(436g) "Invited Talk" the Response of Food-Related Pathogens to Natural Antimicrobials Coupled with Non Thermal Novel Processing Technologies in Structured Food Model Systems

High quality foods retaining characteristics such as taste, texture, natural colouring and nutritional content, that have been minimally processed and/or are ready-to-eat (e.g. fresh smoothies, fruit juices, fresh fruit and vegetables, cheeses and dairy products), are in increasing demand.¹ Traditional microbial inactivation methods such as pasteurization or heat sterilization cannot meet these demands as the properties of the food are often changed, and some food products cannot be processed with these methods. As such, the food industry is increasingly interested in novel non-thermal processing technologies (e.g. ultrasound, cold pasteurisation, cold atmospheric plasma, natural antimicrobials), which are milder than traditional thermal methods and can maintain the desired fresh-like food characteristics.^2–4 Furthermore, processing efficiency may be increased by synergistically combining non-thermal inactivation methods, as the combined technologies can act as hurdles for microbial growth.^5,6 However, the efficacy and mechanisms of action of combined treatments such as natural antimicrobials and non-thermal processing (e.g. ultrasounds), remains unclear.⁷ These technologies could instead represent a mild, sub-lethal stress which could result in stress adaptation, post-treatment survival, and the potential development of antimicrobial resistance.^6,8

Most studies conducted thus far on the inactivation of food-related pathogens by natural antimicrobials and/or ultrasound are conducted in liquid broths, or in/on specific food products e.g. fruit juice, lettuce leaves ^2,9. However, the chemical composition and rheological/structural properties of foods can vary significantly, potentially impacting the efficacy of such treatments, and studies in real foods are informative only for the specific product studied.¹⁰ Natural antimicrobials may be produced in situ by natural microflora (e.g. nisin-producing Lactococcus lactis) or added artificially, thus the production rate, quantity and efficacy of natural antimicrobials may also be affected. Furthermore, many factors are known to influence the efficacy of ultrasound treatment i.e. system frequency, viscosity, sonication time, and equipment set-up.^11,12

A fundamental multi-frequency study into the inactivation effect of ultrasound, alone or combined with nisin, against food-related pathogens in food model systems of controlled rheological and structural complexity is currently lacking: the present work aims to address this gap.

Materials and Methods:

Food model systems were prepared using Tryptic Soy Broth supplemented with 0.6% Yeast Extract (TSBYE), with 0, 0.1, 0.3 or 0.5% Xanthan gum (XG) for a range of viscoelastic immersed systems.^3,4 XG is widely used in the food industry and is stable at a range of temperatures¹³ thus was selected for use in the present study. The rheological stability and characteristics of all XG systems was tested by conducting dynamic oscillatory measurements to assess their viscoelastic behaviour.

Ultrasound inactivation of L. innocua or E. coli stationary phase cells, with/without nisin, was investigated in 0 – 0.5% XG systems (44 kHz, 500 kHz, 1 MHz) with a constant power of 30W and a treatment time of up to 30 min. Cells were grown planktonically in TSBYE and added at 10⁶CFU/mL into the viscoelastic systems before inactivation treatments. For nisin-containing systems, nisin was added as a treatment for 30 minutes before or immediately after ultrasound treatment, at a final concentration of 35 IU/mL (a sublethal concentration which allows the observation of combined effects with ultrasound).

Inactivation kinetics were monitored, and morphological changes of cells following inactivation treatments were studied using scanning electron microscopy (SEM). Samples were fixed in a 3% formaldehyde solution and dehydrated in an ethanol/water series before imaging. Flow cytometry methods were also employed to identify the dead and sublethally injured populations using Propidium iodide (PI) and bis-(1,3-dibutylbarbituric acid) trimethaine oxanol (BOX) stains for dead and injured cells respectively.

Results and Discussion:

The inactivation efficacy of ultrasound and nisin, alone or combined, was observed to depend on many factors including themicrobial species, ultrasound frequency, treatment time, treatment order, and system structure.

No inactivation by ultrasound was observed for Listeria at any of the conditions tested, although an effect of nisin was observed (as expected, as nisin is known to act on gram positive bacteria). For E. coli, inactivation by ultrasound was observed at 500 kHz but not at the other frequencies investigated, with greater reduction in the surviving population as the treatment time was increased. As the concentration of XG was increased i.e. as the system viscosity increased, a reduction in inactivation efficacy was observed i.e. the system structure impacts significantly inactivation by ultrasound. Finally, a combined effect of nisin was observed for E. coli inactivation (despite nisin acting only on gram positive cells), but only when nisin was applied before ultrasound treatment.

SEM imaging and flow cytometry analysis of samples following ultrasound and/or nisin treatments show significant differences in cell death, injury and survival on a cellular level for different orders and combinations of treatments, as observed in the inactivation kinetics. Furthermore, ghost cells (i.e. dead cells with an intact cell membrane but no cellular contents) are observed following ultrasound treatment, suggesting that the mechanism of inactivation by ultrasound in this system is physical rather than chemical and involves the partial destruction or disturbance of the cell membrane, causing efflux of the cell contents.

Significance and Impact:

This work presents a systematic fundamental study on the efficacy of non thermal processing and natural antimicrobials as individual or combined treatments on the inactivation of Listeria or E. coli in/on food systems of different physiochemical and structural complexity. This study sheds light on the combined efficacy and potential mechanisms of novel processing techniques for the inactivation of food-related pathogensin structured food models, and highlights the importance of accounting for structural effects when designing novel inactivation processes for the food industry.

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), the National Biofilm Innovation Centre, R-Processing Ltd and the Royal Society. E.V. is grateful to the Royal Academy of Engineering for an Industrial Fellowship.

References:

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