Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae in relation to membrane permeabilization and subsequent leakage of intracellular compounds due to pulsed electric field processing

Abstract

Membrane permeabilization, caused by pulsed electric field (PEF) processing of microbial cells, was investigated by measurement of propidium iodide (PI) uptake with flow cytometry. Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae was determined by viable counts, and leakage of intracellular compounds, such as ATP and UV-absorbing substances, was measured in the extracellular environment. Electrical field strength and pulse duration influenced membrane permeabilization of all three tested organisms of which S. cerevisiae was the most PEF sensitive, followed by E. coli and L. innocua. It was shown by viable counts, PI uptake and leakage of intracellular compounds that L. innocua was the most resistant. Increased inactivation corresponded to greater numbers of permeabilized cells, which were reflected by increased PI uptake and larger amounts of intracellular compounds leaking from cells. For E. coli and L. innocua, a linear relationship was observed between the number of inactivated cells (determined as CFU) and cells with permeated membranes (determined by PI uptake), with higher number of inactivated cells than permeated cells. Increased leakage of intracellular compounds with increasing treatment severity provided further evidence that cells were permeabilized. For S. cerevisiae, there was higher PI uptake after PEF treatments, although very little or no inactivation was observed. Results suggest that E. coli and L. innocua cells, which took up PI, lost their ability to multiply, whereas cells of S. cerevisiae, which also took up PI, were not necessarily lethally permeabilized.

Introduction

The occurrence of spoilage and/or pathogenic microorganisms in foods is of concern to the entire food production chain. Thermal processing is the most common way to maintain food safety and to increase shelf life by inactivating pathogenic and spoilage microorganisms. However, the heat treatment triggers unwanted reactions and alters the food quality by causing loss of flavors, nutrients and vitamins. Today, not only long shelf life, but also other aspects of food quality are important to consumers. Consequently, nonthermal processing technologies are under intense research to evaluate their potential as an alternative to conventional heat treatment and to eliminate, or at least minimize, the quality degradation of foods by processing.

Pulsed electric field (PEF) processing is a nonthermal technique, which involves the application of short pulses of high electric field intensity to foods placed between two electrodes. The inactivation effect of PEF has been demonstrated on several microorganisms (Hülsheger et al., 1983, Pothakamury et al., 1996, Sale and Hamilton, 1967, Wouters et al., 1999, Zhang et al., 1994a, Zhang et al., 1994b). Shelf-life studies on a range of food products (Dunn and Pearlman, 1987, Jia et al., 1999, Jin and Zhang, 1999, Evrendilek et al., 1999, Evrendilek et al., 2000) in combination with scaling up of the process to a commercial level (Min and Zhang, 2003, Min et al., 2003a, Min et al., 2003b) have enhanced the interest by the food industry and today, the basis for industrial implementation of this technology exists.

The underlying mechanism of inactivation of microorganisms is still not fully understood. The most widely accepted theory involves free charges, which accumulate at both membrane surfaces when a cell is exposed to an electric field, resulting in the formation of a transmembrane potential. Membrane destruction, in the form of increased permeability of the membrane and/or membrane breakdown, occurs when the membrane potential induced exceeds a critical value of electrical field strength (Tsong, 1990). When the electrical field is turned off, the membrane returns to its initial state. Only when the size and number of pores become large in relation to the membrane surface, is irreversible breakdown associated with mechanical destruction of the cells and microbial inactivation (Zimmermann, 1986).

In a previous study by Aronsson et al. (2001), examination with transmission electron microscopy (TEM) could not confirm extensive membrane rupture; however, these results did not rule out the presence of permeabilization or other membrane damage, which could not be revealed by TEM (Chang and Reese, 1990, Tsong, 1990). Wouters et al. (2001) demonstrated membrane permeabilization on Lactobacillus plantarum cells exposed to PEF by studying the uptake of propidium iodide (PI) of individual cells. This compound, which becomes fluorescent when binding to nucleic acids, was shown to be a good indicator of membrane integrity, since it is not normally taken up by intact cells (Breeuwer and Abee, 2000). Furthermore, leakage of intracellular substances can also be used as an indicator of membrane damage (Hurst, 1977) and leakage of UV-absorbing substances has been demonstrated on PEF exposed cells of several researchers (Grahl and Märkl, 1996, Harrison, 1996, Simpson et al., 1999).

In the present study, the relationship between membrane permeabilization and the level of inactivation was evaluated to gain further insight into the mechanisms of inactivation of vegetative microbial cells. Membrane permeabilization was demonstrated by PI uptake of individual cells measured by flow cytometry, and measurement of intracellular substances (ATP and UV-absorbing substances) in the extracellular environment. Three microbial species, Escherichia coli, Listeria innocua and Saccharomyces cerevisiae, with variation in cell morphology, were used to determine the effects of different process conditions (electrical field strength and pulse duration) on membrane permeabilization and inactivation.