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Preservation of liquid foods by high intensity pulsed electric fields—basic concepts for process design
Trends in Food Science & Technology 12 (2002) 103–111
Review
Preservation of liquid foods by high intensity pulsed electric fields—basic concepts for process design V. Heinz,a,* I. Alvarez,b A. Angersbacha and D. Knorra a
Department of Food Biotechnology and Food Process Engineering, TU Berlin, Ko¨nigin-Luise-Str. 22, D-14195 Berlin, Germany (tel: +49-30-314-71250; fax: +49-30-832-7663; e-mail: [email protected]) b Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, ES-50013 Zaragoza, Spain In excess of a critical transmembrane potential ’M of 1 V produced by high intensity pulsed electric fields a rapid electrical breakdown and local conformational changes of cell membranes occur which result in a drastic increase in permeability and an equilibration of the electrochemical and electrical potential differences of the cell plasma and the extracellular medium. As irreverible membrane permeabilization impairs most vital physiological control systems, high intensity pulsed electric fields may be applied as a highly effective process for the microbial decontamination of liquid foods. The efficiency of the treatment is largely influenced by the inherent properties of the foods and of the spoiling microorganisms. In addition a number of technical limitations have to be considered. In this review an * Corresponding author.
approach is presented which reduces the diversity of parameters that affect microbial inactivation during pulsed power treatment. In particular, the required total specific energy input is discussed. # 2001 Published by Elsevier Science Ltd.
Introduction Microbial cells which are exposed to an external electrical field for a few microseconds respond by an electrical breakdown and local structural changes of the cell membrane. In consequence of the so called electroporation, a drastic increase in permeability is observed which in the irreversible case is equivalent to a loss of viability. This type of non-thermal inactivation of microorganisms by high intensity pulsed electric fields might be beneficial for the development of quality retaining preservation processes in the food industry. However, process safety, cost-effectiveness, and consumer benefits of pulsed electric field treatment have to be confirmed. Despite the extensive knowledge in food preservation by heat treatment (Larousse & Brown, 1997; Ramesh, 1999) and despite continued attempts to improve the quality of processed foods (Durance, 1997) there is still a need for technologies that minimize the destructive influence of heat on valuable food compounds (Knorr & Heinz, 2001). Even intelligent concepts like high-temperature–short-time processing fail if heat transfer and/ or heat penetration is limited by intrinsic thermophysical properties of the product. Since the thermal energy which is required to destroy the contaminating microorganisms has to be transmitted across the product itself, the design of fast and uniform heating and cooling steps is one of the primary challenges of industrial preservation by heat. Most of the thermal processing equipment in use consists of systems which transfer heat across an interface driven by a temperature gradient. On the product side only the convective heat transport can be enhanced by external measures, i.e. by the generation of turbulent flow. Compared to thermal inactivation the destruction of microbes by electroporation shows no time delay with respect to the propagation of the lethal treatment intensity.
Fundamental aspects In electrically conductive food placed between a high voltage and a grounded electrode the developing electrical field can be predicted from the Laplace equation
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r2 ’ ¼ 0, where ’ denotes the electrical potential. At the microbial membranes which are electrically insulating the electrical field produces the accumulation of charges with opposite polarity on either side of the bi-layer structure. Pore formation occurs when a certain threshold value of the electric field strength E is exceeded. However, in spite of the subtantial knowledge of the impact of high intensity pulsed electric fields on microorganisms (Barsotti, Merle, & Cheftel, 1999; Ho & Mittal, 2000; Wouters & Smelt, 1997) until now there is no clear evidence on the underlying mechanism of membrane permeabilization at the molecular level. The potential difference ’M at the membrane of a biological cell with spherical shape and a radius R induced by the external electrical field E can be approximated by eqn (1) which is derived from solving Maxwell’s equations in spherical coordinates assuming several simplifying restrictions (Neumann, 1996): 3 ’M ¼ EfðÞRcos 2
ð1Þ
For the calculation of ’M at a particular location at the membrane, the angle a of the radial direction vector has to be specified. Per definition is zero (and cosa=1) when the vector coincides with the direction of the electrical field. Hence, the highest membrane potential differences are assumed to occur at the two poles of the cell in field direction. The factor fðÞ is an explicit function of the electrical conductivities of the suspending medium , the plasma i , the cell membrane M , and the ratio of the membrane thickness and the cell radius (Neumann, 1989): fðÞ ¼
1 1 þ ðM ð2 þ i =Þ=ð2i d=RÞÞ
In literature (Zimmermann, 1996), it is reported that in excess of a critical transmembrane potential ’M of 1 V a rapid electrical breakdown and local conformational changes of bilayer structures occur. A drastic increase in permeability re-establishes the equilibrium of the electrochemical and electrical potential differences of the cell plasma and the extracellular medium. Simultaneously, the neutralization of the transmembrane gradient across the membrane irreversibly impairs vital physiological control systems of the cell like osmoregulation and consequently cell death occurs. Making use of the experimental result for ’M which leads to electroporation eqn (1) can be solved for the critical field strength E. From a practical point of view, this rearrangement is helpful as it allows the prediction of the minimum field strength required for preservation processes. In Fig. 1, the impact of cell size and geometry on E is presented. It is evident that the critical field strength sharply increases when the characteristic dimension of the cell (i.e. the radius of sperical cells or the shorter half-axis of elliptical cells) is shifted to smaller values. Moreover, variations in cell shape produce a considerable raise of E. A rod-shaped cell requires a more than 5 times higher electrical field than a spherical cell with the same characteristic dimension. Due to the large diversity in cell size and shape of a microbial population the prediction of the critical field strength for a number of microorganisms has to be
ð2Þ
However, M being in the range of nS/m for intact membranes it is readily seen from eqn (2) that fðÞ can be approximated by 1, independently from the electrical conductivity of the suspending medium. For cells with non-spheric shape an estimate for ’M can be obtained by solving the Maxwell’s equations in ellipsoidal coordinates. In those situations eqn (3) has to be applied (Zimmermann, Pilwat, & Riemann, 1974): ’M ¼ fðAÞAF E
ð3Þ
This formula yields the local membrane potential difference at the distance AF from the centre in direction of the external electrical field. The shape factor f(A) is a function of the three semi-axis (A1, A2, A3) of elliptical cells: fðAÞ ¼
2
ð1 1=
2 A1 A2 A3 0
sþ
A2F
3 qffiffiffiffiffiffiffiffiffiffiffiffiffiffi X s þ A2n Þ ð n¼1
!
ð4Þ ds
Fig. 1. Critical electroporative field strength as a function of cell dimensions. The membrane potential difference ’M is assumed to be 1 V. Curve A is calculated from eqn (1) where the radius is taken as the characteristic cell dimension. For elliptical cells eqn (3) is used. The critical field strength is plotted against the shorter semi-axis. Curves B–E are calculated for different ratios of the short and the long semi-axis: B, D: 0.5; C, E: 0.2. For each ratio, the critical field strength is presented for two different orientations of the cell relative to the direction of the electric field. Mean size and typical shape of the microorganisms mentioned above is compiled in Table 1.
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Table 1. Geometry and mean size of selected microorganisms. Data taken from Bergey (1986)
1. 2. 3. 4. 5. 6. 7. 8.
Organism
Shape
Size (m)
Listeria monocytogenes Yersinia enterocolitica Lactobacillus brevis Bacillus subtilis (vegetative cells) Lactobacillus plantarum Salmonella senftenberg Escherichia coli Saccharomyces cerevisiae
Short rods Straight rod to cocobacilli Rod with rounded ends Rods with rounded or squared ends Rod with rounded ends Straight rods Straight rods Ellipsoidal shape
0.4–0.5 0.5-2 0.5–0.8 1–3 0.7–1.0 2–4 Small: 0.5 1.2 large: 2.5 10 0.9–1.2 3–8 0.7–1.5 2–5 1–1.5 2–6 3–15 2–8
regarded as an estimate, only. However, Fig. 1 can provide information for a first approach towards the design of pulsed power laboratory or pilot-scale pasteurization units. It can be concluded that a field strength of approximately 30 kV/cm is sufficiently high to trigger electroporation of small bacteria like Listeria monocytogenes. However, due to the random cell orientation relative to the external electrical field, a fraction of the microbial population (which is progressively decreased with increasing cell diameters) will remain unaffected. Since membrane permeabilization is produced by ionic interfacial polarization in conductive media, a change in free enthalpy occurs which gives rise to the assumption that the pulse related energy W is another crucial process parameter beside the field strength E. Figure 2 compiles fundamental considerations concerning capacitor discharges as the most common technical solution for pulse generation. The voltage signal Utc which can be measured at the treatment chamber is mainly determined by the components of the discharge cirquit (Fig. 2a), i.e. the capacitance C and the charging voltage U0 of the storage capacitor, the ohmic resistances of the connectors Rconn and the treatment chamber Rtc, and the inductance L. If these quantities are known, eqn (5) accurately predicts Utc as a function of time. utc ¼ ael1t þ bel2t U0 Rtc a ¼ b ¼ rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 4L ðRconn þ Rtc Þ2 C sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Rconn þ Rtc Rconn þ Rtc 2 1 l1 ¼ þ LC 2L 2L sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2 Rconn þ Rtc Rconn þ Rtc 1 l2 ¼ LC 2L 2L
ð5Þ
This equation holds for aperiodic behaviour of the voltage at the treatment chamber which occurs in overdamped situations (D > 1, see eqn (6)).
Rconn þ Rtc D¼ 2
rffiffiffiffi C L
ð6Þ
For small cubic volume fractions, the field strength E is given by the ratio of voltage drop and edge length in field direction (Fig. 2b). Consequently, the specific current density per unit area perpendicular to the electric field is defined as S ¼ I=A. The electrical conductivity is the proportionality factor in Ohm’s law which relates the field strength and the current density: S ¼ E. The generated electric field in the treatment chamber is defined by the geometry and the electrical potential of the electrodes. During the discharge of the capacitor a pulsation of the electric field strength occurs in the exposed food (Fig. 2c). The specific power dis-
Fig. 2. Basic electrical circuit diagram of pulsed electric field devices. When the switch is open the capacitor (C) is charged across a charging resistor (Rcharge, typically several k) by a high voltage generator to a potential U0 against ground. When the switch is closed, the stored charge Q0 is released to the discharge circuit producing the current I=dQ/dt. The shape of the voltage signal (Utc) that can be measured at the treatment chamber is mainly determined by the components of the resonant cirquit, i.e. C, U0, the ohmic resistances of the connectors Rconn and the treatment chamber Rtc, and the inductance L.
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sideration is calculated by eqn (8). This equation also holds for pulses with other time characteristics like, e.g. square wave pulses which are generated by pulse-forming networks or partial discharges of over-dimensioned storage capacitors using on–off switches. In adiabatic situations the temperature increase t can be predicted from the effective pulse energy W: T ¼
Fig. 3. (Left) Temperature depencence of electrical conductivity of selected liquid foods (Data taken from Reitler, 1990). The linear relationship is also expressed by the slope which is indicated for the individual products. (LWE: liquid whole egg). (Right) Charging time constant of the cell membrane calculated from eqn (9) using the radius or the longer semi-axis for the sperical or ellipsoidal microorganisms from Table 1 (the indicating numbers of the curves refer to the numbering in Table 1). The diagram provides the charging time constant for these microorganisms suspended in different foods in a temperature range from 0 to 90 C. An example is given for for Lactobacillus brevis in beer at 40 C (see arrows).
sipation per unit volume is calculated by eqn (7) using the electrical conductivity of the treated product. ð 1 1 W¼ EðtÞ2 dt ð7Þ V 0 The power dissipation produces a Joule heating effect in the volume fraction under consideration. It is evident from this equation that energy cannot be treated separately from the effective field strength because of the quadratic dependence of W on E. However, as most pulsed power treatments apply repetitive pulses instead of one single pulse, the total specific pulse energy WT provides additional information on the treatment intensity. Hence, the occurring maximum Joule heating of the applied pulse train in the volume fraction under con-
W cp V
ð8Þ
where V and cp denote the volume and the specific heat capacity, respectively. The electrical conductivity which largely determines the crucial process parameters, and consequently affects the treatment chamber design, is an intrinsic property of the treated foods. Due to the ionic conduction of charges in those materials shows a considerable increase with raising temperatures. In Fig. 3 (left) this dependence is presented for six different fluids in a temperature range from 0 to 90 C. It is remarkable that in all examples the impact of temperature on can be approximated by linear fuctions (Reitler, 1990). Additional data on thermophysical and transport properties of these products are compiled in Table 2. From these data and the the slopes of the temperature function of , a quantity d/dw can be derived which provides useful information of the drop in treatment chamber resistance in response to a particular pulse treatment (see also Table 2). This knowledge is important for an appropriate design of the treatment chamber in a way that the damping of the discharge circuit remains well above a value of D=1 (eqn (6)) under operation condition. In poorly damped circuits voltage reversals might occur which substantially reduce the lifetime of the storage capacitor. Another direct effect of the medium conductivity on the efficiency of microbial inactivation can be derived from eqn (9) which yields the time constant of the membrane charging process during the exposure to a constant electrical field (Schwan, 1957):
Table 2. Selected physical properties of foods suitable for high intensity pulsed electric field treatment. Data taken from Kessler (1996), Hayes (1987) and from Food Properties Data Base: www.nel.uk/fooddb Products at 20 C
Thermal Thermal Prandtl Electrical Specific heat Conductivity change Density Viscosity (kg/m3) conductivity diffusivity conductivity capacity due to adiabatic (103 Pa s) number (W/m K) a (107 m2/s) Pr (—) (S/m) Cp (kJ/kg K) Joule heating d/dw ((S/m)/(kJ/kg))
Water (municipal) Beer (lager) Apple juice Orange juice Milk (1.5% fat) Liquid whole egg Egg white Tomato juice
0.06 0.20 0.29 0.44 0.57 0.57 0.65 1.50
4.18 3.80 3.85 3.77 4.14 3.20 3.81 3.98
0.301 103 1.211 103 1.958 103 2.801 103 3.019 103 3.906 103 n.a. 7.915 103
998 1007 1040 1040 1033 1043 1045 1010
0.60 0.52 0.56 0.55 0.58 0.50 0.59 0.50
1.45 1.36 1.40 1.40 1.36 1.50 1.48 1.24
1.00 1.45 1.93 1.78 1.34 6.00 4.31 5.60
7.0 10.6 13.3 12.2 9.6 38.4 27.8 44.6
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M ¼ RCM
i þ 2 2i þ M ði þ 2Þ
ð9Þ
CM denotes the specific membrane capacitance per unit area. For most biological cells this value is in the range of 1 F/cm2. The electrical conductivities of the membrane M and of the cell plasma i can be approximated by 10 nS/m and 1 S/m, respectively. Using these settings for CM, M, and i, the charging time constant M can be plotted as a function of the characteristic cell dimension R and of the medium conductivity . M indicates the time elapsed until an external electrical field E has induced a critical membrane potential difference ’M which is sufficiently high to cause electroporation. As discussed previously, the minimum field strength E is calculated from eqn (1) or eqn (3) assuming a membrane potential difference ’M of 1 V. In Fig. 3 (right), the charging time constant is presented using the characteristic cell dimensions of the previously introduced microorganisms (see Fig. 1). Figure 3 should be read from left to right, i.e. for a selected food material and process temperature the resulting electrical conductivity corresponds to a particular charging time constant of one of the microorganisms considered in this chart. For example, M of Lactobacillus brevis in beer at 40 C is approximately 0.08 S. It is evident from Fig. 3 that for a diversity of microorganisms and food products the required membrane charging time will always be longer than 10 nS and shorter than 0.5 S. Consequently, it appears that for all pulses longer than 1 S the impact of the medium conductivity on the occurance of electroporation has to be denied. However, this result should be used cautiously as it is based on the above described set of approximated boundary conditions and restrictions. Especially the lack of knowledge of the critical membrane potential ’M imposes considerable uncertainity on these considerations. It is reported that various treatment parameters should have an impact on this quantity. For example, it is well known that ’M can substantially be reduced by repetitive pulsing or by an increase in temperature (Neumann, 1989). Additionally, it has to be considered that the treatment chamber resistance Rtc is strongly affected by changes in product conductivity . For example, the resistance of an electrode system which consists of two parallel metal plates of a given area A which are placed in a distance l. The product flow through the gap produces a treatment chamber resistance of: Rtc ¼
I A
ð10Þ
An increase in conductivity, produced for instance by an increase in temperature due to the dissipated pulse
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energy (see d/dw in Table 1) will reduce Rtc. Consequently, the peak field strength between the electrodes and the specific energy input W will drop. In contrast to the negligible cirquit energy losses in the case when the connective resistance Rconn is small compared to Rtc, the drop in field strength can take considerable extents. In laboratory experiments as well as at industrial scale, pulsed power processing units control systems should be used which automatically compensate the field strength in the treatment chamber by changing the charging voltage U0 of the storage capacitor. Additionally, as variations in U0 also impact on pulse energy, the total specific energy input W (eqn (7)) has to be controlled by the pulse repetition rate, i.e. the average number of pulses applied to each volume fraction passing through the treatment chamber. For an experimentor, such a unit design which independently controls the settings of field strength and total specific energy input is suitable to identify reproducable results of microbial inactivation in continuous flow. For the design of high intensity pulsed electric field processing equipment eqn (10) (or similar equations for other geometries) provides important information regarding the dimensions of the electrode area and the gap. For the reasons of energy efficiency, Rtc should be chosen much larger than Rconn. The connective resistance Rconn is an essential part of the discharge cirquit as it limits the current according to the ratings of the high voltage switch in the case of arcing in the treatment chamber. With a given product conductivity k the possible range for dimensioning the gap and the area A is rather limited. Consequently, for pulsed power processing units with a volume flow of approx. 500 L/h the residence time of the product between the electrodes is typically less than 1 s.
Experimental results In Fig. 4, the reduction of Escherichia coli in response to pulsed electric fields is presented as a function of field strength on four different energy levels (left). Due to the experimental design chosen, the same set of data can be plotted versus the total specific energy input W with four constant field strength levels. In both presentations, threshold values for the onset of microbial inactivation are observed. The occurance of a critical field strength is consistent with theoretical considerations as discussed previously, although the experimental threshold level (approx. 5 kV/cm) is significantly lower than the predicted minimum field strength of 8 kV/cm (see Fig. 1). This deviation might be explainded by the comparably high treatment temperature (42 C) of the example shown in Fig. 4. In contrast to the critical field strength, the existence of a minimum energy level is rarely reported in food processing literature related to pulsed electric fields. Shortcomings of conventional pulse generators which
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do not control the field strength and total specific energy input might be the most likely reason. However, some fundamental works on the mechanisms of electroporation consider alterations of free enthalpy which coincide with membrane perturbations on the molecular level during the exposure to external electrical fields. For the design of pulsed power processing units, the knowledge of the threshold energy level is crucial. In excess of this value the extent of microbial inactivation increases linerarly. Further, it is noteworthy to indicate that the efficiency of pulse treatment strongly depends on the field strength applied (Fig. 4, left), i.e. the higher the field strength at constant energy levels, the higher is the lethality of the treatment. This trend was confirmed up to 50 kV/cm (Heinz & Knorr, 2000; Heinz, Phillips, Zenker, & Knorr, 1999; McDonalds, Lloyds, Vitale, Petersson, & Innings, 2000) or even up to 100 kV/cm (Boyko, Tur, Evdoshenko, Zarochentsev, & Ivanov, 1998). However, due to the limited dielectric strength of food materials (Ho & Mittal, 2000) these field strength levels should be considered with caution. Other more practical aspects of process design, discussed below, suggest that the maximum field strength should be restricted to levels in the range of 30 kV/cm. Numerous overviews on the impact of pulsed electric fields on microbial inactivation have been presented (Barbosa-Canovas, Gongora-Nieto, Pothakamury, & Swanson, 1999; Barsotti et al., 1999; Ho & Mittal, 1996; Jeyamkondan, Jayas, & Holley, 1999; Wouters & Smelt, 1997). Apart from these detailed literature studies, a
Fig. 4. Inactivation of E. coli HB5 in response to high intensity pulsed electric field treatment in a continuous flow system (Heinz, Alvarez, & Knorr, 2000). The bacteria were suspended in Ringer solution and the inlet temperature was 42 C. The treatment chamber used consisted of two parallely placed plane electrodes forming a gap of 2.5 mm. The experimental design provided that the effect of field strength has been studied at constant energy levels (left chart). Additionally, the investigated field strength has been approximately fixed to four different levels (right chart).
simplifying approach to review the relevant information might be useful in the context of this paper. In Fig. 5, the relationship between the total specific energy W and the electric field strength E necessary to inactivate 2 log cycles is compiled for several microorganisms obtained by different authors. It is the aim of this chart to provide an estimate of the efficacy of pulsed electric field treatment for the preservation of food. Hence, all details on medium composition, treatment temperature, time characteristics of the pulses, and electrical conductivity have been ignored. The low inactivation ratio of only 2 log cycles has been chosen because of the limited availability of studies which report higher reduction levels of the selected microorganisms. The influence of the cell size on the lethal effect of pulsed electric fields seems to be consistent with the previous theoretical considerations: L. monocytogenes is
Fig. 5. Approximation of crucial pulsed electric field treatment parameters required to produce a 2-log cycle (99%) inactivation of the bacteria, yeasts, and plant cells, listed below. The numbered lines indicate that identical lethal effects are obtained at different settings of the field strength and of the energy input, respectively. Energy levels in excess of the intermittent line (T 65 C) are expected to contribute a prevalent thermal effect on inactivation. Gram positive bacteria: (1) B. subtilis (vegetative cells) (Heinz, et al., 1999); (2) L. brevis (Grahl & Ma¨rkl, 1996); (3) L. plantarum (Raso et al., 1999); (4) L. monocytogenes (Raso et al. 1999). Gram negative bacteria: (5) E. coli (Evrendilek, Jin, Ruhlman, Qiu, Zhang, & Richter, 2000; Grahl & Ma¨rkl, 1996; Martı´n-Belloso, Vega-Mercado, Qin, Chang, Barbosa-Ca´novas, & Swanson, 1997; Qin, Zhang, BarbosaCa´novas, Swanson, & Pedrow, 1994; Zhang, Monsalve-Gonza´lez, Qin et al., 1994); (6) E. coli: continuous pulsed power treatment (Heinz et al., 2000); (7) S. senftenberg (Raso, Alvarez, Condon, & Trepat, 2000); (8) Y. enterocolitica (Raso et al., 1999). Yeast: (9) S. cerevisiae (Grahl & Ma¨rkl, 1996; Qin, Chang, Barbosa-Ca´novas, & Swanson, 1995; Zhang, Monsalve-Gonza´lez, Barbosa-Ca´novas et al., 1994; Zhang, Monsalve-Gonza´lez, Qin, Barbosa-Ca´novas, & Swanson, 1994). Plant cells: (10) Potato tissue (Knorr & Angersbach, 1998).
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the smallest organism in Fig. 5 and shows the highest resistance to high intensity pulsed electric field treatments. Also the high sensitivity of yeast and plant cells can be related to the cell size. However, the structure and the thickness of the cell seems to impact the efficiency of the pulse treatment. At a field strength of 28 kV/cm the gram positive and comparably large Bacillus subtilis requires more energy for the two log reduction than the gram negative and smaller E. coli or Salmonella senftenberg. Also Yersina enterocolitica being one of the smallest cells in this chart, requires less energy than all the other bacteria. The tendency that gram positive bacteria are more resistant than gram negative species has been discussed elsewhere (Hu¨lsheger, Potel, & Niemann, 1983; Qin, Barbosa-Ca´novas, Swanson, Pedrow, & Olsen, 1995; 1998; Sale & Hamilton, 1967; VegaMercado, Pothakamury, Chang, Barbosa-Ca´novas, & Swanson, 1996; Wouters, Dutreux, Smelt, & Lelieveld, 1999; Wouters & Smelt, 1997; Zhang, Monsalve-Gonza´lez, Barbosa-Ca´novas, & Swanson, 1994). Obviously, cell size and cell shape, as well as, the varying morphological and biochemical properties of the cells, are responsibles for the particular behaviour observed. It is evident that, for all microorganisms considered in Fig. 5, the required total energy input decreases when the pulse treatment is performed on higher field strength levels. Although lethal effects of pulse treatment are also observed at field strengths much below 20 kV/cm it is questionable whether an efficient pulsed power process can be designed at those specific energy input levels. Because the electroporative action is strongly reduced at those fieldstrength levels, the treatment is approaching pure ohmic heating. To demonstrate the extent of resistive heating of pulsed power, a threshold line at 250 kJ/ kg was introduced to Fig. 5 assuming that the corresponding warming up of approximately 65 C will primarily cause thermal damage rather than membrane electroporation. However, this consideration only holds for continuous high intensity pulsed electric field treatments when the total specific energy input is applied by one pulse train in the treatment chamber. If several pulse trains are applied to the product flow, e.g. in a multiple electrode setup with intermediate cooling systems, the resistive heating by pulsed power can strongly be reduced. Similar situations occur in batch treatment chambers with sufficiently low pulse repetition rates to allow thermal equilibration with the materials of the chamber. The results for L. monocytogenes (4), E. coli (5), and S. senftenberg (7) are obtained by using batch treatment chambers. In contrast to vegetative cells presented in Fig. 5, the resistance of bacterial spores against pulsed electric fields is much higher. No lethal effects were detected
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with spores of Clostridium tyrobutyricum, Bacillus cereus or ascospores of Bacillus nivea (Grahl & Ma¨rkl, 1996). Spores were resistant to pulsed electric field treatment even after the onset of germination (Paga´n, Esplugas, Go´ngora-Nieto, Barbosa-Ca´novas, & Swanson, 1998). However, Ma´rquez Mittal, and Griffiths (1997) obtained more than 3 log reductions of Bacillus subtilis and B. cereus spores in a salt solution. Ascospores of molds are not inactivated by pulsed electric fields (Raso, Caldero´n, Go´ngora, Barbosa-Ca´novas, & Swanson, 1998).
Conclusions and examples From the previously reported fundamental aspects and experimental data, it may be concluded that pulsed electric field treatment can beneficially be applied as a less severe alternative to thermal pasteurization. The decision whether pulsed power processing is advantageous for product quality and treatment efficacy can be estimated in a first approach by considering the relevant physical properties of the treated food. Those properties are primarily: the electrical conductivity and the specific heat capacity cp (see Table 2 for examples). Although a direct effect of on the mechanism of electric field induced membrane permeabilization can be neglected if the duration of the pulse at supercritical field strength is longer than 1 s (valid for microorganisms which are not smaller than 0.1 m), the dissipated pulse energy and the simultaneous resistive heating of the suspending medium is crucial for the efficiency of the treatment. The electrical load resistance of a pulsed power processing unit is mainly determined by the electrical conductivity of the product. Consequently, the time course of the pulse (eqn (5)) as well as the pulse energy (eqn (7)) are also affected by variations in . Geometrical changes of the electrode arrangement as well as changes of the connective resistance can be used to keep the load resistance at a desired level. However, these measures cannot compensate the decrease of the treatment chamber resistance due to the resistive heating during operation. Under these conditions the required field strength in the treatment chamber has to be maintained by raising the charging voltage. To control the simultaneously increased pulsed energy the repetition rate of the capacitor discharges must be changed. From Figs. 4 and 5, it can be extrapolated that at a field strength of approx. 30 kV/cm the total energy input required for 5 log cycles inactivation odds up to 150 kJ/kg. Since the residence time of the product in the treatment chamber is shorter than 1 s for industrial scale pulsed power processing systems, adiabatic conditions can be assumed. Hence, the corresponding change in electrical conductivity during the passage can be derived from the quantity d/dw, as given for selected foods in Table 2. In apple juice, the conductivity changes by
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0.3 S/m whereas in tomato juice a strong increase of 1.2 S/m can be expected when 150 kJ/kg pulsed power are introduced to the product flow. In the latter case, the control of field strength and total energy input is complicated because of the expectable large deviations of the load resistance. Additionally, the damping of the discharge circuit (eqn (6)) may approach or even fall below the critical value of 1 which produces strongly increased pulse rise times. To assure whether pulsed electric field pasteurization can be applied as a quality retaining process, the effect of the occuring resistive heating related to the estimated minimum energy input of 150 kJ/kg has to be checked. In the case of liquid whole egg this results in a temperature increase of 47 C. From this example, it is evident that the pulsed power process has to be equipped with heat exchangers which adjust the inlet temperature of the treatment chamber and quickly cool down the product after leaving the electrode system. During the passage through the treatment which lasts approx. 0.1 s in the case of a volume flow of 500 L/h and a treatment chamber dimension of 1 cm gap and 15 cm2 area, local overheatings may occur. These effects are due to inhomogenities in the electric field and are more pronounced when the thermal conductivity l is low as it is the case for liquid whole egg. Even if a residence time in the range of 10 s is obtained by suitable dimensions of electrode area and gap, the large Prandtl number Pr indicates that due to the situation within the boundary layer of the flow, thermal equilibration by heat removal across the electrodes is complicated. Consequently, cooling of the treatment chamber does not improve the situation. From Fig. 1, it can be derived that the theoretical critical field strength for small cells like Y. enterocolitica can take extremely high values if the orientation of the short semi-axis of the rod-shaped cells is in direction of the electric field. It appears contradictory that the experimental results in Fig. 5 show much lower field strength (less than 30 kV/cm) to be sufficient for microbial reduction. However, the theoretical value for rod shaped cells with the long end in the direction of the field is in the same range. It can be speculated whether the stochastic spacial orientation of microorganisms impacts on the efficiency of the treatment as it is the case with the diversity in size. The consequence for process design is to apply multiple pulses instead of only one and to support the rotation of the cells during the break times between the pulses. For this consideration, turbulent flow through the treatment chamber may be desirable. Another reason for this demand is that residence time distribution in turbulent flow is more homogeneous than in laminar flow. From this point of view, the treatment of products with low viscosity like, e.g. beer, milk or juices is more advantageous than liquid egg or tomato juice.
Outlook In this article, microbial inactivation in response to high intensity pulsed electric fields has been attributed to membrane permeabilization. Hence, most of the presented results are based on the ‘classical’ research work in the field of electroporation (Neumann, 1989; Zimmermann, 1996). This approach is justified for most of the in-use pulsed power applications in biotechnology and food process engineering. However, modifications of electric field treatment using, e.g. pulsed radio frequencies (Chang, 1989), may yield completely different results regarding threshold field strength and pulse energy requirements. Future work will have to consider these aspects in order to further optimize the preservation of liquid foods by pulsed power.
Acknowledgement Parts of the presented work has been carried out with finacial support of the European Community within the project: FAIR CT97-3044 (HELP) and EVK1-200000596 (WIRES).
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apple juice using pulsed electric fields. Lebensmittelwissenschaft und -Technologie, 28, 564–568. Qin, B.-L., Zhang, Q., Barbosa-Ca´novas, G. V., Swanson, B. G., & Pedrow, P. D. (1994). Inactivation of microorganisms by pulsed electric fields of different voltage waveforms. IEEE Transactions of Dielectrical and Electrical Insulation, 1, 1047–1057. Ramesh, M. N. (1999). Food preservation by heat treatment. In M. S. Rahman (Ed.), Handbook of food preservation (pp. 95–172). New York: Marcel Dekker. Raso, J., Alvarez, I., Condon, S., & Trepat, F. J. S. (2000). Predicting inactivation of Salmonella senftenberg by pulsed electric fields. Innovative Food Science and Emerging Technologies, 1, 21–30. Raso, J., Alvarez, I., Condon, S., & Trepat, F. J. S. (1999). Unpublished data. Raso, J., Caldero´n, M. L., Go´ngora, M., Barbosa-Ca´novas, G. V., & Swanson, B. G. (1998). Inactivation of mold ascospores and conidiospores suspended in fruit juices by pulsed electric fields. Lebensmittelwissenschaft und -Technologie, 31, 668–672. Reitler, W. (1990). Conductive heating of foods. PhD thesis, TU Munich, Munich. Sale, A. J., & Hamilton, W. A. (1967). Effect of high electric fields on micro-organisms. I. Killing of bacteria and yeast. II. Mechanism of action of the lethal effect. Biochimica Biophysica Acta, 148, 781–800. Schwan, H. P. (1957). Electrical properties of tissue and cell suspensions. Advances in Biological and Medical Physics, 5, 147– 209. Vega-Mercado, H., Pothakamury, U. R., Chang, F.-J., BarbosaCanovas, G. V., & Swanson, B. G. (1996). Inactivation of Escherichia coli by combining pH, ionic strength and pulsed electric field hurdles. Food Research International, 29, 117–121. Wouters, P. C., Dutreux, N., Smelt, J. P. P. M., & Lelieveld, H. L. M. (1999). Effects of pulsed electric fields on inactivation kinetics of Listeria innocua. Applied and Environmental Microbiology, 65, 5364–5371. Wouters, P. C., & Smelt, J. P. P. M. (1997). Inactivation of microorganisms with pulsed electric fields: Potential for food preservation. Food Biotechnology, 11, 193–229. Zhang, Q., Monsalve-Gonza´lez, A., Barbosa-Ca´novas, G. V., & Swanson, B. G. (1994a). Inactivation of Escherichia coli and Saccharomyces cerevisiae by pulsed electric fields under controlled temperature conditions. Transactions of the American Society of Agricultural Engineering, 37, 581–587. Zhang, Q., Monsalve-Gonza´lez, A., Qin, B. L., Barbosa-Ca´novas, G. V., & Swanson, B. G. (1994b). Inactivation of Saccharomyces cerevisiae in apple juice by square-wave and exponential-decay pulsed electric fields. Journal of Food Processing and Engineering, 17, 469–478. Zimmermann, U. (1996). The effect of high intensity electric field pulses on eucaryotic cell membranes: fundamentals and applications. In U. Zimmermann, & G. A. Neil (Eds.), Electromanipulation of cells (pp. 1–106). Boca Raton: CRC Press. Zimmermann, U., Pilwat, G., & Riemann, F. (1974). Dielectric breakdown in cell membranes. Biophysical Journal, 881–899.
Review
Preservation of liquid foods by high intensity pulsed electric fields—basic concepts for process design V. Heinz,a,* I. Alvarez,b A. Angersbacha and D. Knorra a
Department of Food Biotechnology and Food Process Engineering, TU Berlin, Ko¨nigin-Luise-Str. 22, D-14195 Berlin, Germany (tel: +49-30-314-71250; fax: +49-30-832-7663; e-mail: [email protected]) b Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, ES-50013 Zaragoza, Spain In excess of a critical transmembrane potential ’M of 1 V produced by high intensity pulsed electric fields a rapid electrical breakdown and local conformational changes of cell membranes occur which result in a drastic increase in permeability and an equilibration of the electrochemical and electrical potential differences of the cell plasma and the extracellular medium. As irreverible membrane permeabilization impairs most vital physiological control systems, high intensity pulsed electric fields may be applied as a highly effective process for the microbial decontamination of liquid foods. The efficiency of the treatment is largely influenced by the inherent properties of the foods and of the spoiling microorganisms. In addition a number of technical limitations have to be considered. In this review an * Corresponding author.
approach is presented which reduces the diversity of parameters that affect microbial inactivation during pulsed power treatment. In particular, the required total specific energy input is discussed. # 2001 Published by Elsevier Science Ltd.
Introduction Microbial cells which are exposed to an external electrical field for a few microseconds respond by an electrical breakdown and local structural changes of the cell membrane. In consequence of the so called electroporation, a drastic increase in permeability is observed which in the irreversible case is equivalent to a loss of viability. This type of non-thermal inactivation of microorganisms by high intensity pulsed electric fields might be beneficial for the development of quality retaining preservation processes in the food industry. However, process safety, cost-effectiveness, and consumer benefits of pulsed electric field treatment have to be confirmed. Despite the extensive knowledge in food preservation by heat treatment (Larousse & Brown, 1997; Ramesh, 1999) and despite continued attempts to improve the quality of processed foods (Durance, 1997) there is still a need for technologies that minimize the destructive influence of heat on valuable food compounds (Knorr & Heinz, 2001). Even intelligent concepts like high-temperature–short-time processing fail if heat transfer and/ or heat penetration is limited by intrinsic thermophysical properties of the product. Since the thermal energy which is required to destroy the contaminating microorganisms has to be transmitted across the product itself, the design of fast and uniform heating and cooling steps is one of the primary challenges of industrial preservation by heat. Most of the thermal processing equipment in use consists of systems which transfer heat across an interface driven by a temperature gradient. On the product side only the convective heat transport can be enhanced by external measures, i.e. by the generation of turbulent flow. Compared to thermal inactivation the destruction of microbes by electroporation shows no time delay with respect to the propagation of the lethal treatment intensity.
Fundamental aspects In electrically conductive food placed between a high voltage and a grounded electrode the developing electrical field can be predicted from the Laplace equation
0924-2244/01/$ - see front matter Copyright # 2001 Published by Elsevier Science Ltd. PII: S 0 92 4 - 2 24 4 ( 0 1 ) 0 0 06 4 - 4
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r2 ’ ¼ 0, where ’ denotes the electrical potential. At the microbial membranes which are electrically insulating the electrical field produces the accumulation of charges with opposite polarity on either side of the bi-layer structure. Pore formation occurs when a certain threshold value of the electric field strength E is exceeded. However, in spite of the subtantial knowledge of the impact of high intensity pulsed electric fields on microorganisms (Barsotti, Merle, & Cheftel, 1999; Ho & Mittal, 2000; Wouters & Smelt, 1997) until now there is no clear evidence on the underlying mechanism of membrane permeabilization at the molecular level. The potential difference ’M at the membrane of a biological cell with spherical shape and a radius R induced by the external electrical field E can be approximated by eqn (1) which is derived from solving Maxwell’s equations in spherical coordinates assuming several simplifying restrictions (Neumann, 1996): 3 ’M ¼ EfðÞRcos 2
ð1Þ
For the calculation of ’M at a particular location at the membrane, the angle a of the radial direction vector has to be specified. Per definition is zero (and cosa=1) when the vector coincides with the direction of the electrical field. Hence, the highest membrane potential differences are assumed to occur at the two poles of the cell in field direction. The factor fðÞ is an explicit function of the electrical conductivities of the suspending medium , the plasma i , the cell membrane M , and the ratio of the membrane thickness and the cell radius (Neumann, 1989): fðÞ ¼
1 1 þ ðM ð2 þ i =Þ=ð2i d=RÞÞ
In literature (Zimmermann, 1996), it is reported that in excess of a critical transmembrane potential ’M of 1 V a rapid electrical breakdown and local conformational changes of bilayer structures occur. A drastic increase in permeability re-establishes the equilibrium of the electrochemical and electrical potential differences of the cell plasma and the extracellular medium. Simultaneously, the neutralization of the transmembrane gradient across the membrane irreversibly impairs vital physiological control systems of the cell like osmoregulation and consequently cell death occurs. Making use of the experimental result for ’M which leads to electroporation eqn (1) can be solved for the critical field strength E. From a practical point of view, this rearrangement is helpful as it allows the prediction of the minimum field strength required for preservation processes. In Fig. 1, the impact of cell size and geometry on E is presented. It is evident that the critical field strength sharply increases when the characteristic dimension of the cell (i.e. the radius of sperical cells or the shorter half-axis of elliptical cells) is shifted to smaller values. Moreover, variations in cell shape produce a considerable raise of E. A rod-shaped cell requires a more than 5 times higher electrical field than a spherical cell with the same characteristic dimension. Due to the large diversity in cell size and shape of a microbial population the prediction of the critical field strength for a number of microorganisms has to be
ð2Þ
However, M being in the range of nS/m for intact membranes it is readily seen from eqn (2) that fðÞ can be approximated by 1, independently from the electrical conductivity of the suspending medium. For cells with non-spheric shape an estimate for ’M can be obtained by solving the Maxwell’s equations in ellipsoidal coordinates. In those situations eqn (3) has to be applied (Zimmermann, Pilwat, & Riemann, 1974): ’M ¼ fðAÞAF E
ð3Þ
This formula yields the local membrane potential difference at the distance AF from the centre in direction of the external electrical field. The shape factor f(A) is a function of the three semi-axis (A1, A2, A3) of elliptical cells: fðAÞ ¼
2
ð1 1=
2 A1 A2 A3 0
sþ
A2F
3 qffiffiffiffiffiffiffiffiffiffiffiffiffiffi X s þ A2n Þ ð n¼1
!
ð4Þ ds
Fig. 1. Critical electroporative field strength as a function of cell dimensions. The membrane potential difference ’M is assumed to be 1 V. Curve A is calculated from eqn (1) where the radius is taken as the characteristic cell dimension. For elliptical cells eqn (3) is used. The critical field strength is plotted against the shorter semi-axis. Curves B–E are calculated for different ratios of the short and the long semi-axis: B, D: 0.5; C, E: 0.2. For each ratio, the critical field strength is presented for two different orientations of the cell relative to the direction of the electric field. Mean size and typical shape of the microorganisms mentioned above is compiled in Table 1.
V. Heinz et al. / Trends in Food Science & Technology 12 (2002) 103–111
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Table 1. Geometry and mean size of selected microorganisms. Data taken from Bergey (1986)
1. 2. 3. 4. 5. 6. 7. 8.
Organism
Shape
Size (m)
Listeria monocytogenes Yersinia enterocolitica Lactobacillus brevis Bacillus subtilis (vegetative cells) Lactobacillus plantarum Salmonella senftenberg Escherichia coli Saccharomyces cerevisiae
Short rods Straight rod to cocobacilli Rod with rounded ends Rods with rounded or squared ends Rod with rounded ends Straight rods Straight rods Ellipsoidal shape
0.4–0.5 0.5-2 0.5–0.8 1–3 0.7–1.0 2–4 Small: 0.5 1.2 large: 2.5 10 0.9–1.2 3–8 0.7–1.5 2–5 1–1.5 2–6 3–15 2–8
regarded as an estimate, only. However, Fig. 1 can provide information for a first approach towards the design of pulsed power laboratory or pilot-scale pasteurization units. It can be concluded that a field strength of approximately 30 kV/cm is sufficiently high to trigger electroporation of small bacteria like Listeria monocytogenes. However, due to the random cell orientation relative to the external electrical field, a fraction of the microbial population (which is progressively decreased with increasing cell diameters) will remain unaffected. Since membrane permeabilization is produced by ionic interfacial polarization in conductive media, a change in free enthalpy occurs which gives rise to the assumption that the pulse related energy W is another crucial process parameter beside the field strength E. Figure 2 compiles fundamental considerations concerning capacitor discharges as the most common technical solution for pulse generation. The voltage signal Utc which can be measured at the treatment chamber is mainly determined by the components of the discharge cirquit (Fig. 2a), i.e. the capacitance C and the charging voltage U0 of the storage capacitor, the ohmic resistances of the connectors Rconn and the treatment chamber Rtc, and the inductance L. If these quantities are known, eqn (5) accurately predicts Utc as a function of time. utc ¼ ael1t þ bel2t U0 Rtc a ¼ b ¼ rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 4L ðRconn þ Rtc Þ2 C sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Rconn þ Rtc Rconn þ Rtc 2 1 l1 ¼ þ LC 2L 2L sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2 Rconn þ Rtc Rconn þ Rtc 1 l2 ¼ LC 2L 2L
ð5Þ
This equation holds for aperiodic behaviour of the voltage at the treatment chamber which occurs in overdamped situations (D > 1, see eqn (6)).
Rconn þ Rtc D¼ 2
rffiffiffiffi C L
ð6Þ
For small cubic volume fractions, the field strength E is given by the ratio of voltage drop and edge length in field direction (Fig. 2b). Consequently, the specific current density per unit area perpendicular to the electric field is defined as S ¼ I=A. The electrical conductivity is the proportionality factor in Ohm’s law which relates the field strength and the current density: S ¼ E. The generated electric field in the treatment chamber is defined by the geometry and the electrical potential of the electrodes. During the discharge of the capacitor a pulsation of the electric field strength occurs in the exposed food (Fig. 2c). The specific power dis-
Fig. 2. Basic electrical circuit diagram of pulsed electric field devices. When the switch is open the capacitor (C) is charged across a charging resistor (Rcharge, typically several k) by a high voltage generator to a potential U0 against ground. When the switch is closed, the stored charge Q0 is released to the discharge circuit producing the current I=dQ/dt. The shape of the voltage signal (Utc) that can be measured at the treatment chamber is mainly determined by the components of the resonant cirquit, i.e. C, U0, the ohmic resistances of the connectors Rconn and the treatment chamber Rtc, and the inductance L.
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sideration is calculated by eqn (8). This equation also holds for pulses with other time characteristics like, e.g. square wave pulses which are generated by pulse-forming networks or partial discharges of over-dimensioned storage capacitors using on–off switches. In adiabatic situations the temperature increase t can be predicted from the effective pulse energy W: T ¼
Fig. 3. (Left) Temperature depencence of electrical conductivity of selected liquid foods (Data taken from Reitler, 1990). The linear relationship is also expressed by the slope which is indicated for the individual products. (LWE: liquid whole egg). (Right) Charging time constant of the cell membrane calculated from eqn (9) using the radius or the longer semi-axis for the sperical or ellipsoidal microorganisms from Table 1 (the indicating numbers of the curves refer to the numbering in Table 1). The diagram provides the charging time constant for these microorganisms suspended in different foods in a temperature range from 0 to 90 C. An example is given for for Lactobacillus brevis in beer at 40 C (see arrows).
sipation per unit volume is calculated by eqn (7) using the electrical conductivity of the treated product. ð 1 1 W¼ EðtÞ2 dt ð7Þ V 0 The power dissipation produces a Joule heating effect in the volume fraction under consideration. It is evident from this equation that energy cannot be treated separately from the effective field strength because of the quadratic dependence of W on E. However, as most pulsed power treatments apply repetitive pulses instead of one single pulse, the total specific pulse energy WT provides additional information on the treatment intensity. Hence, the occurring maximum Joule heating of the applied pulse train in the volume fraction under con-
W cp V
ð8Þ
where V and cp denote the volume and the specific heat capacity, respectively. The electrical conductivity which largely determines the crucial process parameters, and consequently affects the treatment chamber design, is an intrinsic property of the treated foods. Due to the ionic conduction of charges in those materials shows a considerable increase with raising temperatures. In Fig. 3 (left) this dependence is presented for six different fluids in a temperature range from 0 to 90 C. It is remarkable that in all examples the impact of temperature on can be approximated by linear fuctions (Reitler, 1990). Additional data on thermophysical and transport properties of these products are compiled in Table 2. From these data and the the slopes of the temperature function of , a quantity d/dw can be derived which provides useful information of the drop in treatment chamber resistance in response to a particular pulse treatment (see also Table 2). This knowledge is important for an appropriate design of the treatment chamber in a way that the damping of the discharge circuit remains well above a value of D=1 (eqn (6)) under operation condition. In poorly damped circuits voltage reversals might occur which substantially reduce the lifetime of the storage capacitor. Another direct effect of the medium conductivity on the efficiency of microbial inactivation can be derived from eqn (9) which yields the time constant of the membrane charging process during the exposure to a constant electrical field (Schwan, 1957):
Table 2. Selected physical properties of foods suitable for high intensity pulsed electric field treatment. Data taken from Kessler (1996), Hayes (1987) and from Food Properties Data Base: www.nel.uk/fooddb Products at 20 C
Thermal Thermal Prandtl Electrical Specific heat Conductivity change Density Viscosity (kg/m3) conductivity diffusivity conductivity capacity due to adiabatic (103 Pa s) number (W/m K) a (107 m2/s) Pr (—) (S/m) Cp (kJ/kg K) Joule heating d/dw ((S/m)/(kJ/kg))
Water (municipal) Beer (lager) Apple juice Orange juice Milk (1.5% fat) Liquid whole egg Egg white Tomato juice
0.06 0.20 0.29 0.44 0.57 0.57 0.65 1.50
4.18 3.80 3.85 3.77 4.14 3.20 3.81 3.98
0.301 103 1.211 103 1.958 103 2.801 103 3.019 103 3.906 103 n.a. 7.915 103
998 1007 1040 1040 1033 1043 1045 1010
0.60 0.52 0.56 0.55 0.58 0.50 0.59 0.50
1.45 1.36 1.40 1.40 1.36 1.50 1.48 1.24
1.00 1.45 1.93 1.78 1.34 6.00 4.31 5.60
7.0 10.6 13.3 12.2 9.6 38.4 27.8 44.6
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M ¼ RCM
i þ 2 2i þ M ði þ 2Þ
ð9Þ
CM denotes the specific membrane capacitance per unit area. For most biological cells this value is in the range of 1 F/cm2. The electrical conductivities of the membrane M and of the cell plasma i can be approximated by 10 nS/m and 1 S/m, respectively. Using these settings for CM, M, and i, the charging time constant M can be plotted as a function of the characteristic cell dimension R and of the medium conductivity . M indicates the time elapsed until an external electrical field E has induced a critical membrane potential difference ’M which is sufficiently high to cause electroporation. As discussed previously, the minimum field strength E is calculated from eqn (1) or eqn (3) assuming a membrane potential difference ’M of 1 V. In Fig. 3 (right), the charging time constant is presented using the characteristic cell dimensions of the previously introduced microorganisms (see Fig. 1). Figure 3 should be read from left to right, i.e. for a selected food material and process temperature the resulting electrical conductivity corresponds to a particular charging time constant of one of the microorganisms considered in this chart. For example, M of Lactobacillus brevis in beer at 40 C is approximately 0.08 S. It is evident from Fig. 3 that for a diversity of microorganisms and food products the required membrane charging time will always be longer than 10 nS and shorter than 0.5 S. Consequently, it appears that for all pulses longer than 1 S the impact of the medium conductivity on the occurance of electroporation has to be denied. However, this result should be used cautiously as it is based on the above described set of approximated boundary conditions and restrictions. Especially the lack of knowledge of the critical membrane potential ’M imposes considerable uncertainity on these considerations. It is reported that various treatment parameters should have an impact on this quantity. For example, it is well known that ’M can substantially be reduced by repetitive pulsing or by an increase in temperature (Neumann, 1989). Additionally, it has to be considered that the treatment chamber resistance Rtc is strongly affected by changes in product conductivity . For example, the resistance of an electrode system which consists of two parallel metal plates of a given area A which are placed in a distance l. The product flow through the gap produces a treatment chamber resistance of: Rtc ¼
I A
ð10Þ
An increase in conductivity, produced for instance by an increase in temperature due to the dissipated pulse
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energy (see d/dw in Table 1) will reduce Rtc. Consequently, the peak field strength between the electrodes and the specific energy input W will drop. In contrast to the negligible cirquit energy losses in the case when the connective resistance Rconn is small compared to Rtc, the drop in field strength can take considerable extents. In laboratory experiments as well as at industrial scale, pulsed power processing units control systems should be used which automatically compensate the field strength in the treatment chamber by changing the charging voltage U0 of the storage capacitor. Additionally, as variations in U0 also impact on pulse energy, the total specific energy input W (eqn (7)) has to be controlled by the pulse repetition rate, i.e. the average number of pulses applied to each volume fraction passing through the treatment chamber. For an experimentor, such a unit design which independently controls the settings of field strength and total specific energy input is suitable to identify reproducable results of microbial inactivation in continuous flow. For the design of high intensity pulsed electric field processing equipment eqn (10) (or similar equations for other geometries) provides important information regarding the dimensions of the electrode area and the gap. For the reasons of energy efficiency, Rtc should be chosen much larger than Rconn. The connective resistance Rconn is an essential part of the discharge cirquit as it limits the current according to the ratings of the high voltage switch in the case of arcing in the treatment chamber. With a given product conductivity k the possible range for dimensioning the gap and the area A is rather limited. Consequently, for pulsed power processing units with a volume flow of approx. 500 L/h the residence time of the product between the electrodes is typically less than 1 s.
Experimental results In Fig. 4, the reduction of Escherichia coli in response to pulsed electric fields is presented as a function of field strength on four different energy levels (left). Due to the experimental design chosen, the same set of data can be plotted versus the total specific energy input W with four constant field strength levels. In both presentations, threshold values for the onset of microbial inactivation are observed. The occurance of a critical field strength is consistent with theoretical considerations as discussed previously, although the experimental threshold level (approx. 5 kV/cm) is significantly lower than the predicted minimum field strength of 8 kV/cm (see Fig. 1). This deviation might be explainded by the comparably high treatment temperature (42 C) of the example shown in Fig. 4. In contrast to the critical field strength, the existence of a minimum energy level is rarely reported in food processing literature related to pulsed electric fields. Shortcomings of conventional pulse generators which
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do not control the field strength and total specific energy input might be the most likely reason. However, some fundamental works on the mechanisms of electroporation consider alterations of free enthalpy which coincide with membrane perturbations on the molecular level during the exposure to external electrical fields. For the design of pulsed power processing units, the knowledge of the threshold energy level is crucial. In excess of this value the extent of microbial inactivation increases linerarly. Further, it is noteworthy to indicate that the efficiency of pulse treatment strongly depends on the field strength applied (Fig. 4, left), i.e. the higher the field strength at constant energy levels, the higher is the lethality of the treatment. This trend was confirmed up to 50 kV/cm (Heinz & Knorr, 2000; Heinz, Phillips, Zenker, & Knorr, 1999; McDonalds, Lloyds, Vitale, Petersson, & Innings, 2000) or even up to 100 kV/cm (Boyko, Tur, Evdoshenko, Zarochentsev, & Ivanov, 1998). However, due to the limited dielectric strength of food materials (Ho & Mittal, 2000) these field strength levels should be considered with caution. Other more practical aspects of process design, discussed below, suggest that the maximum field strength should be restricted to levels in the range of 30 kV/cm. Numerous overviews on the impact of pulsed electric fields on microbial inactivation have been presented (Barbosa-Canovas, Gongora-Nieto, Pothakamury, & Swanson, 1999; Barsotti et al., 1999; Ho & Mittal, 1996; Jeyamkondan, Jayas, & Holley, 1999; Wouters & Smelt, 1997). Apart from these detailed literature studies, a
Fig. 4. Inactivation of E. coli HB5 in response to high intensity pulsed electric field treatment in a continuous flow system (Heinz, Alvarez, & Knorr, 2000). The bacteria were suspended in Ringer solution and the inlet temperature was 42 C. The treatment chamber used consisted of two parallely placed plane electrodes forming a gap of 2.5 mm. The experimental design provided that the effect of field strength has been studied at constant energy levels (left chart). Additionally, the investigated field strength has been approximately fixed to four different levels (right chart).
simplifying approach to review the relevant information might be useful in the context of this paper. In Fig. 5, the relationship between the total specific energy W and the electric field strength E necessary to inactivate 2 log cycles is compiled for several microorganisms obtained by different authors. It is the aim of this chart to provide an estimate of the efficacy of pulsed electric field treatment for the preservation of food. Hence, all details on medium composition, treatment temperature, time characteristics of the pulses, and electrical conductivity have been ignored. The low inactivation ratio of only 2 log cycles has been chosen because of the limited availability of studies which report higher reduction levels of the selected microorganisms. The influence of the cell size on the lethal effect of pulsed electric fields seems to be consistent with the previous theoretical considerations: L. monocytogenes is
Fig. 5. Approximation of crucial pulsed electric field treatment parameters required to produce a 2-log cycle (99%) inactivation of the bacteria, yeasts, and plant cells, listed below. The numbered lines indicate that identical lethal effects are obtained at different settings of the field strength and of the energy input, respectively. Energy levels in excess of the intermittent line (T 65 C) are expected to contribute a prevalent thermal effect on inactivation. Gram positive bacteria: (1) B. subtilis (vegetative cells) (Heinz, et al., 1999); (2) L. brevis (Grahl & Ma¨rkl, 1996); (3) L. plantarum (Raso et al., 1999); (4) L. monocytogenes (Raso et al. 1999). Gram negative bacteria: (5) E. coli (Evrendilek, Jin, Ruhlman, Qiu, Zhang, & Richter, 2000; Grahl & Ma¨rkl, 1996; Martı´n-Belloso, Vega-Mercado, Qin, Chang, Barbosa-Ca´novas, & Swanson, 1997; Qin, Zhang, BarbosaCa´novas, Swanson, & Pedrow, 1994; Zhang, Monsalve-Gonza´lez, Qin et al., 1994); (6) E. coli: continuous pulsed power treatment (Heinz et al., 2000); (7) S. senftenberg (Raso, Alvarez, Condon, & Trepat, 2000); (8) Y. enterocolitica (Raso et al., 1999). Yeast: (9) S. cerevisiae (Grahl & Ma¨rkl, 1996; Qin, Chang, Barbosa-Ca´novas, & Swanson, 1995; Zhang, Monsalve-Gonza´lez, Barbosa-Ca´novas et al., 1994; Zhang, Monsalve-Gonza´lez, Qin, Barbosa-Ca´novas, & Swanson, 1994). Plant cells: (10) Potato tissue (Knorr & Angersbach, 1998).
V. Heinz et al. / Trends in Food Science & Technology 12 (2002) 103–111
the smallest organism in Fig. 5 and shows the highest resistance to high intensity pulsed electric field treatments. Also the high sensitivity of yeast and plant cells can be related to the cell size. However, the structure and the thickness of the cell seems to impact the efficiency of the pulse treatment. At a field strength of 28 kV/cm the gram positive and comparably large Bacillus subtilis requires more energy for the two log reduction than the gram negative and smaller E. coli or Salmonella senftenberg. Also Yersina enterocolitica being one of the smallest cells in this chart, requires less energy than all the other bacteria. The tendency that gram positive bacteria are more resistant than gram negative species has been discussed elsewhere (Hu¨lsheger, Potel, & Niemann, 1983; Qin, Barbosa-Ca´novas, Swanson, Pedrow, & Olsen, 1995; 1998; Sale & Hamilton, 1967; VegaMercado, Pothakamury, Chang, Barbosa-Ca´novas, & Swanson, 1996; Wouters, Dutreux, Smelt, & Lelieveld, 1999; Wouters & Smelt, 1997; Zhang, Monsalve-Gonza´lez, Barbosa-Ca´novas, & Swanson, 1994). Obviously, cell size and cell shape, as well as, the varying morphological and biochemical properties of the cells, are responsibles for the particular behaviour observed. It is evident that, for all microorganisms considered in Fig. 5, the required total energy input decreases when the pulse treatment is performed on higher field strength levels. Although lethal effects of pulse treatment are also observed at field strengths much below 20 kV/cm it is questionable whether an efficient pulsed power process can be designed at those specific energy input levels. Because the electroporative action is strongly reduced at those fieldstrength levels, the treatment is approaching pure ohmic heating. To demonstrate the extent of resistive heating of pulsed power, a threshold line at 250 kJ/ kg was introduced to Fig. 5 assuming that the corresponding warming up of approximately 65 C will primarily cause thermal damage rather than membrane electroporation. However, this consideration only holds for continuous high intensity pulsed electric field treatments when the total specific energy input is applied by one pulse train in the treatment chamber. If several pulse trains are applied to the product flow, e.g. in a multiple electrode setup with intermediate cooling systems, the resistive heating by pulsed power can strongly be reduced. Similar situations occur in batch treatment chambers with sufficiently low pulse repetition rates to allow thermal equilibration with the materials of the chamber. The results for L. monocytogenes (4), E. coli (5), and S. senftenberg (7) are obtained by using batch treatment chambers. In contrast to vegetative cells presented in Fig. 5, the resistance of bacterial spores against pulsed electric fields is much higher. No lethal effects were detected
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with spores of Clostridium tyrobutyricum, Bacillus cereus or ascospores of Bacillus nivea (Grahl & Ma¨rkl, 1996). Spores were resistant to pulsed electric field treatment even after the onset of germination (Paga´n, Esplugas, Go´ngora-Nieto, Barbosa-Ca´novas, & Swanson, 1998). However, Ma´rquez Mittal, and Griffiths (1997) obtained more than 3 log reductions of Bacillus subtilis and B. cereus spores in a salt solution. Ascospores of molds are not inactivated by pulsed electric fields (Raso, Caldero´n, Go´ngora, Barbosa-Ca´novas, & Swanson, 1998).
Conclusions and examples From the previously reported fundamental aspects and experimental data, it may be concluded that pulsed electric field treatment can beneficially be applied as a less severe alternative to thermal pasteurization. The decision whether pulsed power processing is advantageous for product quality and treatment efficacy can be estimated in a first approach by considering the relevant physical properties of the treated food. Those properties are primarily: the electrical conductivity and the specific heat capacity cp (see Table 2 for examples). Although a direct effect of on the mechanism of electric field induced membrane permeabilization can be neglected if the duration of the pulse at supercritical field strength is longer than 1 s (valid for microorganisms which are not smaller than 0.1 m), the dissipated pulse energy and the simultaneous resistive heating of the suspending medium is crucial for the efficiency of the treatment. The electrical load resistance of a pulsed power processing unit is mainly determined by the electrical conductivity of the product. Consequently, the time course of the pulse (eqn (5)) as well as the pulse energy (eqn (7)) are also affected by variations in . Geometrical changes of the electrode arrangement as well as changes of the connective resistance can be used to keep the load resistance at a desired level. However, these measures cannot compensate the decrease of the treatment chamber resistance due to the resistive heating during operation. Under these conditions the required field strength in the treatment chamber has to be maintained by raising the charging voltage. To control the simultaneously increased pulsed energy the repetition rate of the capacitor discharges must be changed. From Figs. 4 and 5, it can be extrapolated that at a field strength of approx. 30 kV/cm the total energy input required for 5 log cycles inactivation odds up to 150 kJ/kg. Since the residence time of the product in the treatment chamber is shorter than 1 s for industrial scale pulsed power processing systems, adiabatic conditions can be assumed. Hence, the corresponding change in electrical conductivity during the passage can be derived from the quantity d/dw, as given for selected foods in Table 2. In apple juice, the conductivity changes by
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0.3 S/m whereas in tomato juice a strong increase of 1.2 S/m can be expected when 150 kJ/kg pulsed power are introduced to the product flow. In the latter case, the control of field strength and total energy input is complicated because of the expectable large deviations of the load resistance. Additionally, the damping of the discharge circuit (eqn (6)) may approach or even fall below the critical value of 1 which produces strongly increased pulse rise times. To assure whether pulsed electric field pasteurization can be applied as a quality retaining process, the effect of the occuring resistive heating related to the estimated minimum energy input of 150 kJ/kg has to be checked. In the case of liquid whole egg this results in a temperature increase of 47 C. From this example, it is evident that the pulsed power process has to be equipped with heat exchangers which adjust the inlet temperature of the treatment chamber and quickly cool down the product after leaving the electrode system. During the passage through the treatment which lasts approx. 0.1 s in the case of a volume flow of 500 L/h and a treatment chamber dimension of 1 cm gap and 15 cm2 area, local overheatings may occur. These effects are due to inhomogenities in the electric field and are more pronounced when the thermal conductivity l is low as it is the case for liquid whole egg. Even if a residence time in the range of 10 s is obtained by suitable dimensions of electrode area and gap, the large Prandtl number Pr indicates that due to the situation within the boundary layer of the flow, thermal equilibration by heat removal across the electrodes is complicated. Consequently, cooling of the treatment chamber does not improve the situation. From Fig. 1, it can be derived that the theoretical critical field strength for small cells like Y. enterocolitica can take extremely high values if the orientation of the short semi-axis of the rod-shaped cells is in direction of the electric field. It appears contradictory that the experimental results in Fig. 5 show much lower field strength (less than 30 kV/cm) to be sufficient for microbial reduction. However, the theoretical value for rod shaped cells with the long end in the direction of the field is in the same range. It can be speculated whether the stochastic spacial orientation of microorganisms impacts on the efficiency of the treatment as it is the case with the diversity in size. The consequence for process design is to apply multiple pulses instead of only one and to support the rotation of the cells during the break times between the pulses. For this consideration, turbulent flow through the treatment chamber may be desirable. Another reason for this demand is that residence time distribution in turbulent flow is more homogeneous than in laminar flow. From this point of view, the treatment of products with low viscosity like, e.g. beer, milk or juices is more advantageous than liquid egg or tomato juice.
Outlook In this article, microbial inactivation in response to high intensity pulsed electric fields has been attributed to membrane permeabilization. Hence, most of the presented results are based on the ‘classical’ research work in the field of electroporation (Neumann, 1989; Zimmermann, 1996). This approach is justified for most of the in-use pulsed power applications in biotechnology and food process engineering. However, modifications of electric field treatment using, e.g. pulsed radio frequencies (Chang, 1989), may yield completely different results regarding threshold field strength and pulse energy requirements. Future work will have to consider these aspects in order to further optimize the preservation of liquid foods by pulsed power.
Acknowledgement Parts of the presented work has been carried out with finacial support of the European Community within the project: FAIR CT97-3044 (HELP) and EVK1-200000596 (WIRES).
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