(22c) The Role of Sigb on the (stress) Response of the Pathogen Listeria Monocytogenes to Novel Sustainable Processing Technologies.

Consumers, in the recent years, are seeking minimally processed foods that do not contain chemical preservatives or antibiotics and are produced in a more sustainable way. Novel processing technologies i.e. ultrasound, cold atmospheric plasma (CAP), high hydrostatic pressure and natural antimicrobials are such alternative approaches that can be used as mild decontamination methods. However, as these treatments are less harsh than classic heat sterilisation, the desirable microbial inactivation (5 log CFU/ml) might not occur, leading to contaminated and unsafe products. 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 combinatory treatments (the so-called hurdle approach) and their microbial stress response should be investigated².

The principle of the hurdle technology approach on microbial safety, is the utilization of two or more methods/approaches/processes to enable microbial inactivation and consequently to ensure food safety³. A combination of them could lead to synergistic and/or complementary actions against bacteria. For example, one technology/treatment could be used to damage or increase the permeability of the bacterial cell wall and another one to interfere with the intracellular metabolism⁴. To develop novel hurdle technology approaches, it is important to understand the mode of action and the microbial stress response for each treatment. For example, it is known that the gene regulator, SigB, plays a major role in the environmental stress adaptation of L. monocytogenes for multiple type of environmental stresses such as thermal, acid, or osmotic stress. A recent study has found that SigB can be overexpressed as a protective measure, transforming the cells to hyperestistant but susceptible to oxidative stress⁵. The aim of our work is to develop novel green hurdle technology approaches, targeting the weakness of L. monocytogenes stress response. More specifically, acid stress, Cold Atmospheric Plasma (CAP) and the grape seed extracts (GSE-which have antimicrobial properties) were assessed individually and/or combined.

METHODS

L. monocytogenes and its mutant (ΔSigB) was cultured in Tryptone Soy Broth supplemented with 0.6% of yeast extract (TSBYE) until mid-exponential phase of growth (10⁵ CFU/ml). Thereafter, mid-exponential cells were treated with different stress factors, i.e., acid, grape seed extracts or CAP. For the mild acid stress treatment, the cells (cell pellets) were exposed in TSBYE with 0.35ml hydrochloric acid (HCl, 1M) at pH 4.5. The cells were challenged for 30min in this mild acid condition. For the grape seed extract (GSE) treatment the cells were challenged for 2h in TSBYE with 1% GSE. For the CAP treatment 300μl of each tube (10⁵ CFU/ml) were placed in a 12 well plate and CAP treatment was applied using flow rate of 4ml/min for 1 to 3 minutes. Two different combinatory treatments were applied: 1) hydrochloric acid and CAP treatment and 2) GSE and CAP treatment. The viable and sub-lethally damaged cells were enumerated (using the spread plate technique in TSAYE and PALCAM agar) after each individual and combinatory treatment.

RESULTS

Both sets of combined treatments induced a reduction in the cell concentration. In contrast, individual treatments did not affect, at all or as much, the cell survival. The most effective hurdle approach was the combination of GSE and CAP treatment. This is due to the increased numbers of sub-lethal damaged cells caused by the individual GSE treatment. The increased sensitivity of L. monocytogenes to oxidative stress was clearer for 3 minutes of CAP treatment, when the combined treatments were applied, as cells post-treatment were undetectable (<1 log CFU/ml). Lower synergistic effect of the treatments was observed in the mutant strain (SigB deletion) validating the initial hypothesis of the presence of SigB linked with L. monocytogenes susceptibility to oxidative stress.

CONCLUSIONS

This work shows the importance of SigB on L. monocytogenes to novel sustainable processing approaches and their combination. This insight to the microbial stress response is essential for the development of novel and robust green/sustainable hurdle technology 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:

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