(387b) Unravelling the Effect of Waste Based Antimicrobials and Cold Atmospheric Plasma on the Inactivation of Pathogens: A Comparison of 2D and 3D Multiphasic Viscoelastic Models.

INTRODUCTION

In recent years, consumers are showing a preference in minimally processed foods that do not contain chemical preservatives or antibiotics. At the same time the (food) industry is aiming at increasing sustainable production of products. Novel processing technologies such as ultrasound, cold atmospheric plasma (CAP), high hydrostatic pressure and natural antimicrobials can be used as alternative approaches to classic heat sterilisation. However, as these treatments are milder than classic decontamination methods, the desirable microbial inactivation (5 log CFU/ml) might not occur, leading to contaminated and unsafe products. Furthermore, microbial stress adaptation and antimicrobial resistance can develop. This is due to the nature of those mild/alternative technologies, i.e., due to their mode of action, they cause less damage and death to bacteria as compared to classic treatments, e.g., sterilisation¹. For this reason, novel control strategies and their microbial stress response should be thoroughly investigated either individually or in combination (the so-called hurdle approach)², to enable the design of robust and safe decontamination strategies.

Most studies to date, focus on exploring new natural antimicrobials (NA) as an alternative to chemical preservatives are conducted in either real food products on in 2D (liquid broth) in vitro systems. However, real food products vary substantially in terms of their structural and biochemical properties and have significant batch-to-batch variations. In contrast 2D models are very reproducible, however, they are too simplistic and inaccurate in capturing structural complexity as well as the way bacteria grow and self-organise in 3D. More specifically, bacteria behave differently in (3D) solid environments, where they form clusters/aggregates, colonies and/or biofilms, as compared to individual cell growth (planktonic growth) which occurs in 2D³. Such growth in 3D can lead to a completely different response to inactivation approaches due to shelf-induced stress adaptation in colonies or biofilms as well as due to extracellular polymeric substance (EPS) production^2,4–6. Grape seed extracts (GSE) have antimicrobial properties, but there is limited research on their effectiveness, which mainly focuses on specific food items and or simple 2D systems. We have recently reported that 1% w/v GSE was highly effective at inactivating L. monocytogenes (10⁵CFUL/ml) in liquid nutrient broth (3 log CFU/ml after 24h)⁷. Additionally, there are limited studies investigating the combination of GSE with other novel technologies such as CAP⁸. To develop robust novel hurdle technology approaches, it is important to understand the mode of action and the microbial stress response for each treatment in more representative, robustly controlled 3D environments.

The aim of this research is to provide a fundamental understanding of the impact of GSE on the microbial dynamics and stress response of the bacterial pathogens L. monocytogenes and E. coli as affected by structural and biochemical complexity.

More specifically, those bacteria (wild types and knockouts for genes associated to environmental stress adaptation) were inoculated, grown and treated with GSE, in various 3D viscoelastic models, i.e., monophasic xanthan gum-based models of various viscosities as well as biphasic xanthan gum/Whey protein model and a tri-phasic xanthan gum/ Whey protein/lipid model. Moreover, the bacterial response to cold atmospheric plasma (CAP) was assessed individually or in combination as a new minimal processing technique.

METHODS

Multiple 3D viscoelastic model systems of various viscosities and biochemical complexities were developed as previously described^6,9,10. More specifically, three monophasic polysaccharide 3D models (1.5, 2.5 and 5% w/v xanthan gum), a biphasic system (5% polysaccharide + whey protein) and a triphasic system (biphasic + 10% v/v vegetable oil) were developed. For the GSE treatment, 1% w/v GSE was added in all models under study. L. monocytogenes (10⁵ CFU/mL) was inoculated on the surface of the 3D models, and the microbial dynamics were observed for 24 h. Sequentially, CAP was used as combinatory treatment to investigate possible synergistic effects of NA and CAP. Additionally, L. monocytogenes and its knockout mutants (ΔsigB, ΔgadD) were cultured in Tryptone Soy Broth supplemented with 0.6% of yeast extract (TSBYE) until stationary phase of growth (10⁹CFU/ml). The gene regulator, SigB, has an essential impact on the environmental stress adaptation of L. monocytogenes for multiple type of environmental stresses such as thermal, acid, or osmotic stress. The gadD gene encodes a glutamate decarboxylase, an enzyme involved in the bacterial response to acid stress. Thereafter, cells were inoculated (10⁵ CFU/mL) in 2D models and challenged with GSE and CAP individually or in combination, to unravel the role of specific gene regulons of the NA/GSE response and/or adaptation.

RESULTS

The combined treatment of GSE/CAP showed promising microbial inactivation potential in all our 2D and 3D models. 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. However, the GSE antimicrobial effect was decreased in the multiphasic systems, resulting to only slight disturbance of the microbial growth. In contrast, CAP showed better antimicrobial efficacy on the surface of the multiphasic models as compared to the monophasic models. In general, the mutant strains of L. monocytogenes showed increased sensitivity to the individual and combined GSE and CAP treatments.

CONCLUSIONS

This work shows the potential of GSE, CAP, and their combination as sustainable antimicrobial strategies in the food industry. Furthermore, we show how the structural properties and biochemical composition of our 3D models can affect the antimicrobial efficacy of GSE, CAP or their combination against L. monocytogenes. Therefore, our results highlight the importance of exploring the potential mechanism of inactivation and including 3D solid/solid-like systems in food safety studies. This insight to the microbial dynamic and stress response is essential for the development of novel and robust sustainable hurdle approaches, i.e., application of GSE coupled with CAP treatment in a hurdle approach.

ACKNOWLEDGEMENTS

This work was supported by the Doctoral College and the Department of Chemical and Process Engineering of the University of Surrey, United Kingdom. E.V. is grateful to the Royal Academy of Engineering for an Industrial Fellowship and to the Medical Research Council UK for a New Investigator Research Grant (MR/V028553/1).

REFERENCES

1. Bahrami, A., Moaddabdoost Baboli, Z., Schimmel, K., Jafari, S. M. & Williams, L. Efficiency of novel processing technologies for the control of Listeria monocytogenes in food products. Trends Food Sci. Technol. 96, 61–78 (2020).
2. Velliou, E. G. et al. Heat adaptation of Escherichia coli K12: Effect of acid and glucose. Procedia Food Sci. 1, 987–993 (2011).
3. Costello, K. M. et al. The impact of food model system structure on the inactivation of Listeria innocua by cold atmospheric plasma and nisin combined treatments. Int. J. Food Microbiol. 337, 108948 (2021).
4. Costello, K. M. et al. The effect of ultrasound treatment in combination with nisin on the inactivation of Listeria innocua and Escherichia coli. Ultrason. Sonochem. 79, 105776 (2021).
5. El Kadri, H. et al. The antimicrobial efficacy of remote cold atmospheric plasma effluent against single and mixed bacterial biofilms of varying age. Food Res. Int. 141, 110126 (2021).
6. Costello, K. M. et al. Modelling the microbial dynamics and antimicrobial resistance development of Listeria in viscoelastic food model systems of various structural complexities. Int. J. Food Microbiol. 286, 15–30 (2018).
7. Kitsiou, M. et al. A Systematic Quantitative Determination of the Antimicrobial Efficacy of Grape Seed Extract against Foodborne Bacterial Pathogens. Foods 12, 929 (2023).
8. Sivarooban, T., Hettiarachchy, N. S. & Johnson, M. G. Inhibition of Listeria monocytogenes using nisin with grape seed extract on turkey frankfurters stored at 4 and 10°C. J. Food Prot. 70, 1017–1020 (2007).
9. Costello, K. M. et al. A multi‐scale analysis of the effect of complex viscoelastic models on Listeria dynamics and adaptation in co‐culture systems. AIChE J. 66, 1–15 (2019).
10. Velliou, E. G. et al. The effect of colony formation on the heat inactivation dynamics of Escherichia coli K12 and Salmonella typhimurium. Food Res. Int. 54, 1746–1752 (2013).