Based on the consumers' demand for fresh, durable and safe foods it is obligatory for the food industry to preserve their products at the best. To obtain products with longest possible shelf-lives, inactivation of spores is inevitable. This is usually achieved by thermal sterilization, a process mostly realized through autoclaving at 121°C for approximately 15 min. This eliminates all microorganisms and spores although valuable food ingredients are destroyed (Jaeger et al., 2014a). Therefore, a innovative preservation method is the application of pulsed electric fields (PEF), which might by suitable for sterilization of liquid food (Siemer et al., 2014a,b).

Currently, PEF processing is applied in the food industry for enhancing mass transport, increasing the yield of secondary metabolites and juice production, as well as, for pasteurizing liquid foods without applying heat (Heinz et al., 2001; Saldana et al., 2010; Knorr et al., 2011; Jaeger et al., 2012). Energy inputs of 10–100 J kg⁻¹ and pulse widths in the range of 1–100 μs are applied, with time between pulses being considerably longer than the actual pulse time (Knorr et al., 2011; Jaeger et al., 2014b). Although PEF treatment can principally be a non-thermal process, an energy dependent increase in temperature of the treated medium will occur (Van Loey et al., 2001). This phenomenon is termed joule effect (Spilimbergo et al., 2003). A great advantage of the PEF technology is that thermal impact on foods can be reduced. This may lead to fresh-like products with more natural color, texture, taste, and a higher concentration of secondary plant products while the same shelf life can be obtained. However, only a limited inactivation of enzymes is achievable with this treatment, although this problem can be solved by a combination of PEF and other enzyme-inactivating processes (Van Loey et al., 2001; Buckow et al., 2012; Meneses et al., 2013).

PEF treatment is only suitable for liquid foods and its effectiveness is strongly influenced by the electrical conductivity of the product (Devlieghere et al., 2004). The extent of the warming during the treatment depends on the energy input (Van Loey et al., 2001). To achieve consistent results it is important that the distribution of the electric field and consequently the distribution of the temperature field in the treatment chamber are as homogenous as possible (Meneses et al., 2011a; Knoerzer et al., 2012). Numerical simulations of a co-linear chamber done by Meneses et al. (2011a) revealed that temperatures directly on the inside of the electrodes were significantly higher than in-between. As a matter of fact the temperature peaks were about 30–50 K higher than the average temperature. Furthermore, the temperature at the electrode downstream was significantly higher than those at the upstream electrode. The temperature peaks showed considerable maxima and were only obtained for very short times and afterwards declined exponentially (Meneses et al., 2011a).

The effectiveness of PEF treatment concerning inactivation of microorganisms, depends on certain parameters of the treated liquid. One of the most significant parameters is the conductivity σ [S cm⁻¹]. The higher ion concentration a liquid contains, the higher is its conductivity. In terms of PEF treatment, high conductivities are counterproductive, since thereby only smaller field strengths can be built up and consequently critical field intensities for cell permeabilization are more difficult to attain (Jayaram, 2000; Toepfl et al., 2007). Furthermore, conductivity depends on temperature, whereas an increase of temperature evokes an increase of electrical conductivity. As temperature rises during PEF treatment higher conductivities will be implicated.

Up to the present the inactivation of vegetative microorganisms by PEFs is well understood and numerous publications on this topic have been published (Toepfl et al., 2007; Jaeger et al., 2014b). However, only limited data on the impact of PEF treatment on bacterial endospores has been published. Several articles showed that PEF processing does not induce any spore inactivation (Wouters et al., 2001; Devlieghere et al., 2004; Knorr et al., 2011), whereas other publications describe a limited inactivation. Other authors have even showed up to 5 log of spore inactivation Spilimbergo et al. (2003) observed 0.5 log₁₀ inactivation of B. cereus spores with a field intensity of 25 kV cm⁻¹, pulse number of 20 at 40°C. A pulse number and field intensity dependent inactivation of B. cereus and B. subtilis spores at maximally 25°C was described by Marquez et al. (1997). At field strength of 50 kV cm⁻¹ and 30 applied pulses 3.5 log₁₀ of B. subtilis spores were inactivated. A pulse number of 50 led to a 5 log₁₀ inactivation of B. cereus spores. Most of the published data were obtained under different treatment conditions and set-ups and thus the comparison between them is not possible. However, a certain level of spore inactivation could be achieved if the necessary harsh conditions are applied.

Therefore, the impact of different PEF parameters on microbial inactivation as well as the synergistic effect of the electric field and heat was investigated in this study.

To characterize the used PEF system and to ensure reproducible sampling, residence time spectra for different flow rates were determined. The tested flow rates of 4.8 and 7.1 l h⁻¹ had nearly no impact on the residence time distribution (Figure 3) and hence 7 min was selected as sampling time after parameter or medium change was done.

During the trials, energy inputs were successively increased for each experimental trial until electrical breakdowns occurred. This phenomenon emerged earlier for higher preheating temperatures and higher electrical conductivities. The used carrot juice exhibited an electrical conductivity of 7.73 mS cm⁻¹ and therefore PEF processing was more difficult due to earlier appearing electrical breakdowns. For this reason this medium was processed with pulse widths of only 4 μs (Table 2). It was also noticeable that in carrot juice the actually rectangular pulse shape changed to an exponentially declining waveform.

To estimate if a combined PEF-thermal treatment has a beneficial effect on spore inactivation liquid media, D- and z-values for each spore strain in each media were determined (Table 1). With the used method, we could confirm a D_{121° C}-value of 1.6 min given in certificate for G. stearothermophilus. The reason for variations of the D- and z-values for B. subtilis and G. stearothermophilus in the different treatment media, could be explained due to matrix effects. The small solid sediment particles that were present in the carrot juice most probably exerted a protective effect on the spores. The reason for this behavior is the lower heat conductivity of the solids in comparison to the aqueous continuous phase. The deteriorated inactivation of the B. subtilis spores in milk can be explained by a protective effect of the milk fat (Kessler, 2002). However, the obtained D- and z-values correspond to literature data (Reineke et al., 2011). Dogan et al. (2009) reported a D_{121° C} of 0.7 min and a z-value of 13°C in Ringer's solution, whereas Somavat et al. (2012) determined a D_{121° C} of 2.53 min, a D_{130° C} of 0.6 min as well as a z-value of 7.42°C in tomato soup.

In contrast to the nearly linear progress of spore inactivation kinetics in thin glass capillaries, the kinetics for a combined PEF-thermal treatment were non-linear. The shoulder formation in Figures 4, 5 can be explained by the disaggregation of spore agglomerates (Mathys et al., 2007). The occurring tailing in some kinetics can be explained by different resistances of spores among a certain population and the accumulation of more resistant spores toward the end of the treatment. However, in case of the executed experiments the more plausible explanation is that the electrical breakdowns at high energy inputs, which occurred at the end of almost every treatment, led to an altered flow of the electric current through the electrode-surrounding air rather than through the treatment medium and therefore lower inactivation rates were achieved.

Further it can be concluded, that within the tested range of PEF-parameters no parameter solely executed a significant influence on the inactivation of spores (Table 2, Figures 4, 5). However, other, non-electrical parameters like preheating temperature or the respective volume flow had an influence on spore inactivation. Moreover, the pH-value can alter significantly during PEF treatment (Meneses et al., 2011b) and therefore an additional inactivation of spores can occur. Similar findings were reported by Siemer et al. (2014a). They reported a 1.6 log₁₀ inactivation of B. subtilis spores using 167 kJ kg⁻¹ in Ringer's solution with pH 4 compared to a 0.6 log₁₀ reduction in Ringer's solution with neutral pH. Further, under identical process conditions (80°C preheating and 9 kV cm⁻¹ electric field strength) the addition of 10% sugar permitted to reduce the needed energy input to achieve a 3 log₁₀ reduction from 178 to 146 kJ kg⁻¹.

Somavat et al. (2012) showed a dependence of spore inactivation on electric parameters for ohmic heating. A higher inactivation was found for G. stearothermophilus spores treated at 10 kHz compared to samples treated at 60 Hz and therefore revealed a significant influence of the frequency. It is possible that these results can also be applied for PEF treatment, although this hypothesis was not proven by the executed experiments and therefore has to be verified with specific tests.

However, the dominant impact on spore inactivation was the preheating and maximum treatment temperature (Table 2, Figures 4, 5). Whereas, Bermúdez-Aguirre et al. (2012) reported a higher resistance of PEF treated B. cereus spores in milk at ambient temperatures compared to a preheating to 40°C. Temperatures of 50°C resulted in an increased inactivation of 3 log₁₀. This treatment was accomplished at field intensities of 35 kV cm⁻¹ and 10 pulses. Besides, a utilization of skim milk led to higher inactivation rates compared to a treatment in whole milk (Bermúdez-Aguirre et al., 2012). An explanation for this behavior could be a possible spore-protective effect of the milk fat. Further, Somavat et al. (2012) described synergistic effects of electricity and heat to G. stearothermophilus spores by use of ohmic heating at 10 and 60 Hz. At this a maximum inactivation was achieved by an application of 130°C and 60 Hz.

Considering the contrastive statements mentioned above it can generally be stated that spores are significantly more resistant to PEF treatment than vegetative cells. A possible reason for this phenomenon could be the considerably lower electrical conductivity of spores due to the low water content and immobilized ions in the core, as well as their much smaller cross-section, which makes the spores less susceptible to the electric field (Wouters et al., 2001; Jaeger et al., 2014b). However, since application of PEFs is always associated with a temperature dependent heating of the treatment medium, the temperature field in the PEF chamber has always to be taken into account (Meneses, 2011).

Since the aim of this study was to inactivate spores, relatively high energy inputs were necessary and consequently high temperatures emerged (Figure 6). Due to the utilization of a co-linear treatment chamber, inhomogeneous temperature fields occurred with considerable temperature differences between the wall and the center of the treatment chamber (Meneses et al., 2011a). In UHT processes for the preservation of milk temperatures of approximately 140°C are used and applied for circa 4 s (Kessler, 2002). For a solely thermal sterilization of liquid foods by PEFs similar temperature-time regimes have to be achieved. In case of the examined data points temperature peaks of maximum 60 K above the preheating temperature could be achieved between wall and center of the treatment chamber, leading to temperature increases of the treatment liquid of averagely 20 K (Figure 6).

To verify if a combined PEF-thermal treatment has a beneficial effect on spore inactivation, numerical simulations were used, which coupled the reference data for a pure thermal inactivation with the spatial temperature field in the PEF-chamber. The results presented in Table 3 shows no impact on B. subtilis inactivation but an additional or possibly synergistic effect of the PEF treatment on the inactivation of G. stearothermophilus spores.

To consider the possible deviations of the simulated temperature with the measured temperatures we assumed the simulation may fail from reality within ±2°C. Thus, the inactivation was in some cases higher than the experimental one and the calculated theoretical inactivation varies enormous at high energy inputs. Hence, in some cases it was not possible to distinguish between PEF and thermal inactivation as the theoretical inactivation was higher than the experimental one.

For B. subtilis spore inactivation the specific energy input was significantly higher than for G. stearothermophilus, but much lower inactivation rates were achieved (Table 3), even if the additional 105 kJ kg⁻¹ needed to reach 95°C pre-heating temperature are taking into account.

Consequently it is likely that the higher temperatures for the inactivation of G. stearothermophilus contributed to its susceptibility toward the electric field. It could also be possible that the structure of G. stearothermophilus spores is responsible for its greater electrical vulnerability but this is unlikely due to significant similarities of both used spore types (Setlow, 2007). The obtained results imply that the inactivation of bacterial endospores by a combined PEF-thermal process is not solely based on thermal effects, so that it can be concluded that the electric field generates an additional inactivation. This was also reported by Siemer et al. (2014b), who reported an increased total spore inactivation for higher energy inputs (up to 195 kJ kg⁻¹) for a combined PEF-thermal treatment. After applying 195 kJ kg⁻¹ to B. subtilis spores suspended in Ringer's solution (pH 7) a 4.4 log₁₀ inactivation was achieved, which could be separated in 1.15 log₁₀ of thermal and 3.25 log₁₀ of PEF related inactivation. These data are, with regard to the high energy input and the achieved inactivation, in agreement with our data (Table 3).

However, the exact mechanism of the accelerated spore inactivation due to the electric field is completely unclear. Somavat et al. (2012) hypothesized that the accelerated inactivation is achieved by the rotation of temperature-released polar spore components in the electric field. However, it is most likely that this explanation is inappropriate, since it is generally accepted that temperatures that trigger the release of spore components are so high that at this point essential spore components are already denatured so that the spore is already lethally damaged (Coleman et al., 2007). A possible explanation for this synergism could be the greater resistive heating of the spore's core due to its very low electrical conductivity (Bassi et al., 2012) and higher electric resistance. Consequently the core could undergo increased heating in comparison to the surrounding medium. Nevertheless, this theory cannot explain the high inactivation at higher temperatures that was described above. Another reason for this phenomenon could be a possible detachment of ions from the inner spore membrane and the subsequent migration of these ions through the spore's core. This could lead to a possible reduction of the inner spore membrane's barrier function and therefore to a greater susceptibility of the spore. However, the above-mentioned explanations are only hypotheses. The investigation of the exact spore inactivation mechanism by PEFs needs further detailed research.

In conclusion, it can be stated that a combined application of PEFs and thermal treatment can lead to an accelerated inactivation of endospores in comparison to a pure thermal inactivation in an identical temperature field. It is most likely that the additional sporicidal effect of the electric field is temperature dependent, although the reason for this phenomenon is not clarified yet. To investigate this, quantification of possibly leaked dipicolinic acid from the spore core, flow cytometry and high resolution microscopy might be suitable tools. Additionally it is necessary to analyze the influence of the required high energy inputs on other food constituents in order to determine whether desired or undesired modifications have emerged, to use this treatment as an alternative technique of ultra-high temperature processing for liquid foods with a high pH-value like milk, vegetable juices or soups.