Chemical Engineering and Processing 46 (2007) 537–546

               High intensity pulsed electric fields applied for ...
538                                  S. Toepfl et al. / Chemical Engineering and Processing 46 (2007) 537–546

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S. Toepfl et al. / Chemical Engineering and Processing 46 (2007) 537–546                                             539

540                                         S. Toepfl et al. / Chemical Engineering and Processing 46 (2007) 537–546

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S. Toepfl et al. / Chemical Engineering and Processing 46 (2007) 537–546                                            541

542                                           S. Toepfl et al. / Chemical Engineering and Processing 46 (2007) 537–546

S. Toepfl et al. / Chemical Engineering and Processing 46 (2007) 537–546                                            543

544                                   S. Toepfl et al. / Chemical Engineering and Processing 46 (2007) 537–546

of 6 mm the...
S. Toepfl et al. / Chemical Engineering and Processing 46 (2007) 537–546                                             545

546                                          S. Toepfl et al. / Chemical Engineering and Processing 46 (2007) 537–546

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Toepfl Et Al 2007b High intensity pulsed electric fields applied for food preservation


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Preservation of liquid foods by high intensity pulsed electric fields (PEF) is an interesting alternative to traditional techniques like thermal pasteurization. Based on the underlying mechanism of action, in this paper the crucial process parameters electrical field strength, total pulse energy input and treatment temperature were investigated experimentally. Inactivation studies were performed with three bacteria (E. coli, Bacillus megaterium, Listeria innocua) and one yeast (Saccharomyces cerevisiae). Stainless steel and carbon electrodes have been tested to investigate their applicability as electrode material. Simulating the influence of cell size and orientation as well as the presence of agglomerations or insulating particles indicated that the applied field strength has to be increased above the critical one to achieve product safety. It was found that temperatures higher than 40 ◦C can strongly increase the lethality of the PEF process. In this way also small cells like Listeria are easily affected by pulsed fields even at a field strength as low as 16 kVcm−1. In addition, heating of the product prior to PEF has the advantage that most of the required process energy can be recovered using heat exchangers. Exemplary, such a process is analyzed by an enthalpy balance.

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Toepfl Et Al 2007b High intensity pulsed electric fields applied for food preservation

  1. 1. Chemical Engineering and Processing 46 (2007) 537–546 High intensity pulsed electric fields applied for food preservation S. Toepfl a,∗ , V. Heinz b , D. Knorr a a Department of Food Biotechnology and Food Process Engineering, Berlin University of Technology, Koenigin-Luise-Str. 22, D-14195 Berlin, Germany b German Institute of Food Technology (DIL e.V.), Prof.-Klitzing-Str. 7, 49610 Quakenbr¨ ck, Germany u Received 21 July 2006; accepted 26 July 2006 Available online 1 August 2006 Abstract Preservation of liquid foods by high intensity pulsed electric fields (PEF) is an interesting alternative to traditional techniques like thermal pasteurization. Based on the underlying mechanism of action, in this paper the crucial process parameters electrical field strength, total pulse energy input and treatment temperature were investigated experimentally. Inactivation studies were performed with three bacteria (E. coli, Bacillus megaterium, Listeria innocua) and one yeast (Saccharomyces cerevisiae). Stainless steel and carbon electrodes have been tested to investigate their applicability as electrode material. Simulating the influence of cell size and orientation as well as the presence of agglomerations or insulating particles indicated that the applied field strength has to be increased above the critical one to achieve product safety. It was found that temperatures higher than 40 ◦ C can strongly increase the lethality of the PEF process. In this way also small cells like Listeria are easily affected by pulsed fields even at a field strength as low as 16 kV cm−1 . In addition, heating of the product prior to PEF has the advantage that most of the required process energy can be recovered using heat exchangers. Exemplary, such a process is analyzed by an enthalpy balance. © 2006 Elsevier B.V. All rights reserved. Keywords: Pulsed electric fields; Inactivation; Yeast; Bacteria; Enthalpy 1. Introduction that an increase of the 10 mV resting potential across the cell membrane by the exposure to an external electrical field up to Conventional preservation methods such as heat treatment potentials higher than approximately 1 V leads to rapid elec- often fail to produce microbiologically stable food at the desired trical breakdown and local conformational changes of bilayer quality level. It has already been demonstrated that high inten- structures [4] and cell membranes [5]. A drastic increase in sity pulsed electric fields (PEF) processing can alternatively permeability re-establishes the equilibrium of the electrochem- be applied to deliver safe and shelf-stable products such as ical and electric potential differences of the cell plasma and fruit juices or milk with fresh-like character and high nutri- the extracellular medium forming a Donnan-equilibrium [6]. tional value [1]. However, commercial exploitation of PEF as Simultaneously, the neutralization of the transmembrane gradi- an alternative to traditional preservation techniques requires a ent across the membrane irreversibly impairs vital physiological detailed analysis of process safety, cost-effectiveness, and con- control systems of the cell like osmoregulation and consequently sumer benefits. From experimental data [2,3] it is evident that cell death occurs. sufficient microbial reduction can be achieved. However, the Microbial cells which are exposed to an external electrical degree of inactivation strongly depends on the intensity of the field for a few microseconds respond by an electrical breakdown pulses in terms of field strength, energy and number of pulses and local structural changes of the cell membrane. In conse- applied on the microbial strain and on the properties of the food quence of the so called electroporation, a drastic increase in matrix under investigation. Hence, for the optimization of pro- permeability is observed which in the irreversible case is equiv- cess design it is necessary to consider the mechanisms of action alent to a loss of viability. This type of non-thermal inactivation of PEF on the level of microbial cells. It is a well known fact of microorganisms by high intensity pulsed electric fields might be beneficial for the development of quality retaining preser- vation processes in the food industry. However, process safety, ∗ Corresponding author. Tel.: +49 30 314 71250; fax: +49 30 8327663. cost-effectiveness, and consumer benefits of pulsed electric field E-mail address: (S. Toepfl). treatment have to be confirmed. 0255-2701/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cep.2006.07.011
  2. 2. 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 have treatment [7,8] and despite continued attempts to improve the to be used. In this case the pulse generation system, in particular quality of processed foods [9] there is still a need for technolo- the impedance of the line has to be adapted to the resistive load gies that minimize the destructive influence of heat on valuable of the treatment chamber. A comparison of energy performance food compounds [10]. Even more recent concepts like high- of different pulse generation systems has been conducted by temperature-short-time processing fail if heat transfer and/or De Haan et al. [15], concluding an exponential decay system heat penetration is limited by intrinsic thermophysical proper- will not exceed an energy efficiency of 38%. They compared ties of the product. Since the thermal energy which is required square wave pulses with a certain peak voltage Vpeak and to destroy the contaminating microorganisms has to be trans- duration Tp to exponential ones by fitting blocks of Vpeak and mitted across the product itself, the design of fast and uniform Tp under an exponential pulse, assuming that excess voltage of heating and cooling steps is one of the primary challenges of the exponential pulses results in excess losses. Several studies industrial preservation by heat. Most of the thermal processing investigating the relation between field strength and increase equipment in use consists of systems transfering heat across an in inactivation have shown that in particular for microbial cells interface driven by a temperature gradient. On the product side with small diameter or for media containing particles higher only 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 applied thermal inactivation the destruction of microbes by electropora- for continuous product flow, the efficiency of the treatment tion shows no time delay with respect to the propagation of the strongly depends on the design of the flow-through treatment lethal treatment intensity. chamber which basically is composed of two electrodes and an insulating body. Various treatment chamber designs as parallel 2. Theory plates, coaxial cylinders or co-linear configurations have been used for PEF processing [20]. 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 volume dicted 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-linear brane of a biological cell with spherical shape and a radius R configuration is producing a flow pattern which is desirable for induced 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 inner in ellipsoidal coordinates assuming several simplifying [11] 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 slightly at the distance AF from the centre in direction of the external pinched in order to produce a more homogeneous electrical field electrical field. The shape factor f(A) is a function of the three [21]. 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 of f (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 [12] it is reported that in excess of a critical 3.1. PEF treatment system transmembrane 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 voltage to a critical membrane field strength Ecrit of 2000 kV cm−1 ! A charging unit. A thyristor switch (HTS 240-800SCR, Behlke drastic increase in permeability re-establishes the equilibrium Electronic GmbH, Germany) is used to discharge the total stored of the electrochemical and electrical potential differences of the energy across a protective resistor and the treatment chamber to cell plasma and the extracellular medium. Although many dif- ground. The pulse raise time typically was in the range between ferent waveforms are applicable for PEF technology, the pulse 80 and 120 ns, the pulse width, defined as the time needed to shapes commonly used are either exponential decay or square decrease the voltage to 37% of its peak value between 1.5 and wave pulses. Square wave generating systems require a switch 10 s, depending on the number of capacitors, the load voltage with turn-off capability or a pulse forming network. As switches and the electrical properties of the media. The pulse frequency with turn off capability are hardly available for high power was varied between 1 and 100 Hz. For a treatment of ringer solu- applications systems [13], serial or parallel connections of tion with a conductivity of 1.5 mS cm−1 , a capacity of 27.2 nF, switches [14] or lumped or distributed pulse forming networks a load voltage of 12 kV at an initial treatment temperature of
  3. 3. S. Toepfl et al. / Chemical Engineering and Processing 46 (2007) 537–546 539 45 ◦ 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 at 4 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 limit of 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 counts GmbH, 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-value set between 2 and 7 kg h−1 . The calculated mean residence time in the treatment chamber (not the treatment zone) is 1 s at a flow The Cook (C)-value, a key benchmark for the thermal load rate 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 treatment tem, 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 [23] for from the temperature Tin,1 to the initial treatment temperature quality losses and a reference temperature of 100 ◦ C Tin,2 in the range of 35–75 ◦ C. The maximum temperature was t in 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- 0 cups and placed on ice immediately. Due to the short residence The pasteurization unit (PU), a key benchmark for thermal times 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 ◦ C ber a 400 MHz digital oscilloscope, a 75 MHz high voltage and [24]. The z-value is the increase or decrease in temperature a 100 MHz current probe are used. The specific energy input required to increase the decimal reduction time by one order wspecific can be calculated by Eq. (3), where E, κ(T), f and denote of magnitude the electric field strength, the media conductivity, the repetition t rate 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 discussion 3.2. 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 theoretically Bacillus 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 for microbial inactivation as outbreaks of E. coli O157:H7 infec- tions were observed in the USA after consumption of apple juice in 1996. Inocula were prepared from stock cultures 24 h before 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 Extract Broth (Merck KgaA, Darmstadt, D) for S. cerevisae. Cells were incubated for 24 h at 37 ◦ C to obtain cultures in stationary growth phase. Before PEF treatment, 10 ml L−1 cell suspension were added to the treatment media. As treatment media ringer solution diluted to a temperature dependent electrical conductivity in the range 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 the media 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 require treatment 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 dimensions ingstoke, UK). Viable counts of vegetative cells were determined from Bergey [25]). The chart on the left shows the fraction of cells which have using drop plating method [22] 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.
  4. 4. 540 S. Toepfl et al. / Chemical Engineering and Processing 46 (2007) 537–546 is 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 which are considerably different in shape and in size, characteristi- 4.2. Electric field strength distribution cal dimensions have been taken from Bergey [25]. The three semi-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 central Eqs. (1) and (2) denotes the length of the semi-axis in direc- high voltage electrode made from stainless steel with an inner tion of the external field E. In Fig. 1, top right, this is shown drilling diameter of 6 mm has two grounded counterparts at the schematically 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 for microorganisms randomly distributed in orientations which did the upper half of the treatment chamber is the field distribution not 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 numerical natural size variation between different cells of a strain has to simulation of the electric field strength in the treatment zone the be 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 axis fields. Yeast cells of S. cerevisiae are affected already at ca. (between position A and B) the spacial distribution is plotted 2 kV cm−1 if the longer semi-axis A1 is directed in parallel to for the specified treatment conditions (Fig. 2, bottom). Along the 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 the favorable orientations. For yeast it is evident that most of the product, it has to be ensured that all volume elements flowing organisms are hit when the external field applied is in excess of through the chamber receive a lethal dose of electrical energy 4 kV cm−1 . [21]. 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 be about extensive microbial inactivation. Most of the other bacteria impaired by agglomerations among microorganisms or between relevant for food preservation are located between the curves of microbes and insulating particles present in the food. This effect Saccharomyces 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 voltage electrode is separated from two grounded electrodes on either side by an electrical insulator. One example is given which shows the field strength along the central axis at the treatment zone A–B.
  5. 5. 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 justified agglomeration of two bacterial cells; bottom: bacterial cell attached to a fat for most of the in-use pulsed power applications in food process globule. The external electrical field strength is 12.5 kV cm−1 . The white ring engineering. However, modifications of electric field treatment shows the location on the membrane where the pore-formation is expected to using, e.g., pulsed radio frequencies [28], may yield completely happen 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 pulse both 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 of ysis 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). During tribution to approximate the degree of field perturbation. In the electrical discharges temperature is increasing dependent Fig. 3 the membrane potential of small bacterial cells (simi- on energy input in the respective volume element, leading to lar 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 of a critical electrical field strength Ecrit of 2000 kV cm−1 ori- the liquid media determines the voltage drop across it. If the ented along the electric field lines and without presence of resistance of connectors and protective resistor is in the same agglomerations. The white rings show the location where the range than that of the treatment chamber there will be a high membrane 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 low well as the presence of insulating particles can strongly reduce resistance systems might be difficult, as most of the available the lethality of the PEF process, as the required membrane switching systems and in particular semiconductor switches potential 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 a fat globules as well as formation of clusters might result in co-linear electrode configuration with a resistance in the range an even lower factors as 0.45. Dependent on the properties of of several hundred Ohm results in a more effective voltage the food matrix and its contents a worst case scenario has to division and a higher electric field strength can be achieved be developed to choose an appropriate electric field strength to even with highly conductive treatment media. The electric field excess the critical transmembrane potential for all cells. Elec- strength E, which is easily determined in the case of parallel trically insulating gas bubbles which may be produced at the plate electrode configuration was calculated by the measured electrode by electrolysis can cause a similar weakening effect peak voltage U multiplied with a cell factor of 1.33, repre- [26]. 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 electric response to high intensity pulsed electric fields. All considera- field strength for this particular co-linear electrode configuration tions and conclusions are based on the ‘classical’ research work (Fig. 2).
  6. 6. 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 [29]. 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 [35] or polymer coatings have been suggested [36]. 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 current Fig. 5. Inactivation of E. coli in ringer solution in relation to specific energy input at different treatment temperatures, an electric field strength of 16 (– – –) flows via the Faradaic impedances in parallel to the double layer and 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 interface data from two experiments, standard deviations are shown by error bars, the [37]. To transfer a high amount of energy avoiding Faradaic initial 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. Under 4.3. 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 tolerated strength and specific energy input on inactivation of E. coli in maximum level the treatment chamber has to be submitted to ringer solution is shown in Fig. 5. A significant relation of sur- short pulses, so that only a small portion of the applied potential vivor count to specific energy input was observed. Increasing builds up across the two double layer capacitors. Dependent on the initial treatment temperature or the electric field strength the electrochemical properties, mainly the double layer capacity leads 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 significant the range of 15 kV cm−1 , but in a previous study, modeling the damage. Stainless steel has a very low double layer capacity impact of field strength based on inactivation kinetics of E. coli (35 F cm−2 ) compared to graphite (260 F cm−2 ), resulting in in apple juice it was found that increasing field strength above a maximum pulse width avoiding electrochemical reactions as this value is improving treatment efficiency [29]. Raising the low as 0.5 s with current densities of 200 A cm−2 [32], depen- electric field strength from 16 to 20 kV cm−1 at a treatment tem- dent on energy per single pulse. Replacing the high voltage perature 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 between the external threshold field strength the existence of a minimum inactivation with steel and graphite electrode at an electric field energy level required to induce electroporation is rarely reported, strength of 16 kV cm−1 is shown at different initial treatment although 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 by with perturbations on the molecular level as a prerequisite of the an improved homogeneity of the electric field distribution in membrane breakdown [30]. Statistically fluctuating membrane the treatment zone. Due to the higher double layer capacity of defects, 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 [37], possibly resulting in less electrolysis voltage. 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 have treatment 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 field electrical 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 [31] which enables a higher fluc- centration of potential within the bubbles increasing the chance tuation rate of the defects within the membrane. for a dielectric breakdown and arcing [26]. Modeling the electric
  7. 7. 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. [34]. 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 strains of 16 kV cm−1 at different initial treatment temperatures with graphite (- - -) or steel (– – –) 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. 7 from two experiments, standard deviations are shown by error bars, the initial count 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) the field 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. Above a bubble is very low, possibly leading to under-processing, in that the cell membrane constitution has an important influence particular 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 has effects 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 between tion. Investigating the treated media at the outlet of the chamber energy input and inactivation rate, for E. coli a sigmoid curve an reduced amount of bubbles when using graphite instead of a has been obtained. This curve shape is contrary to our earlier steel anode could be confirmed, but still small bubbles ( 1 mm) studies conducted with parallel electrodes and a field strength were found, which might also result from oversaturation of air in the range of 30–40 kV cm−1 [29]. 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 gap Fig. 7. Inactivation of E. coli, Listeria innocua, Saccharomyces cerevisae and Bacillus megaterium in ringer solution with an electrical conductivity of 1.25 mS cm−1 after 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 based on 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 initial treatment temperatures of 35, 45 and 55 ◦ C, respectively.
  8. 8. 544 S. Toepfl et al. / Chemical Engineering and Processing 46 (2007) 537–546 of 6 mm the maximum electric field strength was limited to 20 kV cm−1 . An inhomogeneous distribution of the treatment intensity in the co-linear treatment chamber may have caused this effect, which was found at low treatment intensities only if low impulse frequencies were applied. This may possibly result in under-processing of volume elements with a short residence time in the treatment zone. For inactivation of B. megaterium and S. cerevisiae a very low input of specific energy in the range of 10 and 30 kJ kg−1 is required for a 5 log cycle reduction at 55 ◦ 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 repetitive pulsing 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 juice L. 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 heat process efficacy, as also variations of PEF sensitivity among dif- exchanger was estimated for 5%. ferent strains of L. monocytogenes have been described [40]. It has 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 [41] and in particular ment temperature is the potential to recover the electrical energy endospores show resistance [1]. dissipated into the product after treatment, as there is a need to preheat the media. An enthalpy diagram of a suggested PEF 4.6. 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 of presumption that the advantage of PEF application, to provide a additional energy for preheating or cooling of the product after mild and non-thermal preservation process is lost. Investigating treatment. When operating at ambient temperature there is no the temperature-time-profile of a PEF treatment at an elevated potential to recover the dissipated energy as there is no need treatment temperature of 55 ◦ C using synergetic effects of mild for preheating the media, and if the process shall be conducted heat to reduce the required input of electrical energy showed that under close to isothermal conditions a huge amount of additional compared to a conventional high temperature short time treat- energy is required for cooling. ment the thermal load of the product is strongly reduced [29]. Dependent on the specific energy input and the specific heat 5. Conclusions capacity 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 in with 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 of 40 kJ kg−1 a temperature increase of 11 ◦ C is obtained, leading to PEF and mild heat provides the possibility for a gentle process a process with a maximum temperature of 66 ◦ C for a very short to increase product shelf life with a low maximum temperature residence time in range of seconds. A calculation of the C and and short residence times, resulting in a drastic reduction of the the PU value, key benchmarks for the over all thermal load of the thermal load. Above that the specific energy input required for a product during heat treatment [23,24] showed that a PEF treat- given inactivation is reduced by synergetic effects of temperature ment with a specific energy input of 40 kJ kg−1 , a field strength of on microbial inactivation by PEF. When using an elevated initial 16 kV cm−1 and a initial treatment temperature of 55 ◦ C caused treatment temperature the dissipated electrical energy, causing a 5 log cycle inactivation of E. coli in apple juice with very low a temperature increase of the product, can be recovered in a heat Cook 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 treatment an 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 volume treatment produces a shelf stable product with Cook and PU elements and the selection of an electrode material preventing values 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 in potential to achieve higher inactivation with similar energy input the treatment zone, the impact of particles or agglomerations and thermal load [29]. 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 process allowing high flow rate capability and adequate, high pulse rep- parameters and therefore to ensure product safety. Further work etition rate will be a crucial towards an industrial exploitation. will be necessary for choice of suitable electrode geometry and
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