Elsevier

Bioelectrochemistry

Volume 130, December 2019, 107338
Bioelectrochemistry

Mechanisms of enhanced bacterial endospore inactivation during sterilization by ohmic heating

https://doi.org/10.1016/j.bioelechem.2019.107338Get rights and content

Highlights

  • Investigation of inactivation mechanisms of bacterial endospores by electric fields

  • Use of B. subtilis spores and mutants lacking components involved in resistance

  • Up to 2.4 log10 of additional inactivation by the electric field (wild-type spores)

  • Electric field effects predominantly on the spore core and not outer spore layers

Abstract

During ohmic heating, the electric field may additionally inactivate bacterial endospores. However, the exact mechanism of action is unclear. Thus, a mechanistic study was carried out, investigating the possible target of electric fields inside the spore. Bacillus subtilis spores were heated by conventional and ohmic heating in a capillary system under almost identical thermal conditions. Wild-type (PS533) spores were used, as well as isogenic mutants lacking certain components known for their contribution to spores' heat resistance: small-acid soluble proteins (SASP) protecting DNA (PS578); the coat covering the spore (PS3328); and the spore germination enzyme SleB (FB122(+)). Treatment-dependent release of the spore core's depot of dipicolinic acid (DPA) was further evaluated. Up to 2.4 log10 additional inactivation of PS533 could be achieved by ohmic heating, compared to conventional heating. The difference varied for the mutants, with a decreasing difference indicating a decreased effect of the electric field and vice versa. In particular, mutant spores lacking SASPs showed a behavior more similar to thermal inactivation alone. The combination of heat and electric field was shown to be necessary for enhanced spore inactivation. Thus, it is hypothesized that either the heat treatment makes the spore susceptible to the electric field, or vice versa.

Introduction

Ohmic heating is an alternative thermal process increasingly used in the food and life science industries, as it is capable of overcoming heat transfer limitations usually present during conventional thermal processing [1,2]. Common applications of ohmic heating include processing and preservation of meat, whole vegetables and fruits, as well as corresponding juices and preparations, milk and dairy products, baking and cereal technology, distillation, waste and wastewater treatment [1].

Most heating technologies rely on hot surfaces and conduction to deliver thermal energy to a product, and thus result in a temperature gradient within the product, leading to overprocessed outer layers, while the desired process temperature, e.g. for pasteurization or sterilization, is just reached in the geometric center of the product [3]. This results in an decrease in product quality, accompanied by unnecessary degradation of valuable, thermo-labile food compounds, the formation of so-called neoformed processing contaminants like furan, as well as a possible undesired organoleptic alteration [4]. Ohmic heating, on the other hand, is based on current flow through an electrically conductive matrix, causing an immediate and homogeneous temperature increase due to the electrical resistance of the matrix. Being a volumetric thermal process, ohmic heating is especially suitable for products which are difficult to heat by traditional methods, due to a tendency to adhere and burn on hot surfaces or due to slow heating behavior, e.g. highly viscous or chunky foods [1,2].

Bacterial endospores (spores) are dense dormant structures formed by a variety of Bacillus and Clostridium spp., and are able to withstand most antimicrobial treatments to a certain extent [5,6]. Thus, inactivation of bacterial endospores within the context of a sterilization treatment necessitates severe process intensities [3]. The reason for high spore resistance is due to the layered structure of bacterial endospores, with several components contributing to their resistance against a variety of antibacterial treatments [7]. In case of wet heat treatment, the following spore properties are of special importance: saturation of DNA inside the core by α/β-type small acid soluble proteins (SASPs), the relatively high amounts (up to 10% dry weight) of dipicolinic acid (DPA) inside the core, the low water content of the core, and the core's high content of divalent metal ions [[5], [6], [7], [8], [9]].

Considering ohmic heating of bacterial endospores, some studies suggest effects that go beyond the impact of heat alone, due to the presence of an electric field [10,11]. Similar results have been published for pulsed electric fields (PEF) treatment, which is similar to ohmic heating, but uses higher voltages, lower pulse durations and frequencies [12,13]. However, so far most of the reported studies give only descriptive results showing an increased inactivation effect by ohmic heating, but not investigating the underlying mechanism of this effect in detail.

Thus, the present study focused on mechanisms of inactivation of bacterial endospores by ohmic heating. In this context, the ohmic heat killing of B. subtilis spores was studied, and of isogenic mutant strains lacking components known to contribute to spore resistance, in particular the spore coat, the α/β-type SASPs, and the cortex lytic enzyme SleB which is important in spore germination. DPA release during spore inactivation by conventional and ohmic heating was also compared.

Since comparison of results obtained from conventional and ohmic heating processes can be difficult, due to distinctly different heat transfer mechanisms [10], the present study used a specifically designed experimental setup, enabling the most possible treatment homogeneity for heat transfer mechanisms in ohmic and conventional heating by using thin, conductive and non-conductive glass capillaries immersed in an ohmically heated liquid. Use of this equipment allowed spore treatment by conventional and ohmic heating using equivalent temperature-time profiles.

Section snippets

Materials and methods

During this study, a variety of wild-type and mutant B. subtilis spores were exposed to wet heat, using almost identical temperature-time profiles for both conventional and ohmic heating. Samples were analyzed for survivors before and after treatments and the results from the spores of the various strains were compared (Scheme 1). In order to maximize the non-thermal influence of the electric field, the field strength had to remain constant for all trials. Thus, the implementation of a holding

Characterization of capillary heating

The different preparation of the capillaries enabled conventional heating inside the non-conductively sealed capillaries and ohmic heating inside the conductively sealed capillaries. Having a higher conductivity of the alginate plugs, compared to surrounding liquid and spore suspensions, further ensured that ohmic heating took place inside these capillaries at all times, independent of the individual treatment temperature (Fig. 1). This was confirmed by fact that the temperature curve of ohmic

Experimental differentiation of thermal and electric field effects

The differentiation of thermal and electric field effects for ohmic heating is particularly challenging, as there is an immediate interconnection of both effects and a complete decoupling of heat and electric field is almost impossible [1,10]. Furthermore, the comparison of ohmically heated and conventionally heated samples is difficult considering the experimental design, due to the distinctly different nature of the individual heat transfer mechanisms: conventional heating in a batch process

Conclusion

The present study shows an additional inactivation effect of ohmic heating on B. subtilis endospores, as compared to conventional heat treatment. The use of isogenic mutant spores lacking important individual spore components revealed that the target of the electric field is predominantly within the spore's core. Since a combination of heat and electric field was necessary for the efficacy of the electric field during ohmic heating, it was hypothesized that changes in the spore core due to the

Author contributions

FS, TP, KR, PS, SKS, and HJ contributed to the conception and design of the experiments. PS provided the spore suspensions. FS conducted the experiments, tasks related to microbiological analyses were carried out together with TP. FS acquired and analyzed the data and wrote the manuscript. All authors gave input to the manuscript and contributed to the final revision prior to submission.

Declaration of Competing Interest

The authors declare no conflicts of interest.

Acknowledgements

The authors thank Dr. Ahmed Youssef and Dr. Bala Balasubramaniam (The Ohio State University) for access to the laboratories at the Ohio State University, and Brian F. Heskitt (The Ohio State University) for assistance establishing the experimental setup. Further, we acknowledge Dr. Michael Gänzle (University of Alberta, Canada) for input on the DPA measurements. This study was supported by the Austrian Research Promotion Agency (FFG Project No. 859077, volTECH) and by EQ-BOKU VIBT GmbH (Center

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