Combined Effect of Temperature,pH,and Presence of Nisin on Inactivation of Staphylococcus aureus and Listeria monocytogenes by Pulsed Electric Fields

Abstract

The combined effect of pH (3.5–7.0), temperature (4°C–50°C), and the presence of nisin (0–200 μg/mL) on the inactivation caused by pulsed electric fields (PEF) in two PEF-resistant Gram-positive microorganisms, Staphylococcus aureus and Listeria monocytogenes, was investigated. A response surface model using a central composite design was developed for the purpose of understanding the individual effects and interactions of these factors and to identify the most promising combinations for microbial inactivation. According to the developed models, temperature was the factor showing the greatest influence on PEF inactivation in the two strains investigated. A temperature increment from 4°C to 50°C increased the lethality of PEF by 2 and 3 log₁₀ cycles in S. aureus and L. monocytogenes, respectively. PEF inactivation in both microorganisms decreased with increased pH in the treatment medium from 3.5 to 7. The effect of the presence of nisin on the increment of PEF lethality for L. monocytogenes was additive or slightly synergistic. For S. aureus, this effect was synergistic at low temperatures and tended to disappear with increasing temperature. An inactivation of 4.5 and 5.5 log₁₀ cycles was achieved in the populations of S. aureus and L. monocytogenes, respectively, in a medium of pH 3.5 in the presence of 200 μg/mL of nisin at 50°C. Therefore, the application of PEF at moderate temperatures in the presence of antimicrobials such as nisin has great potential for achieving effective control of the vegetative forms of the two PEF-resistant Gram-positive strains investigated, especially in foods of low pH, such as fruit juices.

Introduction

T he potential of pulsed electric fields (PEF) to destroy undesirable microorganisms at temperatures below those used in thermal processing is very attractive for the food industry. It has been widely demonstrated that the application of an external electric field induces changes that cause a drastic increase in the permeability of the cytoplasmic membrane of the microorganisms (Aronsson et al., 2005; García et al., 2007). Although several theoretical models have been proposed, the underlying mechanisms of the electroporation of the cytoplasmic membrane at the molecular level have not yet been fully elucidated (Saulis, 2010).

The food industry requires microbial inactivation technologies to guarantee the safety and stability of foods. The efficacy of PEF to inactivate the vegetative cells of bacteria, yeasts, and molds has been widely demonstrated (Álvarez et al., 2006). However, microbial resistance to PEF is extremely variable (García et al., 2005; Saldaña et al., 2009). For the most resistant microorganisms, obtaining microbial lethality equivalent to thermal processing requires treatments of long duration or very high electric field strengths (Maresellés-Fontanet et al., 2009). Several technical and economic limitations exist to upgrade the treatment chamber of the PEF apparatus to a large capacity to increase the residence time or to apply electric field strengths above 30 kV/cm (Toepfl et al., 2007). An approach to increase the lethal effect of PEF with short treatment times at moderate electric field strengths involves combining this technology with other preservation factors in an overall preservation strategy (Raso and Barbosa-Cánovas, 2003).

The temperature and pH of the treatment medium are two factors that have been demonstrated to influence microbial inactivation by PEF (Wouters et al., 2001). Generally, the highest microbial resistance to PEF is observed at temperatures below room temperature, and mild temperature elevation from this temperature causes a greater level of microbial inactivation even at temperatures that are not lethal for the microorganism (Heinz et al., 2003; Sepúlveda et al., 2005). The influence of pH on microbial inactivation by PEF is not as clear as the influence of temperature. While some microorganisms are more PEF resistant at neutral pH, others are more PEF resistant at acidic pH, and, in some cases, the microbial PEF resistance is not affected by the pH (García et al., 2005). However, the combined effects of pH and temperature on microbial inactivation by PEF have not yet been investigated.

The presence of naturally occurring antimicrobials such as nisin has been proven effective in increasing the lethality of PEF in both buffer systems and foods (Liang et al., 2002; Gallo et al., 2007). The presence of this antimicrobial agent offers interesting possibilities for increasing the efficiency of PEF in the media of different pH at different temperatures.

In this article, the effects of pH, temperature, and nisin on the inactivation caused by PEF in two PEF-resistant Gram-positive microorganisms, Staphylococcus aureus and Listeria monocytogenes, by PEF were investigated. A response surface model using a central composite design was developed for the purpose of understanding the individual effects and interactions of these factors and to identify the most promising combinations of these factors to achieve optimal microbial inactivation by PEF treatments that are applicable to an industrial scale.

Materials and Methods

Microorganisms and growth conditions

The strains of L. monocytogenes (STCC 5672) and S. aureus (STCC 4459) used in this investigation were supplied by the Spanish Type Culture Collection. In a previous study, it was demonstrated that these two strains were especially resistant to PEF (Saldaña et al., 2009).

During this investigation, the cultures were maintained on slants of tryptic soy agar (Biolife, Milan, Italy) with 0.6% yeast extract added (Biolife) (TSAYE). A broth subculture was prepared by inoculating a test tube containing 5 mL of tryptic soy broth (Biolife) with 0.6% yeast extract (TSBYE) with a single colony, followed by incubation at 37°C for 18 h. With this subculture, a flask containing 50 mL of sterile TSBYE was inoculated to a final concentration of ∼10⁶ cells/mL. The culture was incubated under agitation (125 rpm) at 37°C until the stationary growth phase was reached (12 h for the S. aureus strain and 24 h for the L. monocytogenes strain).

PEF unit

The PEF unit used in this investigation was previously described by Saldaña et al. (2010). To obtain microbial inactivation data at different temperatures under a uniform electric field strength distribution and quasi-isothermal conditions, a batch parallel electrode treatment chamber with tempered electrodes, previously described, was used (Saldaña et al., 2010). This chamber consists of a cylindrical polypropylene tube closed with two polished stainless steel cylinders of 2.01 cm² in surface area and 4 cm in length. The distance between electrodes was 0.25 cm and the volume of the treatment zone 0.5 mL.

Experimental design

A central composite design with three factors and face centered was the experimental design used to determine the effects and interactions of the temperature (T), pH, and nisin concentration (Nis) on the inactivation of both microorganisms by a PEF at 30 kV/cm for 99 μs (33 pulses of 3 μs). The independent variables were pH (from 3.5 to 7.0), temperature (from 4°C to 50°C), and nisin concentration (from 0 to 200 μg/mL). The central point was replicated several times to estimate the variance due to experimental and random variability. The rest of the experiments were repeated twice for each combination.

Microbial inactivation experiments

Before treatments, microorganisms were centrifuged at 6,000 g for 5 min at 4°C and resuspended in a citrate-phosphate McIlvaine buffer of an electrical conductivity of 0.10 ± 0.01 S/m and different pH values (3.5, 5.25, and 7.0). Nisin was added to the corresponding media to obtain a concentration of 100 or 200 μg/mL. The microbial suspension (0.5 mL) at a concentration of ∼10⁸ CFU/mL was placed into the treatment chamber with a sterile syringe.

Enumeration of viable cells

PEF-treated cell suspensions were serially diluted in 0.1% sterile peptone solution (Biolife). The medium used for enumeration of viable cells of all the strains was Tryptic Soy Agar (Biolife) plus 0.6% (w/v) of Yeast Extract (Biolife) (TSAYE), whose pH is 6.8 ± 0.1. From the selected dilutions, 0.1 mL was pour plated into TSAYE and then plates were incubated at 37°C for 48 h to detect viable cells.

Statistical analysis

To determine the influence of temperature, pH, and the presence of nisin on the inactivation by PEF, the results obtained were analyzed by multiple regression applying response surface methodology (RSM). The results were fitted to a general quadratic model that accounted for the influence of the individual factors, T (X ₁), pH (X ₂), nisin concentration (X ₃), interaction effects (X ₁ × X ₂, X ₁ × X ₃, and X ₂ × X ₃), and quadratic effects (X ₁ ², X ₂ ², and X ₃ ²) of the investigated factors on the response (Eq. 1), where Y is the response variable to be modeled. \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*} Y &= \beta_0 + \beta_1 X_1 + \beta_2 X_2 + \beta_3 X_3 + \beta_{1 \times 2} ( X_1 \times X_2 ) + \beta_{1 \times 3} ( X_1 \times X_3 ) & \\ & \quad+ \beta_{2 \times 3} ( X_2 \times X_3 ) + \beta_1 X_1^2 + \beta_2 X_2^2 + \beta_3 X_3^2 &( 1 ) \end{align*} \end{document}

A backward regression procedure was used that systematically removes the effects that were not significantly associated (p > 0.05) with the response until a model with only a significant effect was obtained.

The central composite design, response fit analysis, regression coefficient estimations, and model significance evaluations were conducted using the software package Design-Expert 6.0.6 (Stat-Ease, Inc., Minneapolis, MN).

Results

Effect of temperature, pH, and nisin on inactivation of L. monocytogenes and S. aureus by PEF

The log₁₀ reductions caused by the PEF treatment under the experimental conditions investigated for the two strains are shown in Table 1. Inactivation of L. monocytogenes after applying a PEF treatment at 30 kV/cm for 99 μs ranged from 0.1 to 5.9 log₁₀ reductions. PEF treatment at 4°C and pH 7.0 without nisin had little effect on viability of L. monocytogenes. However, 4.86 log₁₀ reduction or higher was achieved when the treatments were applied at 50°C in a medium of pH lower than 7.0 in the presence of nisin.

Table 1.

Response Surface Design and Experimental Results for Listeria monocytogenes 5672 and Staphylococcus aureus 4459

Temperature (°C)	pH	Nisin (μg/mL)	S_t _Listeria ^a	S_t _{Staphylococcus} ^a
4.0	3.5	0	1.21 ± 0.03	2.87 ± 0.15
4.0	3.5	200	2.94 ± 0.08	3.48 ± 0.41
4.0	5.25	100	1.18 ± 0.08	3.20 ± 0.31
4.0	7.0	0	0.10 ± 0.05	1.31 ± 0.23
4.0	7.0	200	0.93 ± 0.28	3.36 ± 0.18
27.0	3.5	100	4.40 ± 0.06	3.80 ± 0.48
27.0	5.25	0	0.91 ± 0.21	3.76 ± 0.24
27.0	5.25	100	3.42 ± 0.15	4.03 ± 0.09
27.0	5.25	100	3.10 ± 0.32	4.01 ± 0.20
27.0	5.25	100	3.15 ± 0.25	3.90 ± 0.35
27.0	5.25	100	3.45 ± 0.02	4.30 ± 0.62
27.0	5.25	100	3.25 ± 0.26	3.90 ± 0.79
27.0	5.25	100	3.52 ± 0.01	4.19 ± 0.05
27.0	5.25	200	4.14 ± 0.88	4.12 ± 0.68
27.0	7.0	100	2.16 ± 0.07	3.59 ± 0.94
50.0	3.5	0	4.76 ± 0.19	4.45 ± 0.13
50.0	3.5	200	5.94 ± 0.30	4.90 ± 0.28
50.0	5.25	100	4.86 ± 0.10	4.56 ± 0.15
50.0	7.0	0	2.77 ± 0.15	4.33 ± 0.47
50.0	7.0	200	3.66 ± 0.36	4.53 ± 0.09

Pulsed electric field treatment conditions: electric field strength, 30 kV/cm; treatment time, 99 μs.

Microbial inactivation in log₁₀ reductions ± standard deviation.

The viability loss of S. aureus, after the application of a PEF treatment at 30 kV/cm for 99 μs, ranged from 1.3 to 4.5 log₁₀ reductions. S. aureus was more sensitive than L. monocytogenes when treatments were applied at 4°C. At 50°C, S. aureus was more sensitive than L. monocytogenes when treated in a medium of pH 7.0, but at lower pH, the resistance of this microorganism was similar to or even higher than the L. monocytogenes resistance.

Mathematical modeling

The application of the multiple regression analysis to the experimental data corresponding to the log₁₀ cycles of inactivation resulted in the following second-order polynomial equations for L. monocytogenes (Eq. 2) and S. aureus (Eq. 3): \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}{ \rm Log \ S} &= 3.01 + 0.07 \times { \rm T} - 0.55 \times { \rm pH} + 0.02 \times [ { \rm Nis} ] \\ & \quad - ( 5.13 \times 10^{ - 5} ) \times [ { \rm Nis} ] ^2 & ( 2 ) \end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}{ \rm Log \ S} &= 0.03 + 0.05 \times { \rm T} - 1.01 \times { \rm pH} + ( 6.62 \times 10^{ - 3} \times [ { \rm Nis} ] \\ &\quad- 0.12 \times { \rm pH}^2 - ( 1.09 \times 10^{ - 4} ) \times { \rm T} \times [ { \rm Nis} ] & ( 3 ) \end{align*} \end{document}

where S represents the survival fraction; T represents the temperature (°C); pH represents the pH of the treatment medium; and [Nis] represents the concentration of nisin (μg/mL).

The determination coefficient for each model was higher than 0.90. This means that less than 10% of the total response variation remained unexplained by the models developed.

According to the model F-values obtained, 55.42 for L. monocytogenes and 26.07 for S. aureus from the ANOVA analysis, the quadratic models were significant (p < 0.001).

The F-values for model parameters are very useful to indicate the significance of the effects of the variables and their interactions. In both microorganisms, the most significant effect on inactivation by PEF was the temperature, followed by pH, in the case of L. monocytogenes and nisin concentration in the case of S. aureus. This means that changes in these factors will have a very significant effect on microbial inactivation by PEF. The square of nisin and the square of pH were also significant values for the inactivation of L. monocytogenes and S. aureus, respectively. The presence of these square terms in Equations 2 and 3 means that when the nisin concentration or pH changes, their effects on the lethality of PEF are nonlinear; that is, an increase in nisin concentration from 0 to 100 μg/mL will produce a greater change in PEF lethality that an increase form 100 to 200 μg/mL.

To validate the predictive performance of the quadratic models, they were validated with 27 different experiments for each microorganism within the treatment condition range used to generate the models. The values obtained in these experiments were graphically compared to the predicted values obtained from the quadratic models (Fig. 1). The bias factors (Bf) were 1.032 and 0.979 for Equations 2 and 3, respectively, and the accuracy factors (Af) were 1.107 and 1.119 for Equations 2 and 3, respectively. The results of the validation analysis indicated that the experimental values were in agreement with the predicted ones and that the experimental points were evenly distributed around the equivalence line, so the quadratic models obtained were satisfactory and accurate in predicting the PEF inactivation of the strains of L. monocytogenes and S. aureus used in this study.

FIG. 1.

Correlation between experimental and estimated data obtained with Eq. 2 for Listeria monocytogenes 5672 (A) and Eq. 3 for Staphylococcus aureus 4459 (B) treated by pulsed electric fields. Af, accuracy factor; Bf, bias factor.

Predicting the combined effect of temperature, pH, and presence of nisin in PEF inactivation of S. aureus and L. monocytogenes

Figures 2 and 3 illustrate the influence of the factors investigated on the inactivation of both microorganisms by PEF. The graphical representations were obtained using the corresponding regression models for each microorganism (Eq. 2 and 3). As the influence of the factors investigated was different for both microorganisms, the most suitable representation was selected to show their effects. Figure 2A shows the influence of temperature on the PEF inactivation of L. monocytogenes at different pH in the absence and in the presence of 100 μg/mL of nisin. In the absence of nisin, the PEF inactivation of L. monocytogenes increased with increased treatment temperature and decreased pH. In the range of conditions investigated, the increment of lethality by increasing temperature was not influenced by pH. At any pH, a temperature increment of 20°C increased the lethality of the treatment at around 1 log₁₀ reduction. The influence of the temperature and pH in the presence of nisin was similar. The only effect of nisin was that it increased the lethality of the treatments at around 1 log₁₀ reduction independently of the treatment temperature and pH. This effect was similar in the presence of 100 or 200 μg/mL of nisin. The inactivation effect of nisin without applying PEF treatments at different temperatures in media of different pH for the time in which microorganisms were in contact with nisin during the application of the PEF treatment (2.5 min) was between 0.5 and 1 log₁₀ reduction (data not shown). Therefore, the effect of the presence of nisin on the increment of PEF lethality was additive or slightly synergistic.

FIG. 2.

Influence of temperature on the pulsed electric fields (PEF) inactivation predicted by Eq. 2 for L. monocytogenes 5672 (A) and Eq. 3 for S. aureus 4459 (B) at different pH in the absence and in the presence of nisin at pH from 3.5 to 7.0. PEF treatment conditions: field strength, 30 kV/cm; treatment time, 99 μs.

FIG. 3.

Comparison of the PEF inactivation predicted by Eq. 2 for L. monocytogenes 5672 and Eq. 3 for S. aureus 4459 in media of pH 7.0 (A) and 3.5 (B) in the absence or in the presence of 100 μg/mL of nisin. PEF treatment conditions: field strength, 30 kV/cm; treatment time, 99 μs.

Figure 2B shows the influence of temperature on the PEF inactivation of S. aureus in the presence of 100 and 200 μg/mL of nisin in media of pH 3.5 and 7.0. PEF inactivation of S. aureus increased by increasing the treatment temperature in the range of pH and nisin concentration investigated. The influence of pH on the PEF inactivation of S. aureus was lower than that of L. monocytogenes. When the pH decreased from 7.0 to 3.5, the inactivation only increased by around 0.5 log₁₀ reduction for any condition investigated. At both pH levels, the increment of the lethality of the PEF treatment in the presence of nisin decreased with the increments of temperature. No significant (p > 0.05) differences in PEF lethality were observed at 50°C in the absence or presence of this compound at a concentration of 100 or 200 μg/mL. The inactivation effect of nisin for the duration of the PEF treatment at 4°C was negligible (data not shown). Therefore, the effect of the presence of nisin on the increment of PEF lethality at these low temperatures was synergistic. This synergistic effect tended to disappear with increasing temperature.

Figure 3 compares the PEF resistance of both microorganisms in media of pH 7.0 (Fig. 3A) and 3.5 (Fig. 3B) in the absence or presence of 100 μg/mL of nisin. At pH 7.0, L. monocytogenes was more PEF resistant than S. aureus, but differences in resistance tended to disappear at higher temperatures in the presence of nisin. At pH 3.5, when nisin was not added, L. monocytogenes was sligltly more resistant than S. aureus, especially at low temperatures. However, when 100 μg/mL of nisin was added, the lethality of the treatment was higher for L. monocytogenes at temperatures above 23°C. Under these conditions, more than 5 log₁₀ reductions in the population of L. monocytogenes were observed at temperatures greater than 35°C.

Discussion

In general, most studies published investigate the influence of only one single factor, such as pH, the presence of an antimicrobial agent, etc., on microbial inactivation by PEF (Aronsson and Rönner, 2001; García et al., 2005; Gallo et al., 2007). In this study it was evaluated and modeled the combined effect of temperature, pH, and the presence of nisin on the inactivation of L. monocytogenes and S. aureus by a PEF treatment at an electric field strength and number of pulses applicable from an industrial point of view. This is the first attempt to model the influence of the presence of nisin on microbial inactivation by PEF in the range of pH of most foods in a wide range of temperatures.

According to the developed models, temperature showed the greatest influence on microbial inactivation by PEF. Incremental effect of PEF with temperature has been attributed to the temperature-related phase transition of the membrane phospholipids from gel to liquid-crystalline, which causes membranes to lose their elastic properties as temperature increases, making them more fluid and therefore more easily disrupted by the application of PEF (Stanley, 1991). In the range of experimental conditions investigated, an increase of the treatment temperature from 4°C to 50°C increased the lethality of the treatment by 3 log₁₀ reductions in L. monocytogenes and by 2 log₁₀ reductions in S. aureus. These results are in agreement with those of other studies in which the influence of the temperature on bacterial inactivation by PEF has been investigated (Heinz et al., 2003; Sepúlveda et al., 2005; Amiali et al., 2007). However, in those investigations, it was difficult to quantify the effect of the temperature because they were conducted in continuous flow processes in which both treatment medium temperature and distribution of the electric field strength in the treatment chamber were not uniform, and, in some experiments, lethal temperatures were reached after the treatment.

Treatment medium pH and the nisin concentration had a greater effect on the inactivation of L. monocytogenes than on the inactivation of S. aureus. In this investigation it has been demonstrated that the pH effect did not depend on the temperature of treatment. Independently of the temperature, the PEF resistance of both microorganisms increased by increasing pH. Results on influence of pH are in agreement with observations of other authors finding that, unlike the inactivation of Gram-negative bacteria, the PEF inactivation of Gram-positive microorganisms increases by decreasing the pH in the treatment medium (García et al., 2005). The lower PEF resistance of Gram-positive bacteria in acidic conditions was related with the lower ability of these bacteria to repair the cytoplasmatic membrane injury caused by PEF when treated at low pH (García et al., 2005).

Since the cytoplasmatic membrane is a common target of the antimicrobial peptide nisin and PEF treatment, the combination of PEF and nisin has been investigated to find synergistic effects that improve microbial lethality. However, as observed in this investigation, in general, an additive or slightly synergistic effect has been reported by other authors in different Gram-positive microorganisms when nisin is added before the PEF treatment (Calderon-Miranda et al., 1999; Gallo et al. 2007). In this investigation, it has been observed for the first time that this effect may depend on the temperature of the applied PEF treatments. In the case of S. aureus, the influence of nisin in the treatment medium tended to disappear when the treatment temperature was increased from 4°C to 50°C.

To establish treatment conditions for PEF food pasteurization, it is necessary to identify the most PEF-resistant microorganisms of public health concern with respect to food (Lado and Yousef, 2003). The results obtained in this investigation demonstrated that the microorganisms of concern not only depend on the intrinsic microbial PEF resistance but also on treatment conditions applied and characteristics of the treatment medium. Our results indicate that, in general, L. monocytogenes was more PEF-resistant than S. aureus; however, when the highest inactivation was observed in both microorganisms (temperature 50°C, pH 3.5, and nisin concentration 200 μg/mL), the strain of S. aureus was more resistant than the strain of L. monocytogenes (Fig. 3A). Thus, before considering the use of PEF technology for preserving any specific food, it is necessary to evaluate the process conditions in the food of interest and with the microorganisms of concern.

In a previous study conducted in our laboratory, it was observed that the level of inactivation of these two Gram-positive pathogenic strains caused by PEF treatments applied at room temperature was inadequate to guarantee food safety. In this research study, it has been demonstrated that the application of PEF at temperatures above room temperature in the presence of antimicrobials such as nisin has great potential for achieving effective control of vegetative forms of the two PEF-resistant Gram-positive pathogenic strains investigated, especially in foods of low pH, such as fruit juices.

Footnotes

Acknowledgments

G.S. gratefully acknowledges the financial support for his doctoral studies from the Spanish Ministry for Science and Innovation. H.M. thanks the financial support of National Council of Science and Technology (CONACyT) and Tecnológico de Estudios Superiores de Ecatepec (TESE). This investigation has been funded by the European Commission (FP6, 015710-2NOVELQ) and CICYT (project AGL 2007-62738).

Disclosure Statement

No competing financial interests exist.

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