by the Department of Chemical and Process Engineering of the University of Surrey as well as an Impact Acceleration Grant (IAA-KN9149C) of the University of Surrey, an IAA-EPSRC Grant (RN0281J), and the Royal Society.

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