538 S. Toepﬂ et al. / Chemical Engineering and Processing 46 (2007) 537–546 Despite the extensive knowledge in food preservation by heat with several sections of capacitors and inductive elements havetreatment [7,8] and despite continued attempts to improve the to be used. In this case the pulse generation system, in particularquality of processed foods  there is still a need for technolo- the impedance of the line has to be adapted to the resistive loadgies that minimize the destructive inﬂuence of heat on valuable of the treatment chamber. A comparison of energy performancefood compounds . Even more recent concepts like high- of different pulse generation systems has been conducted bytemperature-short-time processing fail if heat transfer and/or De Haan et al. , concluding an exponential decay systemheat penetration is limited by intrinsic thermophysical proper- will not exceed an energy efﬁciency of 38%. They comparedties of the product. Since the thermal energy which is required square wave pulses with a certain peak voltage Vpeak andto destroy the contaminating microorganisms has to be trans- duration Tp to exponential ones by ﬁtting blocks of Vpeak andmitted across the product itself, the design of fast and uniform Tp under an exponential pulse, assuming that excess voltage ofheating and cooling steps is one of the primary challenges of the exponential pulses results in excess losses. Several studiesindustrial preservation by heat. Most of the thermal processing investigating the relation between ﬁeld strength and increaseequipment in use consists of systems transfering heat across an in inactivation have shown that in particular for microbial cellsinterface driven by a temperature gradient. On the product side with small diameter or for media containing particles higheronly the convective heat transport can be enhanced by external ﬁeld strength leads to a better inactivation efﬁciency [16–19].measures, i.e. by the generation of turbulent ﬂow. Compared to Since PEF-processing on an industrial scale will be appliedthermal inactivation the destruction of microbes by electropora- for continuous product ﬂow, the efﬁciency of the treatmenttion shows no time delay with respect to the propagation of the strongly depends on the design of the ﬂow-through treatmentlethal treatment intensity. chamber which basically is composed of two electrodes and an insulating body. Various treatment chamber designs as parallel2. Theory plates, coaxial cylinders or co-linear conﬁgurations have been used for PEF processing . The applicability of a particular In electrically conductive food placed between a high voltage design is determined by several crucial properties of the cham-and a grounded electrode the resulting electrical ﬁeld can be pre- ber. To achieve a sufﬁcient treatment intensity for all volumedicted from the Laplace equation 2 ϕ = 0, where ϕ denotes the elements as well to prevent over-processing or arching the elec-electrical potential. The potential difference ϕM at the mem- tric ﬁeld should be free of local intensity peaks. A co-linearbrane of a biological cell with spherical shape and a radius R conﬁguration is producing a ﬂow pattern which is desirable forinduced by the external electrical ﬁeld E can be approximated food processing and cleaning in place. In such chambers hol-by Eq. (1) which is derived from solving Maxwell’s equations low high voltage and grounded electrodes with circular innerin ellipsoidal coordinates assuming several simplifying  hole are kept on a well deﬁned distance by an insulating spacer. The product is pumped through the drilling forming the elec- ϕM = −f (A)AF E (1) trical load of the high voltage discharge circuit. Relative to the This formula yields the local membrane potential difference electrodes, the inner diameter of the insulator should be slightlyat the distance AF from the centre in direction of the external pinched in order to produce a more homogeneous electrical ﬁeldelectrical ﬁeld. The shape factor f(A) is a function of the three . The treatment chamber conﬁguration determines the resis-semi-axis (A1 , A2 , A3 ) of elliptical cells tive load and therefore the properties of the discharging circuit. The load resistance of a chamber is dependent on the conductiv- 2 ity of the treated media, resulting in limitations in the range off (A) = ∞ 3 media conductivity to which the electric pulses can be applied. 2 − A1 A2 A3 0 1/ (s + A2 ) F n=1 s + A2 n ds (2) 3. Experimental In literature  it is reported that in excess of a critical 3.1. PEF treatment systemtransmembrane potential ϕM of 1 V a rapid electrical break-down and local conformational changes of bilayer structures The exponential decay pulse system used consists of a vari-occur. Considering a membrane thickness of 5 nm this translates able storage capacity of 6.8–27.2 nF charged by a high voltageto a critical membrane ﬁeld strength Ecrit of 2000 kV cm−1 ! A charging unit. A thyristor switch (HTS 240-800SCR, Behlkedrastic increase in permeability re-establishes the equilibrium Electronic GmbH, Germany) is used to discharge the total storedof the electrochemical and electrical potential differences of the energy across a protective resistor and the treatment chamber tocell plasma and the extracellular medium. Although many dif- ground. The pulse raise time typically was in the range betweenferent waveforms are applicable for PEF technology, the pulse 80 and 120 ns, the pulse width, deﬁned as the time needed toshapes commonly used are either exponential decay or square decrease the voltage to 37% of its peak value between 1.5 andwave pulses. Square wave generating systems require a switch 10 s, depending on the number of capacitors, the load voltagewith turn-off capability or a pulse forming network. As switches and the electrical properties of the media. The pulse frequencywith turn off capability are hardly available for high power was varied between 1 and 100 Hz. For a treatment of ringer solu-applications systems , serial or parallel connections of tion with a conductivity of 1.5 mS cm−1 , a capacity of 27.2 nF,switches  or lumped or distributed pulse forming networks a load voltage of 12 kV at an initial treatment temperature of
S. Toepﬂ et al. / Chemical Engineering and Processing 46 (2007) 537–546 53945 ◦ C the pulse width was 6.2 s. At a ﬂow rate of 5 kg h−1 the megaterium and Malt Extract Agar (Merck KgaA, Darmstadt,medium residence time in one treatment zone with a diameter of D) for S. cerevisiae. These plates were incubated for 24 h at4 mm and a length of 6 mm (within the insulator) was 0.06 s, the 37 ◦ C, S. cerevisiae was incubated for 48 h at 30 ◦ C. All micro-medium pulse number per volume element was 6 at a frequency bial analysis were done in duplicate, the lower detection limitof 50 Hz, translating to an energy input of 60 kJ kg−1 . was 20 CFU ml−1 . The inactivation of vegetative organisms was A micro toothed wheel pump (mzr 4600, HNP-Mikrosysteme evaluated by calculating the log reduction in viable cell countsGmbH, Parchim, Germany) was used to pump the media through compared to the untreated sample.silicone tubes to a coiled metal tube inversed into a heating bath,the treatment chamber and a cooling coil. The mass ﬂow rate was 3.3. Calculation of Cook- and PU-valueset between 2 and 7 kg h−1 . The calculated mean residence timein the treatment chamber (not the treatment zone) is 1 s at a ﬂow The Cook (C)-value, a key benchmark for the thermal loadrate of 5 kg h−1 , 25 s in the heating and 15 s in the cooling sys- and degradation of ascorbic acid and ﬂavor during a treatmenttem, calculated by the unit volume and the ﬂow rate. The total with variable temperature-time-regime changes can be calcu-residence time was approximately 31 s. The media was heated lated using Eq. (4) with a medium z-value of 25 ◦ C  forfrom the temperature Tin,1 to the initial treatment temperature quality losses and a reference temperature of 100 ◦ CTin,2 in the range of 35–75 ◦ C. The maximum temperature was tin the range of 45–80 ◦ C, after treatment the media was cooled to C-value = 10T −Tref /z dt (4)Tout below 20 ◦ C. Treated samples were collected in Eppendorf- 0cups and placed on ice immediately. Due to the short residence The pasteurization unit (PU), a key benchmark for thermaltimes at high temperatures the thermal load of the media is very load in relation to microorganisms can be calculated using Eq.low. For acquisition of voltage and current at the treatment cham- (5) with a z-value of 10 ◦ C and a reference temperature of 80 ◦ Cber a 400 MHz digital oscilloscope, a 75 MHz high voltage and . The z-value is the increase or decrease in temperaturea 100 MHz current probe are used. The speciﬁc energy input required to increase the decimal reduction time by one orderwspeciﬁc can be calculated by Eq. (3), where E, κ(T), f and denote of magnitudethe electric ﬁeld strength, the media conductivity, the repetition trate and the mass ﬂow rate, respectively PU = 10T −Tref /z dt (5) 0 1 ∞wspeciﬁc = f κ(T )E(t)2 dt (3) m ˙ 0 4. Results and discussion3.2. Sample preparation and microbial analysis 4.1. Impact of cell size and orientation E. coli K12 DH5␣, Saccharomyces cerevisae DSM 70451, In Fig. 1 Eqs. (1) and (2) have been used to theoreticallyBacillus megaterium DSM 322 and Listeria innocua NCTC predict the external electrical ﬁeld E required to induce a trans-11289 where obtained from the German culture collection membrane potential ϕM = 1 V which in many publications(DSM) Braunschweig. E. coli was chosen as main indicator formicrobial inactivation as outbreaks of E. coli O157:H7 infec-tions were observed in the USA after consumption of applejuice in 1996. Inocula were prepared from stock cultures 24 hbefore each experiment by inoculating 50 ml of nutrition broth.As media were used: Standard I Nutrient Broth (Oxoid, Bas-ingstoke, UK) for E. coli and B. megaterium, Tryptose-Soy-Broth (Oxoid, Basingstoke, UK) for L. innocua and Malt ExtractBroth (Merck KgaA, Darmstadt, D) for S. cerevisae. Cells wereincubated for 24 h at 37 ◦ C to obtain cultures in stationary growthphase. Before PEF treatment, 10 ml L−1 cell suspension wereadded to the treatment media. As treatment media ringer solutiondiluted to a temperature dependent electrical conductivity in therange of 1.25–1.5 mS cm−1 was used. (CG 858, Schott Geraete Fig. 1. Impact of orientation of ellipsoidal microorganisms relative to the elec-GmbH, Hofheim, D). Before treatment a sample of untreated trical ﬁeld E. At a cell speciﬁc threshold level the ﬁeld strength inside themedia was taken to determine the initial cell number. cell membrane exceeds a threshold level Ecrit . Those cells are electroporated The collected samples were placed on ice immediately after which have their longer semi-axis in parallel to E. Other orientations requiretreatment and dilutions were made right after ﬁnishing the ﬁeld strengths in excess of Ecrit . By Eqs. (1) and (2) the required external ﬁeld strength has been calculated for all spacial orientations. Three organisms, dif-experiment using sterile Standard I Nutrient Broth (Oxoid, Bas- ferent in geometry have been chosen as examples (characteristical dimensionsingstoke, UK). Viable counts of vegetative cells were determined from Bergey ). The chart on the left shows the fraction of cells which haveusing drop plating method  on Standard I Nutrient Agar an orientation which does not cause electroporation in response to the given(Merck KgaA, Darmstadt, D) for E. coli, L. innocua and B. external ﬁeld strength.
540 S. Toepﬂ et al. / Chemical Engineering and Processing 46 (2007) 537–546is regarded to be the precondition for irreversible membrane is predicted to be sufﬁcient to lethally damage most of the organ-breakdown and cell death in response to single pulse treat- isms in a bacterial suspension exposed to PEF.ment. Exemplary, three microorganisms have been chosen whichare considerably different in shape and in size, characteristi- 4.2. Electric ﬁeld strength distributioncal dimensions have been taken from Bergey . The threesemi-axes (A1 , A2 , A3 ) deﬁne the geometry of the equivalent The electrode conﬁguration of the colinear treatment cham-ellipsoidal bodies used to calculate the shape factor f(A). AF in ber is presented schematically in Fig. 2. One cylindrical centralEqs. (1) and (2) denotes the length of the semi-axis in direc- high voltage electrode made from stainless steel with an innertion of the external ﬁeld E. In Fig. 1, top right, this is shown drilling diameter of 6 mm has two grounded counterparts at theschematically for two-dimensional situations. The diagram on inlet and outlet of the cuvette separated by insulating ceram-the left side in Fig. 1 shows the fraction of cells in populations of ics, producing a gap of 6 mm. For reason of symmetry only formicroorganisms randomly distributed in orientations which did the upper half of the treatment chamber is the ﬁeld distributionnot reach the critical membrane ﬁeld strength Ecrit and, hence, shown. Depending on the position inside the treatment zone,will not be electroporated. Above the orientation of cells the the peak ﬁeld strength can take different levels. For numericalnatural size variation between different cells of a strain has to simulation of the electric ﬁeld strength in the treatment zone thebe taken into account. Quickﬁeld® FEM code (Tera Analysis Ltd., Denmark) has been It is evident that larger cells are more susceptible to electrical used, the modelation is shown in Fig. 2. For the central axisﬁelds. Yeast cells of S. cerevisiae are affected already at ca. (between position A and B) the spacial distribution is plotted2 kV cm−1 if the longer semi-axis A1 is directed in parallel to for the speciﬁed treatment conditions (Fig. 2, bottom). Alongthe external electrical ﬁeld E. However, a ﬁeld strength higher this line, the maximum ﬁeld strength is located in the mid posi-than 2 kV cm−1 is needed to affect also those cells having less tion of the insulator. In combination with the ﬂow pattern of thefavorable orientations. For yeast it is evident that most of the product, it has to be ensured that all volume elements ﬂowingorganisms are hit when the external ﬁeld applied is in excess of through the chamber receive a lethal dose of electrical energy4 kV cm−1 . . Due to the process speciﬁc pulsed energy delivery this is In contrast, smaller cells like L. innocua require 15 kV cm−1 of most importance for the efﬁciency of the PEF process.in minimum and theoretically more than 35 kV cm−1 to bring In addition the theoretical lethal effect of PEF may beabout extensive microbial inactivation. Most of the other bacteria impaired by agglomerations among microorganisms or betweenrelevant for food preservation are located between the curves of microbes and insulating particles present in the food. This effectSaccharomyces and Listeria. For E. coli for example 15 kV cm−1 was investigated by a 2D modeling of the electric ﬁeld dis-Fig. 2. Features of co-linear PEF treatment chambers. Top: sectional view of the electrode conﬁguration and the resulting electrical ﬁeld. The central high voltageelectrode is separated from two grounded electrodes on either side by an electrical insulator. One example is given which shows the ﬁeld strength along the centralaxis at the treatment zone A–B.
S. Toepﬂ et al. / Chemical Engineering and Processing 46 (2007) 537–546 541 Fig. 4. Electric conductivity of different liquid food systems as function of the temperature, determined by conductivity measurement.Fig. 3. Factors which may cause inhomogenities in the electrical ﬁeld—top: in the ﬁeld of electroporation [12,27]. This approach is justiﬁedagglomeration of two bacterial cells; bottom: bacterial cell attached to a fat for most of the in-use pulsed power applications in food processglobule. The external electrical ﬁeld strength is 12.5 kV cm−1 . The white ring engineering. However, modiﬁcations of electric ﬁeld treatmentshows the location on the membrane where the pore-formation is expected to using, e.g., pulsed radio frequencies , may yield completelyhappen when the ﬁeld strength is high enough to produce 1 V membrane poten-tial difference (=2000 kV cm−1 ). However, due to the conﬁgurations shown, in different results regarding threshold ﬁeld strength and pulseboth cases the required critical membrane potential Ecrit is not reached. Finite energy requirements.elements modeling has been performed by the Software Quick Field (Tera Anal- The conductivity of liquid food, caused by the presence ofysis Ltd., Denmark). ions, has been determined for different liquid food by a conduc- tivity measurement (CG 858, Schott Geraete GmbH, Hofheim, D) and shows a linear relation to temperature (Fig. 4). Duringtribution to approximate the degree of ﬁeld perturbation. In the electrical discharges temperature is increasing dependentFig. 3 the membrane potential of small bacterial cells (simi- on energy input in the respective volume element, leading tolar to Listeria) was simulated by application of the Quickﬁeld® changes in media conductivity and electric ﬁeld distribution.FEM code (Tera Analysis Ltd., Denmark). The exposure to Besides that the total resistance of the treatment chamber, deter-an external electrical ﬁeld of 12.5 kV cm−1 which can induce mined by the electrode conﬁguration and the conductivity ofa critical electrical ﬁeld strength Ecrit of 2000 kV cm−1 ori- the liquid media determines the voltage drop across it. If theented along the electric ﬁeld lines and without presence of resistance of connectors and protective resistor is in the sameagglomerations. The white rings show the location where the range than that of the treatment chamber there will be a highmembrane ﬁeld strength is maximized. The reduced peak val- voltage drop, leading to a reduced peak voltage across the elec-ues of 0.84 and 0.45 Ecrit show that agglomeration of cells as trodes. Adaptation of the limiting, protective resistors to lowwell as the presence of insulating particles can strongly reduce resistance systems might be difﬁcult, as most of the availablethe lethality of the PEF process, as the required membrane switching systems and in particular semiconductor switchespotential for an electroporation is not achieved. Variations of require a maximum current limitation in case of short circuit.cell orientation, and in particular the presence of particles or Usage of a treatment chamber with a high resistance like afat globules as well as formation of clusters might result in co-linear electrode conﬁguration with a resistance in the rangean even lower factors as 0.45. Dependent on the properties of of several hundred Ohm results in a more effective voltagethe food matrix and its contents a worst case scenario has to division and a higher electric ﬁeld strength can be achievedbe developed to choose an appropriate electric ﬁeld strength to even with highly conductive treatment media. The electric ﬁeldexcess the critical transmembrane potential for all cells. Elec- strength E, which is easily determined in the case of paralleltrically insulating gas bubbles which may be produced at the plate electrode conﬁguration was calculated by the measuredelectrode by electrolysis can cause a similar weakening effect peak voltage U multiplied with a cell factor of 1.33, repre-. senting the electric ﬁeld strength in the center of the treat- In this article we focus on membrane permeabilization in ment zone. This factor was determined by modeling the electricresponse to high intensity pulsed electric ﬁelds. All considera- ﬁeld strength for this particular co-linear electrode conﬁgurationtions and conclusions are based on the ‘classical’ research work (Fig. 2).
542 S. Toepﬂ et al. / Chemical Engineering and Processing 46 (2007) 537–546 From a processing point of view this behavior may be exploited by splitting the total required energy input into (recov- erable) thermal energy which makes the microbes more suscep- tible to PEF and electrical pulse energy which brings about the electroporation . 4.4. Impact of different electrode materials As electrode material stainless steel is commonly used, but problems with electrolysis, formation of deposits, electrode cor- rosion and transfer of particles into the treated media have been reported [32–34]. To avoid electrochemical reactions also other materials like platin or metal oxides  or polymer coatings have been suggested . Faradaic reactions taking place at the interface electrode|media may result in partial electrolysis of the treated media as well as in electrode corrosion. Transfer- ring electrical energy across this interface leads to formation of charged double layers at the electrode surface. Using steady con- ditions, as used for electrolysis this layers remain charged during the whole process, acting as capacitance. The transferred currentFig. 5. Inactivation of E. coli in ringer solution in relation to speciﬁc energyinput at different treatment temperatures, an electric ﬁeld strength of 16 (– – –) ﬂows via the Faradaic impedances in parallel to the double layerand 20 kV cm−1 (- - -) and a ﬂow rate of 5 kg h−1 . Results are means based on capacitance leading to Faradaic redox reactions at the interfacedata from two experiments, standard deviations are shown by error bars, the . To transfer a high amount of energy avoiding Faradaicinitial count number was 1.58 × 107 CFU ml−1 . processes it is sufﬁcient that the potential drop across each dou- ble layer capacitor remains smaller than the threshold voltage above which signiﬁcant electrochemical reactions occur. Under4.3. Impact of different process parameters such circumstances the current ﬂow would be purely capaci- tive, avoiding oxidative and reductive reactions at the electrode The effect of initial treatment temperature, electric ﬁeld interfaces. To minimize the extent of this reaction to a toleratedstrength and speciﬁc energy input on inactivation of E. coli in maximum level the treatment chamber has to be submitted toringer solution is shown in Fig. 5. A signiﬁcant relation of sur- short pulses, so that only a small portion of the applied potentialvivor count to speciﬁc energy input was observed. Increasing builds up across the two double layer capacitors. Dependent onthe initial treatment temperature or the electric ﬁeld strength the electrochemical properties, mainly the double layer capacityleads to a further improvement of treatment efﬁciency. The crit- of its material, an electrode can withstand a pulse with a cer-ical electric ﬁeld strength for E. coli in ringer solution is in tain current density and an impulse length without signiﬁcantthe range of 15 kV cm−1 , but in a previous study, modeling the damage. Stainless steel has a very low double layer capacityimpact of ﬁeld strength based on inactivation kinetics of E. coli (35 F cm−2 ) compared to graphite (260 F cm−2 ), resulting inin apple juice it was found that increasing ﬁeld strength above a maximum pulse width avoiding electrochemical reactions asthis value is improving treatment efﬁciency . Raising the low as 0.5 s with current densities of 200 A cm−2 , depen-electric ﬁeld strength from 16 to 20 kV cm−1 at a treatment tem- dent on energy per single pulse. Replacing the high voltageperature of 35 ◦ C increased the microbial inactivation from 2 to anode made of stainless steel with a graphite electrode the inac-3.3 log cycles at an energy input of 80 kJ kg−1 . In contrast to tivation study was repeated. In Fig. 6 a comparison betweenthe external threshold ﬁeld strength the existence of a minimum inactivation with steel and graphite electrode at an electric ﬁeldenergy level required to induce electroporation is rarely reported, strength of 16 kV cm−1 is shown at different initial treatmentalthough some fundamental works on the mechanisms of elec- temperatures, indicating a signiﬁcant increase in treatment efﬁ-troporation consider alterations in free enthalpy which coincide ciency when using graphite. This ﬁnding might be caused bywith perturbations on the molecular level as a prerequisite of the an improved homogeneity of the electric ﬁeld distribution inmembrane breakdown . Statistically ﬂuctuating membrane the treatment zone. Due to the higher double layer capacity ofdefects, which naturally occur even in absence of PEF, deter- graphite the occurrence of Faradaic reactions at the electrode sur-mine the membrane resistance and the critical transmembrane face might be reduced , possibly resulting in less electrolysisvoltage. and bubble formation. Gas bubbles, formed by electrolysis, cav- In the same way the temperature level at which the PEF ity effects or release of dissolved gasses caused by heating havetreatment is performed can strongly reduce the stability of the a lower dielectric breakdown strength than the liquid media,membrane against dielectric breakdown induced by external their presence will lead to perturbations of the electric ﬁeldelectrical ﬁelds. This is mainly due to the transition from the distribution. The lower dielectric permittivity of air causes a con-crystalline to the gel-like state  which enables a higher ﬂuc- centration of potential within the bubbles increasing the chancetuation rate of the defects within the membrane. for a dielectric breakdown and arcing . Modeling the electric
S. Toepﬂ et al. / Chemical Engineering and Processing 46 (2007) 537–546 543 cation of short pulses to avoid electrochemical reactions and electrode erosion has been investigated by Morren et al. . Local discharges and dielectric breakdown as well as perturba- tions of electric ﬁeld homogeneity due to the presence of bubbles presumably might have caused the lower microbial inactivation in case of the stainless steel anode. Arcing, as often observed in presence of a big single air bubble in the treatment chamber was not observed in both cases. Usage of graphite increased inactiva- tion of E. coli more than 1 log cycle at many treatment intensities as for example at 45 ◦ C and 60 kJ kg−1 . Combining applica- tion of electrodes with low amount of electrochemical reactions and pressurizing the treatment system to inhibit bubble forma- tion as well as the application of higher electric ﬁeld strengths should lead to further improvements in treatment efﬁciency. Bubbles should be avoided by degassing the treatment media before treatment, processing under pressure particularly in case of sparkling products and avoiding electrochemical effects at the electrode/media interface.Fig. 6. Comparison of inactivation of E. coli in ringer solution at a ﬁeld strength 4.5. Inactivation of different microbial strainsof 16 kV cm−1 at different initial treatment temperatures with graphite (- - -) orsteel (– – –) anode. The ﬂow rate was 5 kg h−1 , results are means based on data The inactivation of four microbial strains is plotted in Fig. 7from two experiments, standard deviations are shown by error bars, the initialcount number was 1.58 × 107 CFU ml−1 . dependent on speciﬁc energy input, emphasizing the differences in PEF resistance between different microorganisms. It can be seen that consistent to the mathematic modeling (see Fig. 1) theﬁeld distribution with bubbles present in the treatment chamber smallest organism, L. innocua has a higher resistivity than organ-it was shown that the ﬁeld strength in the boundary region of isms with higher cell size like E. coli or B. megaterium. Abovea bubble is very low, possibly leading to under-processing, in that the cell membrane constitution has an important inﬂuenceparticular between several bubbles. By using an electrode mate- on the stability of the membrane. The tendency that gram pos-rial with higher double layer capacity like graphite electrolytic itive bacteria are more resistant than gram negative species haseffects should be reduced. Under-treatment in boundary regions frequently been reported [2,36,38,39].of bubbles can be avoided, resulting in higher microbial inactiva- Whereas L. innocua showed a close to linear relation betweention. Investigating the treated media at the outlet of the chamber energy input and inactivation rate, for E. coli a sigmoid curvean reduced amount of bubbles when using graphite instead of a has been obtained. This curve shape is contrary to our earliersteel anode could be conﬁrmed, but still small bubbles ( 1 mm) studies conducted with parallel electrodes and a ﬁeld strengthwere found, which might also result from oversaturation of air in the range of 30–40 kV cm−1 . In this study, using a treat-due to heating by energy dissipation into the media. The appli- ment chamber with co-linear electrode conﬁguration with a gapFig. 7. Inactivation of E. coli, Listeria innocua, Saccharomyces cerevisae and Bacillus megaterium in ringer solution with an electrical conductivity of 1.25 mS cm−1after PEF treatment with graphite anode and a ﬁeld strength of 16 kV cm−1 at different initial temperatures. The ﬂow rate was 5 kg h−1 , results are means basedon two experiments, standard deviations are shown by error bars. The initial count number was 1.58 × 107 for E. coli, 7.9 × 106 for L. innocua, 1.6 × 107 for S.cerevisae and 2.1 × 107 for B. megaterium. To avoid temperature increase above 70 ◦ C the maximum energy input was set to 50, 100 and 120 kJ kg−1 for initialtreatment temperatures of 35, 45 and 55 ◦ C, respectively.
544 S. Toepﬂ et al. / Chemical Engineering and Processing 46 (2007) 537–546of 6 mm the maximum electric ﬁeld strength was limited to20 kV cm−1 . An inhomogeneous distribution of the treatmentintensity in the co-linear treatment chamber may have causedthis effect, which was found at low treatment intensities only iflow impulse frequencies were applied. This may possibly resultin under-processing of volume elements with a short residencetime in the treatment zone. For inactivation of B. megateriumand S. cerevisiae a very low input of speciﬁc energy in the rangeof 10 and 30 kJ kg−1 is required for a 5 log cycle reduction at55 ◦ C initial treatment temperature. Further increase of the spe-ciﬁc energy input led, due to synergetic effects of PEF and heat,to an inactivation below detection limit for these PEF sensi-tive organisms. Temperatures higher than ambient and repetitivepulsing may have led to a reduction of the required transmem-brane potential below 1 V, resulting in a higher inactivation of Fig. 8. Enthalpy diagram of a suggested PEF treatment system for apple juiceL. innocua than predicted by Eq. (1) (see Fig. 1). An important with an initial temperature of 55 ◦ C and a speciﬁc energy input of 40 kJ kg−1 .task will be the selection of resistant target strains to evaluate As speciﬁc heat capacity 3.8 kJ kg−1 K−1 was used, the heat loss in the heatprocess efﬁcacy, as also variations of PEF sensitivity among dif- exchanger was estimated for 5%.ferent strains of L. monocytogenes have been described . Ithas to be taken into account that only vegetative cells are affected An important advantage of operating at elevated initial treat-by a PEF treatment, whereas ascospores  and in particular ment temperature is the potential to recover the electrical energyendospores show resistance . dissipated into the product after treatment, as there is a need to preheat the media. An enthalpy diagram of a suggested PEF4.6. Thermal load and enthalpy balance of a PEF treatment treatment is shown in Fig. 8. Assuming a heat loss of 5% for heat recovery, the process can be operated by the input of the An initial treatment temperature of 55 ◦ C may lead to the electrical energy of 40 kJ kg−1 only, avoiding the necessity ofpresumption that the advantage of PEF application, to provide a additional energy for preheating or cooling of the product aftermild and non-thermal preservation process is lost. Investigating treatment. When operating at ambient temperature there is nothe temperature-time-proﬁle of a PEF treatment at an elevated potential to recover the dissipated energy as there is no needtreatment temperature of 55 ◦ C using synergetic effects of mild for preheating the media, and if the process shall be conductedheat to reduce the required input of electrical energy showed that under close to isothermal conditions a huge amount of additionalcompared to a conventional high temperature short time treat- energy is required for cooling.ment the thermal load of the product is strongly reduced .Dependent on the speciﬁc energy input and the speciﬁc heat 5. Conclusionscapacity of the medium the PEF treatment will cause a temper-ature increase. For example for a pasteurization of apple juice The potential of PEF application for microbial inactivation inwith a heat capacity of 3.8 kJ kg−1 K−1 and an energy input of liquid food to increase shelf life was shown. A combination of40 kJ kg−1 a temperature increase of 11 ◦ C is obtained, leading to PEF and mild heat provides the possibility for a gentle processa process with a maximum temperature of 66 ◦ C for a very short to increase product shelf life with a low maximum temperatureresidence time in range of seconds. A calculation of the C and and short residence times, resulting in a drastic reduction of thethe PU value, key benchmarks for the over all thermal load of the thermal load. Above that the speciﬁc energy input required for aproduct during heat treatment [23,24] showed that a PEF treat- given inactivation is reduced by synergetic effects of temperaturement with a speciﬁc energy input of 40 kJ kg−1 , a ﬁeld strength of on microbial inactivation by PEF. When using an elevated initial16 kV cm−1 and a initial treatment temperature of 55 ◦ C caused treatment temperature the dissipated electrical energy, causinga 5 log cycle inactivation of E. coli in apple juice with very low a temperature increase of the product, can be recovered in a heatCook and PU values of 6.5 and 2.23 × 10−3 , respectively. A PEF exchanger to preheat the untreated media resulting in a dras-treatment at elevated treatment temperature could therefore be tic reduction in costs of operation. The design of a treatmentan interesting, gentle alternative to increase shelf life of a high chamber with a homogenous electric ﬁeld distribution, ensur-acid product as for example fruit juice. A conventional HTST ing a highly uniform and sufﬁcient treatment for all volumetreatment produces a shelf stable product with Cook and PU elements and the selection of an electrode material preventingvalues of 6 × 102 and 0.45. Increasing the maximum electrical electrochemical reactions leads to a further increase of treat-ﬁeld strength of the impulse generating system would provide a ment efﬁciency. Modeling of the electric ﬁeld distribution inpotential to achieve higher inactivation with similar energy input the treatment zone, the impact of particles or agglomerationsand thermal load . Development of impulse generating sys- and the size and orientation of microbial cells on their sensitiv-tems to achieve high electric ﬁeld strength to operate with a gap ity against PEF provides a tool to choose appropriate processallowing high ﬂow rate capability and adequate, high pulse rep- parameters and therefore to ensure product safety. Further worketition rate will be a crucial towards an industrial exploitation. will be necessary for choice of suitable electrode geometry and
S. Toepﬂ et al. / Chemical Engineering and Processing 46 (2007) 537–546 545material and, in particular the development of impulse gener-  D. Knorr, V. Heinz, Development of non-thermal methods for micro-ating systems with sufﬁcient output voltage and repetition rate bial control, in: S.S. Block (Ed.), Disinfection, Sterilization, and Preser-for high capacity treatment chambers, crucial prerequisites for vation, Lippincott Williams & Wilkins, Philadelphia, 2001, pp. 853– 877.a desirable industrial scale up of this mild preservation process.  U. Zimmermann, G. Pilwat, F. Riemann, Dielectric breakdown in cell mem- branes, Biophys. J. 14 (1974) 881–899.Appendix A. Nomenclature  U. Zimmermann, The effect of high intensity electric ﬁeld pulses on eucary- otic cell membranes: fundamentals and applications, in: U. Zimmermann, G.A. Neil (Eds.), Electromanipulation of Cells, CRC Press, Boca Raton, 1996, pp. 1–106.A length of semi-axis of ellipsoid (m)  N. Mohan, T.N. Undeland, W.P. Robbins, Power Electronics; ConvertersAF length of semi-axis in ﬁeld direction (m) and Design, Wiley, New York, 1995.E electric ﬁeld strength (kV cm−1 )  M.P.J. Gaudreau, T. Hawkey, J. Petry, M.A. Kempkes, A solid state pulsed power system for food processing, in: Proceedings of the 24th Pulses Powerf frequency (Hz) Plasma Science Conference, Las Vegas, USA, 2001.f(A) shape factor  S.W.H. De Haan, P.R. Willcock, Comparison of the energy performance ofm˙ mass ﬂow rate (kg h−1 ) pulse generation circuits for PEF, Innov. Food Sci. Emerg.Technol. (2002)PU pasteurization unit 349–356.T temperature (◦ C)  V. Heinz, D. Knorr, Effect of pH ethanol addition and high hydrostatic pressure on the inactivation of Bacillus subtilis by pulsed electric ﬁelds,Tp pulse duration of square wave pulse (s) Innov. Food Sci. Emerg.Technol. 1 (2000) 151–159.Vpeak peak voltage (kV)  C.J. McDonald, S.W. Lloyd, M.A. Vitale, K. Petersson, F. Innings, Effectwspeciﬁc speciﬁc energy input (kJ kg−1 ) of pulsed electric ﬁelds on microorganisms in orange juice using electricz z-value ﬁeld strengths of 30 and 50 kV/cm, J. Food Sci. 65 (2000) 984–989.  N.I. Boyko, A.N. Tur, L.S. Evdoshenko, A.I. Zarochentsev, V.M. Ivanov, A high-voltage pulse generator with mean power up to 50 kW for the treatmentGreek letters of foodstuffs, Instrum. Exp. Tech. 41 (1998) 111–117.κ electrical conductivity (S m−1 )  L. Picart, E. Dumay, C. Cheftel, Inactivation of Listeria innocua inτ charging time constant (s) dairy ﬂuids by pulsed electric ﬁelds: inﬂuence of electric parameters and food composition, Innov. Food Sci. Emerg. Technol. 3 (2002) 357–ϕ electrical potential (V) 369.  G.V. Barbosa-Canovas, M.M. Gongora-Nieto, U.R. Pothakamury, B.G.Indexes Swanson, Preservation of foods with pulsed electric ﬁelds., in: S.L. Taylorcrit critical (Ed.), Food Science and Technology, Academic Press, San Diego, 1999.  M. Lindgren, Pulsed electric ﬁeld food treatment and low frequency bio-in,1 inlet temperature electromagnetics, Dissertation Thesis, Chalmers University of Technology,in,2 initial treatment temperature Gotenburg, 2001.max maximum  J. Baumgart, Mikrobiologische Untersuchung von Lebensmitteln, Behr’sM membrane Verlag, Hamburg, 1986.out outlet temperature  T. Ohlson, Optimal temperatures for sterilization of ﬂat containers, J. Food Sci. 45 (1980) 848–852/859.ref reference  D.A. Shapton, D.W. Lovelock, R. Laurita-Longo, The evaluation of ster- ilization and pasteurization processes from temperature measurements inReferences ◦ C, J. Appl. Bacteriol. 34 (1971) 491–500.  Bergey, Manual of Systematic Bacteriology, 9th ed., Williams and Wilkins, T. Grahl, H. M¨ rkl, Killing of microorganisms by pulsed electric ﬁelds, a Baltimore, 1986. Appl. Microbiol. Biotechnol. 45 (1996) 148–157.  M.M. G´ ngora-Nieto, P.D. Pedrow, B.G. Swanson, G.V. Barbosa-C´ novas, o a P.C. Wouters, N. Dutreux, J.P.P.M. Smelt, H.L.M. Lelieveld, Effects of Impact of air bubbles in a dielectric liquid when subjected to high electric pulsed electric ﬁelds on inactivation kinetics of Listeria innocua, Appl. ﬁeld strengths, Innov. Food Sci. Emerg. Technol. 4 (2003) 57–67. Environ. Microbiol. 65 (1999) 5364–5371.  E. Neumann, The relaxation hysteresis of membrane electroporation, in: R.K. Simpson, R. Whittington, R.G. Earnshaw, N.J. Russel, Pulsed high E. Neumann, A.E. Sowers, C.A. Jordan (Eds.), Electroporation and Elec- electric ﬁeld causes ‘all or nothing’ membrane damage in Listeria mono- trofusion in Cell Biology, PlenumPress, New York, 1989, pp. 61–82. cytogenes and Salmonella typhimurium, but membrane H+-ATPase is not  D.C. Chang, Cell fusion and cell poration by pulsed radio-frequency electric a primary target, Int. J. Food Microbiol. 48 (1999) 1–10. ﬁelds, in: E. Neumann, A.E. Sowers, C.A. Jordan (Eds.), Electroporation E. Neumann, K. Rosenheck, Permeability changes induced by electric and Electrofusion in Cell Biology, Plenum Press, New York, 1989, pp. impulses in vesicular membranes, J. Membr. Biol. 10 (1972) 279–290. 215–227. U. Zimmermann, J. Schulz, G. Pilwat, Transcellular ion ﬂow in E. coli and  V. Heinz, S. Toepﬂ, D. Knorr, Impact of temperature on lethality and energy electrical sizing of bacteria, Biophys. J. 13 (1973) 1005–1013. efﬁciency of apple juice pasteurization by pulsed electric ﬁelds treatment, R. Glaser, Electric properties of the membrane and the cell surface, in: U. Innov. Food Sci. Emerg. Technol. 4 (2003) 167–175. Zimmermann, G.A. Neil (Eds.), Electromanipulation of Cells, CRC Press,  K.T. Powell, E.G. Derrick, D.C.A. Weaver, quantitative theory of reversible Boca Raton, 1996, pp. 329–364. electrical breakdown in bilayer membranes, Bioelectrochem. Bioenerg. 15 J. Larousse, B.E. Brown, Food Canning Technology, Wiley-VCH, New (1986) 243–255. York, 1997.  D.W. Stanley, Biological membrane deterioration and associated quality M.N. Ramesh, Food preservation by heat treatment, in: M.S. Rahman (Ed.), losses in food tissues, in: F.M. Clydesdale (Ed.), Critical Reviews in Food Handbook of Food Preservation, Marcel Dekker, Inc., New York, 1999, pp. Science and Nutrition, CRC Press, New York, 1991. 95–172.  M. Caplot, G. Cote, Les technologies n´ cessaires aux machines de traitment e T.D. Durance, Improving canned food quality with variable retort temper- par Champs Electriques Puls´ s, in: Formation Technologique en Agro- e ature processes, Trends Food Sci.Technol. 8 (1997) 113–118. Alimentaire, La conservation de demain, Bordeaux Pessac, France, 1999.
546 S. Toepﬂ et al. / Chemical Engineering and Processing 46 (2007) 537–546 M.M. G´ ngora-Nieto, Food preservation by pulsed electric ﬁelds: eval- o  H. H¨ lsheger, J. Potel, E.G. Niemann, Killing of bacteria with elec- u uation of critical processing parameters and process optimization, Ph.D. tric pulses of high ﬁeld strength, Radiat. Environ. Biophys. 20 (1983) Dissertation, Washington State University, Pullman, WA, 2000. 53–65. J. Morren, B. Roodenburg, S.W.H. de Haan, Electrochemical reactions  H. Vega-Mercado, U.R. Pothakamury, F.-J. Chang, G.V. Barbosa-C´ novas, a and electrode corrosion in pulsed electric ﬁeld (PEF) treatment chambers, B.G. Swanson, Inactivation of Escherichia coli by combining pH, ionic Innov. Food Sci. Emerg. Technol. 4 (2003) 285–295. strength and pulsed electric ﬁeld hurdles, Food Res. Int. 29 (1996) 117– A.H. Bushnell, R.W. Clark, J.E. Dunn, S.W. Lloyd, Process for reducing 121. levels of microorganisms in pumpable food products using a high pulsed  B.H. Lado, A.E. Yousef, Selection and Identiﬁcation of a Listeria mono- voltage system (1996) US Patent 5,514,391. cytogenes target strain for pulsed electric ﬁeld process optimisation, Appl. B.L. Qin, et al., Continuous ﬂow electrical treatment of ﬂowable food prod- Environ. Microbiol. 69 (2003) 2223–2229. ucts (1997) US Patent 566,203.  J. Raso, M.L. Calder´ n, M. G´ ngora, G.V. Barbosa-C´ novas, B.G. Swan- o o a C. Amatore, M. Berthou, S. H´ bert, Fundamental Principles of elec- e son, Inactivation of mold ascospores and conidiospores suspended in trochemical ohmic heating of solutions, J. Electroanal. Chem. (1998) fruit juices by pulsed electric ﬁelds, Lebensm. Wiss. Technol. 31 (1998) 191–203. 668–672.