Saccharomyces bayanus (CECT 1969) and Brettanomyces bruxellensis (CECT 11045) strains used in this study were supplied by the Spanish Type Culture Collection (CECT) and kept on slants of potato dextrose agar (PDA) (Oxoid, Basingstoke, Hampshire, United Kingdom) and Brettanomyces agar (Scharlab, Barcelona, Spain), respectively. Precultures were obtained by inoculating a single colony into a 10-mL test tube containing 5 mL of Sabouraud Dextrose (SD) broth (Oxoid), followed by incubation for 24 h at 25°C.

To obtain a S. bayanus suspension, a commercial red grape must (GREIP, PepsiCo, Vitoria, Spain) supplemented with 70 g/L of sucrose (Oxoid) was subjected to an alcoholic fermentation procedure for 9 days at 25°C to achieve a final concentration of 220 g of sucrose per liter, which corresponds to a potential ethanol content of 12.9% (v/v). The supplemented grape must was inoculated with S. bayanus at a concentration of 2 × 10⁴ UFC/mL. PEF inactivation experiments on S. bayanus were conducted in the same wine after fermentation. On the other hand, to obtain B. bruxellensis cells for PEF inactivation experiments, 50 mL of SD was incubated 5 days at 25°C under agitation after inoculation with the B. bruxellensis preculture (1.8 × 10⁴ UFC/mL). Cells destined for PEF inactivation experiments were recovered by centrifugation (10.000 g for 4 min) and suspended in the wine after removing S. bayanus cells by centrifugation (10.000 g–10 min). The wine’s pH and conductivity were 3.1 and 1.3 m/cm, respectively.

A commercial VITAVE PEF generator (VPGU 20/500/9, Vitave, Prague, Czech Republic) with a maximum voltage of 20 kV and a current of 500 A was used. For inactivation experiments, wine containing the yeast strains was pumped through a parallel titanium electrode treatment chamber (0.4 cm gap × 3 cm length × 0.5 cm width) at a flow of 5 L/h, corresponding to a residence time of 0.43 s. Wine was tempered to 20°C using a heat exchanger located immediately before the treatment chamber. A cooling system located after the treatment chamber cooled the wine’s temperature to under 20°C in less than 5 s. Actual voltage during treatments was measured using a high-voltage probe (Tektronix, P6015A, Wilsonville, Oregon, United States) connected to an oscilloscope (Tektronix, TDS 220). Inlet and outlet temperatures were measured using type K thermocouples inserted in the circuit (Ahlborn, Holzkirchen, Germany).

Square pulses of 5 µs width at different electric field strengths (10, 15, 20, and 25 kV/cm) were delivered at different repetition rates up to 200 Hz to apply treatments of different duration (number of pulses x pulse width) ranging from 25 to 750 µs, which corresponded to outlet temperatures of 30, 35, 40, 45, 50, 55, and 60ºC ± 2.0°C. Total specific energies of the applied treatments ranged from 19.5 to 156 kJ/kg. The total specific energy (W) of treatments was estimated from the temperature increase during pulses delivering assuming adiabatic conditions (Heinz et al., 2001) and according to the average values of the specific heat capacity of wine obtained from Genc et al. (2017).

A stock solution of 25 g/L of SO₂ was prepared from potassium bisulfite (Sigma, Burlington, MA, United States). Immediately after the PEF treatments, the corresponding volume of the stock solution was added to the untreated and PEF-treated wine samples to obtain final concentrations of 10, 25, and 50 ppm of SO₂.

The viability of S. bayanus and B. bruxellensis was evaluated by plate counting 30 min and 24 h after the application of PEF treatments. S. bayanus was recovered for 48 h at 25°C onto PDA. On the other hand, B. bruxellensis was recovered after 5 days at 25°C onto Brettanomyces agar. The number of colonies counted after incubation corresponded to the number of viable microorganisms expressed as colony forming units per milliliter (CFU/mL). The survival fraction was calculated by dividing the number of microorganisms that survived the treatment (N _t ) by the initial number of viable cells (N ₀ ). Survival curves were obtained by plotting Log₁₀ of the survival fraction vs. treatment time for each electric field strength.

Mathematical modeling was used to evaluate the effect and interactions of the total specific energy (from 0 to 156 kJ/kg) of PEF treatments, the concentration of SO₂ (0, 10, 25, and 50 ppm), and the incubation period (0, 24 h) on the inactivation of B. bruxellensis. The obtained inactivation data obtained after applying the treatment conditions described in Section 2.2 were fitted to the following polynomial equation: Y = β 0 + ∑ i = 1 n β i X i + ∑ i = 1 n β i i X i n + ∑ i > j n β i j X i X j , (1) where Y is the predicted response, and β₀, β_i, β_ii, and β_ij are the intercept, linear, quadratic, and cross-product coefficients, respectively. X_i and X_j represent the independent factors, and n is the total number of independent factors. Data analysis was performed with Design-Expert software (version 10.0, Stat-Ease, Inc., Minneapolis MN, United States). To determine the model’s significant parameters and to remove the non-significant effects (p > 0.05), backward regression was applied.

For validation experiments, red wine supplemented with 20 g/L of sucrose was contaminated with B. bruxellensis to an initial concentration of 10² CFU/mL. After 2 months of incubation, the wine containing 10⁵ CFU/mL of B. bruxellensis was subjected to PEF treatments (15 kV/cm, 5 µs) of varying duration and specific energies. PEF-treated wines were added with different SO₂ doses, and the number of survivors was determined after 30 min and 24 h of incubation.

These independent data were used to validate the model obtained for the prediction of microbial inactivation of B. bruxellensis. The root mean square error (RMSE) between estimated and the actual experimental data was calculated.

At least three samples were analyzed for each condition. Data are expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) and Tukey tests using GraphPad Prism (GraphPad Software, San Diego, California, United States) were performed to evaluate the significance of differences among the mean values. Differences were considered significant at p < 0.05. Multiple regression analysis was conducted to fit Eq. 1 to the experimental data, and Design-Expert software (version 10.0, Stat-Ease Inc., Minneapolis, MN, United States) was used to determine the model’s significant terms (p > 0.005) by ANOVA.

The survival curves of S. bayanus and B. bruxellensis in wine at different electric field strengths are shown in Figure 1. Residence time in the treatment chamber (0.43 s) and pulse width (5 µs) were kept constant. Therefore, to modify the treatment time (number of pulses × pulse width) at the different electric field strengths tested, the number of pulses delivered in the treatment chamber was increased by raising the frequency of the treatments (number of pulses delivered per second). For a given electric field strength, an increase in the number of pulses delivered to the treatment chamber leads to a rise in the total specific energy supplied to the sample. Consequently, the sample’s temperature increases due to the Joule effect. The outlet temperature of samples subjected to PEF treatments of various durations applied at different electric field strengths is indicated in Figure 1 next to the corresponding symbol.

The survival curves of both microorganisms (S. bayanus and B. bruxellensis) showed an initial shoulder whose width was greater at lower electric field strengths. As the electric field strength increased, the shoulder decreased in size and the survival curves became progressively steeper. The survival curves’ shape can be explained by the increase in the lethal effect of PEF treatments at higher temperatures, as demonstrated in several previous studies (Saldaña et al., 2012; Timmermans et al., 2019). The existence of a shoulder in the survival curves implies the presence of a time threshold that appears to depend on the electric field strength and the exit temperature (total specific energy). At lower electric field strengths, longer treatments are necessary to deliver the total specific energy required to reach the exit temperature, from which inactivation increases rapidly with treatment time.

In terms of efficacy, more pulses were required to achieve a given level of inactivation at lower electric field strengths. For example, the treatment time required to achieve an inactivation of 3.0 Log₁₀ cycles in S. bayanus and B. bruxellensis was three times longer at 15 kV/cm than at 25 kV/cm. However, a comparison of treatments at the same outlet temperature but with varying electric field strengths shows that the increment in terms of electric field strength had little effect on microbial inactivation. For instance, an inactivation rate lying between 3.6 and 4.0 Log₁₀ cycles was achieved in S. bayanus at 15, 20, and 25 kV/cm for an outlet temperature of 50°C (117 kJ/kg). Meanwhile, for an outlet temperature of 45°C (97 kJ/kg), the inactivation of B. bruxellensis ranged between 4.1 and 4.6 Log₁₀ cycles when the electric field strength was increased from 15 to 25 kV/cm. In studies of this nature, it is therefore essential to specify the electric field strength, treatment time, and the total specific energy applied when describing PEF treatments, especially in continuous processing where the energy introduced into the system increases the temperature of the sample and the efficacy of the PEF treatment.

Regarding the resistance of the two yeast strains to PEF, B. bruxellensis was generally more sensitive to PEF treatments than S. bayanus. For instance, PEF treatments of the same intensity (20 kV/cm, 150 µs, outlet temperature 45°C) inactivated 4.8 Log₁₀ cycles of B. bruxellensis but only 2.0 Log₁₀ cycles of S. bayanus. Previous studies have likewise observed a greater PEF resistance of Saccharomyces strains in wine compared to Brettanomyces (Puértolas et al., 2009; González-Arenzana et al., 2015). Differences in the composition of the cytoplasmic membrane could explain the observed differences among the two yeast strains in terms of resistance, given that they are of similar shape and size. Saccharomyces has a well-known capacity to perform alcoholic fermentation without suppression of metabolic activity due to the presence of ethanol. Certain studies have suggested that this ethanol tolerance of the Saccharomyces genus is directly related to changes in the cytoplasmic membrane composition triggered by the presence of ethanol (Aguilera et al., 2006; Henderson and Block, 2014).

Our study found that PEF treatments were highly effective in inactivating S. bayanus and B. bruxellensis, with inactivation levels of around 4.0 Log₁₀ cycles or even higher, achieved at total specific energies ranging from 97 to 117 kJ/kg. These values are lower than those reported by previous studies, in which treatments of 121 kJ/kg at 50 kV/cm or 300 kJ/kg at 20 kV/cm were required (Puértolas et al., 2009; Delsart et al., 2015; van Wyk et al., 2018). The higher degree of lethality observed in our study may be due to the improved efficacy of PEF treatments when applied at moderate temperatures (30°C–60°C) (Saldaña et al., 2012; Timmermans et al., 2019). Although PEF is often viewed as a non-thermal technology, our findings suggest that PEF treatments at moderate temperatures may enhance lethality. However, this strategy may only be feasible for the food industry if the increase in temperature during the short PEF treatment duration does not negatively affect food’s sensory or nutritional quality. In the wine industry, maintaining quality properties is of utmost importance and thermal treatments for microbial inactivation tend to be avoided altogether. However, recent research by our team has shown that PEF treatments of up to 156 kJ/kg (resulting in an outlet temperature of 60°C) did not have a negative impact on the sensory profile of a high-value commercial wine (Delso et al., 2023).

The efficacy of PEF treatments on S. bayanus and B. bruxellensis yeasts in wine after 24 h of incubation was also evaluated. Figure 2 shows the total inactivation of S. bayanus and B. bruxellensis, evaluating the number of survivors to the PEF treatments after 24 h. The dark gray area of the column corresponds to the inactivation obtained immediately after PEF, and the light gray area corresponds to the additional inactivation produced after 24 h of incubation in wine. Differences between inactivation achieved immediately after the PEF treatment and after 24 h of incubation are indicated by dots joined by a black line.

A significant increase in efficacy against both yeasts was observed after 24 h at different electric field strengths, although the effect tended to disappear in the case of PEF treatments that achieved a greater degree of efficacy immediately after application. For example, PEF treatment with an electric field strength of 15 kV/cm, 173 µs, and 35°C of the outlet temperature that showed very low efficacy immediately after treatment (<1.0 Log₁₀ reduction) exhibited an increase in inactivation of 3.0 and 2.3 Log₁₀ for S. bayanus and B. bruxellensis, respectively, after 24 h of incubation. In contrast, PEF treatments of higher intensity and efficacy (4.0 Log₁₀ cycles or higher) showed little improvement in inactivation after 24 h of incubation.

The observed differences between immediate inactivation and 24-h inactivation may be due to the presence of a sublethally injured population, the recovery of which may depend on environmental conditions. Electroporation triggered by PEF treatments is a purely physical process in which stable pores in the cytoplasmic membrane can be created if the induced transmembrane voltage lies above the cell’s threshold (Kotnik et al., 2019). However, recovery of cell viability after PEF treatment depends on intrinsic microbial features coupled with external factors that affect membrane integrity repair (Saulis, 2010). Recovery is more likely to occur when cells are transferred to an agar medium immediately after treatment, but the low pH (3.1) and high ethanol content (12.9% v/v) in wine may impair this recovery process, resulting in a lower number of survivors after 24 h of incubation. These results confirm previous findings obtained by Somolinos et al. (2007, 2008) on the subject of sublethal damage in PEF-treated S. cerevisiae and B. bruxellensis cells.

The loss of viability in a significant proportion of PEF-treated cells after 24 h of incubation allowed for a 3.0–4.0 Log₁₀ reduction with very moderate treatments (58 kJ/kg, 35°C outlet temperature) at all the electric field strengths we studied. These results suggest that on account of the additional inactivation that takes place in wine even after moderate PEF treatments (10–15 kV/cm), the total specific energy required for a 3.0–4.0 Log₁₀ reduction of the yeast population can be halved, making PEF technology more feasible to apply in wineries.

The addition of sulfur dioxide (SO₂) is the method most commonly applied for controlling the proliferation of yeast in wine. After confirming that PEF caused sublethal damages in yeasts suspended in wine, we performed a further experiment combining PEF with different doses of SO₂ with the aim of finding a way to further increase the lethal outcome of PEF treatments. For this purpose, 0, 10, 25, or 50 ppm of SO₂ was combined with two moderate PEF treatments that had a low lethal effect but which managed to sublethally damage a high proportion of the yeast population: these treatments were PEF₁ (15 kV/cm, 39 kJ/kg, 30°C outlet temp.) and PEF₂ (15 kV/cm, 78 kJ/kg, 40°C outlet temp.). Figure 3 shows the inactivation of S. bayanus and B. bruxellensis obtained by applying these combined treatments immediately after the PEF treatment and after 24 h of incubation in the same wine. For comparison, we also dosed untreated wine samples with the same varying concentrations of SO₂.

The addition of sulfur dioxide did not affect the population of untreated S. bayanus cells, even after 24 h of incubation. For B. bruxellensis, a loss of viability of 1.1 and 2.4 Log₁₀ cycles was only observed when 25 and 50 ppm of SO₂ were added, respectively, after 24 h of incubation. The presence of sulfites scarcely increased the efficacy of the PEF treatments when the number of survivors was determined just after processing. However, a pronounced increase in inactivation was observed after 24 h of incubation when 25 and 50 ppm of SO₂ were present. In all cases, the combination of the two procedures had a synergistic effect rather than an additive effect. Somolinos et al. (2007) reported a similar synergistic effect on S. cerevisiae and B. bruxellensis by combining a PEF treatment with sublethal doses of sorbic acid. The combination of PEF with further antimicrobial compounds such as nisin, lysozyme, and organic acids has also resulted in synergistic effects in the inactivation of different bacteria (Smith et al., 2002; Mosqueda-Melgar et al., 2008; Saldaña et al., 2012). These synergistic effects have been mainly attributed to the easier accessibility these compounds gain into the electroporated cells, whose target is located in the cytoplasm; another explanation is that the added compound facilitates the electroporation of the cytoplasmic membrane. SO₂ needs to reach the cytoplasm, where it reacts with a number of molecules such as coenzymes, cofactors, vitamins, nucleic acid, and the enzymatic system. PEF treatment could therefore sensitize a proportion of the cells that were able to survive sublethal amounts of SO₂ in the wine after the PEF treatment. The great tolerance of yeast to SO₂ has been chiefly attributed to a sulfite efflux pumping system through the plasma membrane or to the microorganism’s massive production of acetaldehyde, which binds and blocks intracellular SO₂ (García-Ríos and Guillamón, 2019). A lack of lipid membrane integrity in an electroporated cell could directly cancel the efflux pump effect. Furthermore, stress mechanisms triggered by homeostatic imbalance in permeabilized cells (García et al., 2006) could also be responsible for hindering intracellular acetaldehyde production or fixation.

Inactivation of B. bruxellensis was conducted on cells cultivated in a culture medium and then inoculated in red wine. However, growth conditions can influence the physiology of microorganisms and their resistance to different lethal effects. We, therefore, decided to investigate whether the data on resistance of B. bruxellensis cells inoculated in wine could be used to predict the inactivation of B. bruxellensis cells grown in wine. To accomplish this, we developed a multiple regression model featuring the total specific energy of the PEF treatment and sulfite concentration as independent variables. To develop the model, we used the data for B. bruxellensis obtained after the application of PEF treatments (from 0 to 168 kJ/kg), SO₂ concentration (0, 10, 25, and 50 ppm) and incubation time (0, 24 h). This model was validated with inactivation data on the same microorganism after growth in wine for 2 months. The culture of B. bruxellensis in wine was extended during that time to reach a sufficiently large concentration of cells sufficient to evaluate the effectiveness of inactivation treatment.

Table 1 shows the terms of the equations with their coefficients after fitting the experimental data obtained immediately after the PEF treatment and after 24 h of incubation to a quadratic equation after removing non-significant terms (p < 0.05). The table also shows the results of analysis of variance (ANOVA) for the model’s significant terms and the statistics used to test its adequacy. F-value calculation indicates that the overall model was significant (p < 0.05): the model’s terms thus have a significant effect on the response. The model’s R ² value was 0.90, indicating that only 10% of the total response variation could not be explained by the model. The RMSE value confirms the model’s fitness for describing the experimental inactivation data. According to the F-values, total specific energy was the most significant variable with an F-value of 170.7, followed by the linear term of the sulfite concentration.

Figure 4 shows the comparison of the inactivation data predicted by the model with actual data obtained from the observed inactivation of B. bruxellensis cells grown in wine. For data obtained immediately after the PEF treatment (closed symbols), the model predicted lower inactivation levels than those achieved in the B. bruxellensis cells grown in wine. These results indicate that the cells cultivated in the same wine were more sensitive to the PEF treatments than those cultivated in a culture medium and subsequently inoculated in the wine. However, after comparison of the results obtained after 24 h of incubation (open symbols), a close correspondence between predicted and actual inactivation can be observed. Remarkably, for inactivation levels higher than 2.0 Log₁₀ cycles, most of the points lie below the equivalency line, thereby indicating that the model is reliable as it predicts less inactivation than that obtained with the cells grown in the wine.

Our results, therefore, reinforce the potential of applying PEF in combination with reduced concentrations of SO₂ to decontaminate yeast in wine. This could contribute to an overall reduction of the final concentration of sulfites in winemaking and improve other processing stages, such as mutage or the implantation of lactic acid bacteria for the performance of malolactic fermentation after alcoholic fermentation.