- Email: [email protected]
Inactivation effect of an 18-T pulsed magnetic field combined with other technologies on Escherichia coli
Innovative Food Science & Emerging Technologies 2 Ž2001. 273᎐277
Inactivation effect of an 18-T pulsed magnetic field combined with other technologies on Escherichia coli M. Fernanda San Martın ´ a , Federico M. Harte a , Huub Lelieveld b, a,U Gustavo V. Barbosa-Canovas , Barry G. Swansonc ´ a
Department of Biological Systems Engineering, Washington State Uni¨ ersity, Pullman, WA 99164-6120, USA b Unile¨ er Research Vlaardingen, P.O. Box 114, 3130 AC Vlaardingen, The Netherlands c Department of Food Science & Human Nutrition, Washington State Uni¨ ersity, Pullman, WA 99164-6120, USA Received 9 October 2000; accepted 29 August 2001
Abstract The inactivation effect of 18 T pulsed magnetic fields in combination with selected non-thermal technologies was studied on Escherichia coli ATCC 11775. The bacteria were subjected to a treatment of either ultrasound Ž20 kHz, 70 W, 242 m., high hydrostatic pressure Ž207 MPa, 5 min., pulsed electric field Ž6.25 kVrcm, 5.6 ms., or anti-microbials ŽNisin 77.5 mgrl, lysozyme 1 mgrml. and 50 magnetic field pulses Ž18 T, 30 s.. No additional inactivation or cell damage due to exposure to the pulsed magnetic field at 42 ⬚C was observed. 䊚 2001 Elsevier Science Ltd. All rights reserved. Keywords: 18 T Pulsed magnetic field; Escherichia coli; Inactivation
1. Introduction Food processing by non-thermal technologies is finding increased application in the food industry because of the advantages it presents over conventional thermal treatment. The use of high temperatures in food processing often results in products with degraded sensory and quality attributes ŽMertens & Knorr, 1992; Pothakamury, Barbosa-Canovas & Swanson, 1993; Wouters & ´ Smelt, 1997.. However, modern food preservation technologies focus on producing higher quality products than those currently available by using methods that require smaller thermal inputs or by combining methods that could have synergistic or hurdle effects ŽByrne, 2000; Leistner, 1994.. U
Corresponding author. Tel.: q1-509-335-6188; fax: q1-509-3352722. .. E-mail address: [email protected] ŽG.V. Barbosa-Canovas ´
The use of high hydrostatic pressure ŽHHP. in the inactivation of vegetative microorganisms has been well documented ŽHoover, Metrick, Papineau, Farkas & Knorr, 1989; Styles, Hoover & Farkas, 1991; Gervilla, Felipe, Ferragut & Guamis, 1997; Berlin, Herson, Hicks & Hoover, 1999.. It has been demonstrated that HHP has a synergistic effect when combined with mild heat treatments in the inactivation of yeast and bacterial cells ŽPandya, Jewett & Hoover, 1995; Linton, McClements & Patterson, 1999.. The addition of nisin and other bacteriocins may also enhance the bactericidal effect of HHP treatments depending on the pressure used ŽGarcıa-Graells, Masschalck & Michiels, 1999.. ´ Irradiation D-values for Clostridium sporogenes spores on chicken breast were lowered when irradiation treatment was followed by HHP and thermal treatment ŽCrawford, Murano, Olson & Shenoy, 1996.. Pulsed electric fields ŽPEF. is another non-thermal technology capable of inactivating microorganisms. The degree of inactivation depends on the field intensity,
1466-8564r01r$ - see front matter 䊚 2001 Elsevier Science Ltd. All rights reserved. PII: S 1 4 6 6 - 8 5 6 4 Ž 0 1 . 0 0 0 4 9 - 2
274
M. Fernanda San Martın ´ et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 273᎐277
pulse wave-shape, treatment time, environmental factors such as ionic concentration, temperature and pH, and physiological factors such as microbial cell concentration and population growth stage ŽQin, Pothakamury, Barbosa-Canovas & Swanson, 1996; Jeyamkon´ dan, Jayas & Holley, 1999; Barsotti, Merle & Cheftel, 1999.. Different theories exist to explain the electroporation effect of PEF on cellular membranes. Despite the specific mechanism promoting pore formation, alteration of the membrane’s permeability properties can occur ŽZimmerman, 1986; Angersbach, Heinz & Knorr, 2000; Barbosa-Canovas, Pierson, Zhang & Schaffner, ´ 2000.. Synergistic effects have been reported for PEF treatments combined with organic acids or mild thermal treatments ŽBarbosa-Canovas, Gongora-Nieto, ´ ´ Pothakamury & Swanson, 1999.. The use of ultrasound ŽUS. has also been effective in inactivating microorganisms. Ultrasonic waves of sufficiently high amplitude produce cavitation, which is the formation of bubbles or cavities in the medium. Stable cavitation causes a stream of bubbles within the sonic field that are abrasive to membrane surfaces of cells and cause them to fracture or leak. The type of microorganism as well as its shape and size will determine its susceptibility to ultrasonic treatments ŽEarnshaw, 1998.. Synergistic effects in the inactivation of Bacillus subtilis spores by the combination of US with thermal treatments and high pressure have been reported ŽRaso, Palop, Pagan ´ & Condon, ´ 1998.. Several theories exist to explain the interaction mechanism between living organisms and low intensity ŽmT. ᎏ extremely low frequency ŽHz. magnetic fields derived from household appliances and high voltage lines. Ion Cyclotron Resonance ŽLiboff, 1985., Ion Parametric Resonance ŽLednev, 1991. and Radical Recombination Model ŽBlackman, Blanchard, Benane & House, 1994. theories have been developed in an attempt to explain such interaction. Results on the effect of high intensity magnetic fields on microorganisms are scarce and inconsistent. Whereas some researchers report inactivation effects ŽHoffman, 1985., others report no effect ŽMalko, Constantinidis, Dillehay & Fajman, 1994., enhancement of microbial growth ŽOkuno, Tuchiya, Ano & Shoda, 1993. or altered growth rates ŽVan Nostrand, Reynolds & Hedrick, 1967.. The formation of metastable pores by the presence of natural magnetite or contaminant magnetic particles on cell membranes has been suggested as an effect of magnetic fields ŽVaughan & Weaver, 1998.. Altered protein synthesis has been observed by Mittenzwey, Submuth ¨ and Mei Ž1996. and Blank Ž1993.. Our group conducted exploratory research and found no inactivation effect of high intensity pulsed and static magnetic fields on E. coli and S. cere¨ isiae ŽHarte, San Martın, ´ Lacerda, Lelieveld, Swanson & Barbosa-Canovas, 2001.. ´
The objective of the present work was to determine if pulsed magnetic fields in combination with either HHP, PEF, US or anti-microbials Žnisin and lysozyme. were able to cause any inactivation of Escherichia coli.
2. Materials and methods 2.1. Preparation of pellets for inacti¨ ation studies: Freeze-dried E. coli ATCC 11775 was rehydrated with 0.4 ml nutrient broth ŽNB. ŽDIFCO, Sparks, MD.. After 30 min the cell suspension was inoculated into 6 ml of NB. Two milliliters of this suspension were then inoculated into 100 ml of NB and incubated at 37⬚C and 225 rev.rmin for 16 h in an orbital shaking bath ŽModel 3545, Lab Line instruments, Inc., Melrose Park, IL.. Finally, 20 ml of this culture were transferred to 500 ml of NB and incubated under the same conditions for 6 h. One-milliliter samples were taken every hour and absorbance was monitored at 540 nm using a spectrophotometer Ž8452A Hewlett-Packard, Palo Alto, CA, USA. calibrated with NB as the blank. When absorbance readings were approximately 0.7, 1:1 dilutions using NB were made. The stationary phase was determined when no further increase in absorbance was observed in diluted samples. Pour-plating in nutrient agar was done every hour so as to standardize absorbance readings. Bacteria were recovered by centrifugation of the culture broth at 2460 = g for 10 min at 10⬚C. After centrifugation supernatant was discarded, and cultures were re-suspended in 50 ml of NB. One milliliter of sterile 20% glycerol was added to cryogenic vials containing 1 ml of the culture and stored in liquid nitrogen until use in inactivation studies. The vials were thawed at room temperature for 2 min and treated either by US, HHP, PEF, AM and followed by pulsed magnetic fields ŽPMF. treatment. 2.2. Ultrasound treatment One milliliter of thawed culture was diluted 1:9 using 0.1% peptone solution ŽDIFCO, Sparks, MD, USA. to yield a starting inoculum of approximately 10 8 CFUrml. Ten milliliters of this suspension were exposed to five US cycles Ž20 kHz, 70 W. for 5 minrcycle followed by 1 min without US treatment, using a sonicator ŽModel 450, Branson Ultrasonics, Co., CT, USA. with 300 W maximum output. Amplitude was kept constant by automatically regulating power consumption and was set at 242 m. Samples were kept in an ice-water bath during US treatment to prevent heating. After US treatment, samples were subjected to PMF treatment.
M. Fernanda San Martın ´ et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 273᎐277
2.3. Pulsed electric field treatment Thawed culture Ž0.8 ml. with a concentration of 10 10 CFUrml were placed in a 0.4-cm gap Gene Pulser 䊛 cuvette ŽBio-Rad Laboratories, Hercules, CA, USA. and exposed to 10 electric field pulses of 2500 V using an electroporation system ŽGeneZapper TM 2500 IBI, CT.. The average pulse width was 5.6 ms and the energy input was 65.6 Jrpulse. After the PEF treatment samples were subjected to PMF treatment. 2.4. High hydrostatic pressure treatment One milliliter of thawed culture was diluted 1:9 using 0.1% peptone solution ŽDIFCO, Sparks, MD, USA.. Ten milliliters of this suspension were placed in a sterile Whirl-Pack bags ŽNasco, Fort Atkinson, WI, USA.. A pressure of 207 MPa for 5 min at room temperature was applied in a pressure vessel ŽEngineered Pressure Systems, Inc., MA, USA.. After HHP treatment, the samples were exposed to the PMF treatment.
275
pour-plate method in nutrient agar, and violet red bile ŽVRB. agar in the high pressure treatment. The combination of each technology plus the magnetic field treatment constituted one experiment and was analyzed as a completely random design with three replicates. Confidence intervals were established at ␣ s 0.05.
3. Results and discussion Few reports exist on the ability of MF to cause microbial inactivation ŽHoffman, 1985.; however, in a previous work, Harte et al. Ž2000. reported that an 18-T PMF was not effective in inactivating E. coli or S. cere¨ isiae cells. As an attempt to study whether a PMF could inactivate previously stressed microorganisms, four treatments were selected and applied to E. coli cells as stressing factors to study if the combination of any of them and the pulsed magnetic fields treatment would cause any microbial inactivation or cell damage. 3.1. Ultrasound treatment
2.5. Treatment EDTA and lysozyme or nisin Two anti-microbials, nisin and lysozyme were selected based in their ability to damage cell membranes. One milliliter of thawed culture was diluted Ž1:9. in pH 7.0 phosphate buffer. The initial dilution was divided in three equal parts and the following treatments were applied: Ž1. EDTA Ž5 mgrml.; Ž2. EDTA Ž5 mgrml. q nisin Ž77.5 ppm.; and Ž3. EDTA Ž5 mgrml. q lysozyme Ž1 mgrml.. Bacteria were allowed to stand in contact with the EDTA, EDTA and nisin or lysozyme for 15 min prior to PMF treatment. 2.6. Pulsed magnetic field treatment Each of the pre-treated samples Ž0.4 ml. was transferred to a glass culture tube Ž6 mm OD= 50 mm length. and sealed and placed inside a hose that passed through the magnetic field shaper and was connected to a controlled temperature circulating bath at 42 ⬚C ŽModel 9501, Fisher scientific, Niles, IL, USA.. Another vial, used as control, was kept inside the water bath for the same time with the only difference being that it was not exposed to the PMF. The PMF was applied using a 7000 series Magneform ŽMaxwell Laboratories, San Diego, CA, USA. with two capacitors Ž60 F, 8 kJ.. The average pulse duration was 30 s and fundamental frequency was 10᎐15 kHz. Magnetic field intensity was 18 Tesla ŽT.. Microbial enumeration was, in all cases, done by serial dilution of samples in 0.1% peptone solution and
The ultrasound treatment by itself caused a 0.8 log cycle reduction in the concentration of microbial cells as compared to the control sample. However, subsequent treatment with 50 MF pulses did not have any significant effect in increasing the reduction achieved by the ultrasound treatment alone. These results are shown in Fig. 1. 3.2. Pulsed electric fields The results for treatment of E. coli by PEF and MF can be seen in Fig. 2. A PEF treatment that caused reversible poration of the cell membrane without inactivating the microorganism was selected, since the purpose of this work was to study if the use of a magnetic field could actually inactivate cells previously stressed by other factors. Schoenbach, Peterkin, Alden III and Beebe Ž1997. reported that for inactivation of E. coli a
Fig. 1. Effect of 5 US Ž20 kHz, 70 W. cycles and 50 PMF at 42 ⬚C on the inactivation of Escherichia coli ATCC 11775.
276
M. Fernanda San Martın ´ et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 273᎐277
Fig. 2. Effect of 10 PEF Ž6.25 kVrcm. and 50 PMF at 42 ⬚C on the inactivation of Escherichia coli ATCC 11775.
critical electric field of 4.9 kVrcm was needed when bacterial cells were suspended in tap water and when pulses in the order of milliseconds were applied. In our work, an electric field of 6.25 kVrcm was applied in the form of exponential decay pulses with an average duration of 5.6 ms. Our results showed that no inactivation due to the PEF treatment occurred after 10 pulses, and that the further application of 50 MF pulses did not have any effect on the inactivation of E. coli cells. 3.3. High hydrostatic pressure A mild HHP treatment was applied to induce mechanical stress in the cell membrane without significantly reducing the bacterial population since the objective of this work was to study whether a magnetic field treatment could cause inactivation in already damaged cells. Zhou Ž1997. reported that after a treatment of 135 MPa for 10 min, E. coli cells exhibited pores on the cell surface and clumped cytoplasm. In our study, a HHP treatment of 207 MPa for 5 min resulted in a 1.9 log cycle reduction in E. coli population when plated on VRB agar Žselective medium. as compared to a 0.7 log cycle reduction when plated in nutrient agar Žgeneral medium.. The difference in counts suggests a sublethal damage to the cells due to the high pressure treatment. However, as shown in Figs. 3 and 4, no additional inactivation or cell damage of 50 magnetic field pulses at 18 T was observed.
Fig. 3. Effect of HHP Ž207 MPar5 min. and 50 PMF at 42 ⬚C in VRB agar on the inactivation of Escherichia coli ATCC 11775.
further stress promoted by the action of EDTAq antimicrobial treatments were not increased by exposure to PMF Ždata not shown.. Our results for an 18-T PMF treatment on E. coli ATCC 11775 are similar to those reported by Caubet Ž1999. who observed that Listeria innocua, E. coli and Bacillus cereus exposed to 1᎐6 pulses of a 7-T MF did not affect significantly their growth parameters. On the other hand, he observed that exposure of Penicillum cyclopium spores to only one pulse modified the aspect of the colonies when compared to unexposed spores.
4. Conclusion A pulsed magnetic field of 18 T is not capable of causing inactivation of Escherichia coli when applied after ‘mild treatments’ Žtreatments that by themselves do not cause inactivation. of other technologies ŽHHP, PEF, US, anti-microbials . are used as stressing factors. However, higher magnetic field strengths should be
3.4. EDTA and nisin or lysozyme Since E. coli is a Gram-negative bacterium, EDTA was used to remove Ca2q and Mg 2q ions from the cell wall. The loss of these ions increases the permeability of the cell wall and allows anti-microbial agents to act on the cytoplasmic membrane ŽDelves-Broughton, Blackburn, Evans & Hugenholtz, 1996; Cutter & Siragusa, 1995.. Therefore, a bacterial suspension was pretreated by exposure to EDTA either alone, or in the presence of nisin or lysozyme, and then exposed to 50 MF pulses at 18 T. The damage induced by EDTA or
Fig. 4. Effect of HHP Ž207 MPar5 min. and 50 PMF at 42 ⬚C in nutrient agar on the inactivation of Escherichia coli ATCC 11775.
M. Fernanda San Martın ´ et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 273᎐277
tested to draw general conclusions on the ability of this technology to inactivate microorganisms. References Angersbach, A., Heinz, V., & Knorr, D. Ž2000.. Effects of pulsed electric fields on cell membranes in real food systems. Inno¨ ati¨ e Food Science and Emerging Technologies, 1, 135᎐149. Barbosa-Canovas, G. V., Gongora-Nieto, M. M., Pothakamury, U. R., ´ ´ & Swanson, B. G. Ž1999.. Preser¨ ation of Foods with Pulsed Electric Fields. San Diego, CA: Academic Press. Barbosa-Canovas, G. V., Pierson, M. D., Zhang, Q. H., & Schaffner, ´ D. W. Ž2000.. Pulsed electric fields. Journal of Food Science Special Supplement: Kinetics of Microbial Inacti¨ ation for Alternati¨ e Food Processing Technologies, 65, 65᎐79. Barsotti, L., Merle, P., & Cheftel, J. C. Ž1999.. Food processing by pulsed electric fields. 1. Physical aspects. Food Re¨ iews International, 15Ž2., 163᎐180. Blackman, C. F., Blanchard, J. P., Benane, S. G., & House, D. E. Ž1994.. Empirical test of an ion parametric resonance model for magnetic field interactions with PC-12 cells. Bioelectromagnetics, 15, 239᎐260. Berlin, D. L., Herson, D. S., Hicks, D. T., & Hoover, D. G. Ž1999.. Response of pathogenic Vibrio species to high hydrostatic pressure. Applied and En¨ ironmental Microbiology, 65Ž6., 2776᎐2780. Blank, M. Ž1993.. Biological effects of electromagnetic fields. Bioelectrochemistry and Bioenergetics, 32, 203᎐210. Byrne, M. Ž2000.. Food Processing: Emerging Technologies. Anuga Food Tec. Caubet, R. Ž1999.. Effets des champs magnetiques pulses ´ ´ `a haute densite ´ de flux sur les micro-organismes. Proceedings of the Technological Training in the Food Industry, conservation for tomorrow, 2nd Edition. Bordeaux Pessac, France. Crawford, Y. J., Murano, E. A., Olson, D. G., & Shenoy, K. Ž1996.. Use of high hydrostatic pressure and irradiation to eliminate Clostridium sporogenes spores in chicken breast. Journal of Food Protection, 59 Ž7., 711᎐715. Cutter, C. N., & Siragusa, G. R. Ž1995.. Population reduction of Gram-negative pathogens following treatments with nisin and chelators under various conditions. Journal of Food Protection, 58 Ž9., 977᎐983. Delves-Broughton, J., Blackburn, P., Evans, R. E., & Hugenholtz, J. Ž1996.. Applications of the bacteriocin nisin. Antonie Van Leeuwenhoeck, 69 Ž2., 193. Earnshaw, R. G. Ž1998.. Ultrasound: a new opportunity for food preservation. Ch. 10. In M. J. W. Povey, & T. J. Mason, Ultrasound in Food Processing. Lodnon, UK: Blackie Academic and Professional. Garcıa-Graells, D., Masschalck, B., & Michiels, C. W. Ž1999.. Inacti´ vation of Escherichia coli in milk by high-hydrostatic-pressure treatment in combination with antimicrobial peptides. Journal of Food Protection, 62 Ž11., 1248᎐1254. Gervilla, R., Felipe, X., Ferragut, V., & Guamis, B. Ž1997.. Effect of high hydrostatic pressure on Escherichia coli and Pseudomonas fluorescens strains in ovine milk. Journal of Dairy Science, 80, 2297᎐2303. Harte, F., San Martın, ´ M. F., Lacerda, A. H., Lelieveld, H. L. M., Swanson, B. G., & Barbosa-Canovas, G. V. Ž2001.. Inactivation ´ effect of 18 T static and pulsed magnetic fields on Escherichia coli and Saccharomyces cere¨ isiae. Journal of Food Processing and Preser¨ ation, 25Ž3., 223᎐235. Hoffman G. A. Ž1985.. Deactivation of microorganisms by an oscillating magnetic field. US Patent 4,524,079 Hoover, D. G., Metrick, C., Papineau, A. M., Farkas, D. F., & Knorr, D. Ž1989.. Biological effects of high hydrostatic pressure on food microorganisms. Food Technology, 43Ž3., 99᎐107.
277
Jeyamkondan, S., Jayas, D. S., & Holley, R. A. Ž1999.. Pulsed electric field processing of foods: a review. Journal of Food Protection, 62 Ž9., 1088᎐1096. Lednev, V. V. Ž1991.. Possible mechanism for the influence of weak magnetic fields on biological systems. Bioelectromagnetics, 12, 71᎐75. Leistner, L. Ž1994.. Further developments in the utilization of hurdle technology for food preservation. Journal of Food Engineering, 22 Ž1᎐4., 421᎐432. Liboff, A. R. Ž1985.. Cyclotron resonance in membrane transport. In A. Chiabrera, C. Nicolini, & H. P. Schwan, Interactions Between Electromagnetic Fields and Cells Žpp. 281᎐293.. NATO ASI Series. Series A: Life Sciences 97. Linton, M., McClements, J. M. J., & Patterson, M. F. Ž1999.. Inactivation of Escherichia coli O157:H7 in orange juice using a combination of high pressure and mild heat. Journal of Food Protection, 62 Ž3., 277᎐279. Malko, J. A., Constantinidis, I., Dillehay, D., & Fajman, W. A. Ž1994.. Search for influence of 1.5 Tesla magnetic field on growth of yeast cells. Bioelectromagnetics, 15, 495᎐501. Mertens, B., & Knorr, D. Ž1992.. Developments of nonthermal processes for food preservation. Food Technology, 46 Ž5., 124᎐133. Mittenzwey, R., Submuth, R., & Mei, W. Ž1996.. Effects of extremely ¨ low-frequency electromagnetic fields in bacteria ᎏ the question of a co-stressing factor. Bioelectromagnetics, 40, 21᎐27. Okuno, K., Tuchiya, K., Ano, T., & Shoda, M. Ž1993.. Effect of super high magnetic field on the growth of Escherichia coli under various medium compositions and temperatures. Journal of Fermentation and Bioengineering, 75Ž2., 103᎐106. Pandya, Y., Jewett, F. F., & Hoover, D. G. Ž1995.. Concurrent effects of high hydrostatic pressure, acidity and heat on the destruction and injury of cells. Journal of Food Protection, 58 Ž3., 301᎐304. Pothakamury, U. R., Barbosa-Canovas, G. V., & Swanson, B. G. ´ Ž1993.. Magnetic field inactivation of microorganisms and generation of biological changes. Food Technology, 47 Ž12., 85᎐93. Qin, B. L., Pothakamury, U. R., Barbosa-Canovas, G. V., & Swanson, ´ B. G. Ž1996.. Nonthermal Pasteurization of Liquid Foods Using High-Intensity Pulsed Electric Fields. Raso, J., Palop, A., Pagan, ´ R., & Condon, ´ S. Ž1998.. Inactivation of Bacillus subtilis spores by combining ultrasonic waves under pressure and mild heat treatment. Journal of Applied Microbiology, 85, 849᎐854. Schoenbach, K. H., Peterkin, F. E., Alden III, R. W., & Beebe, S. J. Ž1997.. The effect of pulsed electric fields on biological cells: experiments and applications. IEEE Transactions on Plasma Science, 25Ž2., 284᎐292. Styles, M. F., Hoover, D. G., & Farkas, D. F. Ž1991.. Response of Listeria monocytogenes and Vibrio parahaemolyticus to high hydrostatic pressure. Journal of Food Science, 56 Ž5., 1991. Van Nostrand, F. E., Reynolds, R. J., & Hedrick, H. G. Ž1967.. Effects of a high magnetic field at different osmotic pressures and temperatures on multiplication of Saccharomyces cere¨ isiae. Applied Microbiology, 15Ž3., 561᎐563. Vaughan, T. E., & Weaver, J. C. Ž1998.. Molecular change due to biomagnetic simulation and transient magnetic fields: mechanical interference constrains on possible effects by cell membrane pore creation via magnetic particles. Bioelectrochemistry and Bioenergetics, 46, 121᎐128. Wouters, P. C., & Smelt, J. P. P. M. Ž1997.. Inactivation of microorganisms with pulse electric fields: potential for food preservation. Food Biotechnology, 11 Ž3., 193᎐229. Zhou, J. Ž1997.. Inactivation, Recovery, Nucleic Acid Leakage and Cell Disruption of Escherichia coli by High Hydrostatic Pressure. MS Thesis. Washington State University. Zimmerman, U. Ž1986.. Electrical breakdown, electropermeabilization and electrofusion. Re¨ iews of Physiology, Biochemistry and Pharmacology, 105, 175᎐256.
Inactivation effect of an 18-T pulsed magnetic field combined with other technologies on Escherichia coli M. Fernanda San Martın ´ a , Federico M. Harte a , Huub Lelieveld b, a,U Gustavo V. Barbosa-Canovas , Barry G. Swansonc ´ a
Department of Biological Systems Engineering, Washington State Uni¨ ersity, Pullman, WA 99164-6120, USA b Unile¨ er Research Vlaardingen, P.O. Box 114, 3130 AC Vlaardingen, The Netherlands c Department of Food Science & Human Nutrition, Washington State Uni¨ ersity, Pullman, WA 99164-6120, USA Received 9 October 2000; accepted 29 August 2001
Abstract The inactivation effect of 18 T pulsed magnetic fields in combination with selected non-thermal technologies was studied on Escherichia coli ATCC 11775. The bacteria were subjected to a treatment of either ultrasound Ž20 kHz, 70 W, 242 m., high hydrostatic pressure Ž207 MPa, 5 min., pulsed electric field Ž6.25 kVrcm, 5.6 ms., or anti-microbials ŽNisin 77.5 mgrl, lysozyme 1 mgrml. and 50 magnetic field pulses Ž18 T, 30 s.. No additional inactivation or cell damage due to exposure to the pulsed magnetic field at 42 ⬚C was observed. 䊚 2001 Elsevier Science Ltd. All rights reserved. Keywords: 18 T Pulsed magnetic field; Escherichia coli; Inactivation
1. Introduction Food processing by non-thermal technologies is finding increased application in the food industry because of the advantages it presents over conventional thermal treatment. The use of high temperatures in food processing often results in products with degraded sensory and quality attributes ŽMertens & Knorr, 1992; Pothakamury, Barbosa-Canovas & Swanson, 1993; Wouters & ´ Smelt, 1997.. However, modern food preservation technologies focus on producing higher quality products than those currently available by using methods that require smaller thermal inputs or by combining methods that could have synergistic or hurdle effects ŽByrne, 2000; Leistner, 1994.. U
Corresponding author. Tel.: q1-509-335-6188; fax: q1-509-3352722. .. E-mail address: [email protected] ŽG.V. Barbosa-Canovas ´
The use of high hydrostatic pressure ŽHHP. in the inactivation of vegetative microorganisms has been well documented ŽHoover, Metrick, Papineau, Farkas & Knorr, 1989; Styles, Hoover & Farkas, 1991; Gervilla, Felipe, Ferragut & Guamis, 1997; Berlin, Herson, Hicks & Hoover, 1999.. It has been demonstrated that HHP has a synergistic effect when combined with mild heat treatments in the inactivation of yeast and bacterial cells ŽPandya, Jewett & Hoover, 1995; Linton, McClements & Patterson, 1999.. The addition of nisin and other bacteriocins may also enhance the bactericidal effect of HHP treatments depending on the pressure used ŽGarcıa-Graells, Masschalck & Michiels, 1999.. ´ Irradiation D-values for Clostridium sporogenes spores on chicken breast were lowered when irradiation treatment was followed by HHP and thermal treatment ŽCrawford, Murano, Olson & Shenoy, 1996.. Pulsed electric fields ŽPEF. is another non-thermal technology capable of inactivating microorganisms. The degree of inactivation depends on the field intensity,
1466-8564r01r$ - see front matter 䊚 2001 Elsevier Science Ltd. All rights reserved. PII: S 1 4 6 6 - 8 5 6 4 Ž 0 1 . 0 0 0 4 9 - 2
274
M. Fernanda San Martın ´ et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 273᎐277
pulse wave-shape, treatment time, environmental factors such as ionic concentration, temperature and pH, and physiological factors such as microbial cell concentration and population growth stage ŽQin, Pothakamury, Barbosa-Canovas & Swanson, 1996; Jeyamkon´ dan, Jayas & Holley, 1999; Barsotti, Merle & Cheftel, 1999.. Different theories exist to explain the electroporation effect of PEF on cellular membranes. Despite the specific mechanism promoting pore formation, alteration of the membrane’s permeability properties can occur ŽZimmerman, 1986; Angersbach, Heinz & Knorr, 2000; Barbosa-Canovas, Pierson, Zhang & Schaffner, ´ 2000.. Synergistic effects have been reported for PEF treatments combined with organic acids or mild thermal treatments ŽBarbosa-Canovas, Gongora-Nieto, ´ ´ Pothakamury & Swanson, 1999.. The use of ultrasound ŽUS. has also been effective in inactivating microorganisms. Ultrasonic waves of sufficiently high amplitude produce cavitation, which is the formation of bubbles or cavities in the medium. Stable cavitation causes a stream of bubbles within the sonic field that are abrasive to membrane surfaces of cells and cause them to fracture or leak. The type of microorganism as well as its shape and size will determine its susceptibility to ultrasonic treatments ŽEarnshaw, 1998.. Synergistic effects in the inactivation of Bacillus subtilis spores by the combination of US with thermal treatments and high pressure have been reported ŽRaso, Palop, Pagan ´ & Condon, ´ 1998.. Several theories exist to explain the interaction mechanism between living organisms and low intensity ŽmT. ᎏ extremely low frequency ŽHz. magnetic fields derived from household appliances and high voltage lines. Ion Cyclotron Resonance ŽLiboff, 1985., Ion Parametric Resonance ŽLednev, 1991. and Radical Recombination Model ŽBlackman, Blanchard, Benane & House, 1994. theories have been developed in an attempt to explain such interaction. Results on the effect of high intensity magnetic fields on microorganisms are scarce and inconsistent. Whereas some researchers report inactivation effects ŽHoffman, 1985., others report no effect ŽMalko, Constantinidis, Dillehay & Fajman, 1994., enhancement of microbial growth ŽOkuno, Tuchiya, Ano & Shoda, 1993. or altered growth rates ŽVan Nostrand, Reynolds & Hedrick, 1967.. The formation of metastable pores by the presence of natural magnetite or contaminant magnetic particles on cell membranes has been suggested as an effect of magnetic fields ŽVaughan & Weaver, 1998.. Altered protein synthesis has been observed by Mittenzwey, Submuth ¨ and Mei Ž1996. and Blank Ž1993.. Our group conducted exploratory research and found no inactivation effect of high intensity pulsed and static magnetic fields on E. coli and S. cere¨ isiae ŽHarte, San Martın, ´ Lacerda, Lelieveld, Swanson & Barbosa-Canovas, 2001.. ´
The objective of the present work was to determine if pulsed magnetic fields in combination with either HHP, PEF, US or anti-microbials Žnisin and lysozyme. were able to cause any inactivation of Escherichia coli.
2. Materials and methods 2.1. Preparation of pellets for inacti¨ ation studies: Freeze-dried E. coli ATCC 11775 was rehydrated with 0.4 ml nutrient broth ŽNB. ŽDIFCO, Sparks, MD.. After 30 min the cell suspension was inoculated into 6 ml of NB. Two milliliters of this suspension were then inoculated into 100 ml of NB and incubated at 37⬚C and 225 rev.rmin for 16 h in an orbital shaking bath ŽModel 3545, Lab Line instruments, Inc., Melrose Park, IL.. Finally, 20 ml of this culture were transferred to 500 ml of NB and incubated under the same conditions for 6 h. One-milliliter samples were taken every hour and absorbance was monitored at 540 nm using a spectrophotometer Ž8452A Hewlett-Packard, Palo Alto, CA, USA. calibrated with NB as the blank. When absorbance readings were approximately 0.7, 1:1 dilutions using NB were made. The stationary phase was determined when no further increase in absorbance was observed in diluted samples. Pour-plating in nutrient agar was done every hour so as to standardize absorbance readings. Bacteria were recovered by centrifugation of the culture broth at 2460 = g for 10 min at 10⬚C. After centrifugation supernatant was discarded, and cultures were re-suspended in 50 ml of NB. One milliliter of sterile 20% glycerol was added to cryogenic vials containing 1 ml of the culture and stored in liquid nitrogen until use in inactivation studies. The vials were thawed at room temperature for 2 min and treated either by US, HHP, PEF, AM and followed by pulsed magnetic fields ŽPMF. treatment. 2.2. Ultrasound treatment One milliliter of thawed culture was diluted 1:9 using 0.1% peptone solution ŽDIFCO, Sparks, MD, USA. to yield a starting inoculum of approximately 10 8 CFUrml. Ten milliliters of this suspension were exposed to five US cycles Ž20 kHz, 70 W. for 5 minrcycle followed by 1 min without US treatment, using a sonicator ŽModel 450, Branson Ultrasonics, Co., CT, USA. with 300 W maximum output. Amplitude was kept constant by automatically regulating power consumption and was set at 242 m. Samples were kept in an ice-water bath during US treatment to prevent heating. After US treatment, samples were subjected to PMF treatment.
M. Fernanda San Martın ´ et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 273᎐277
2.3. Pulsed electric field treatment Thawed culture Ž0.8 ml. with a concentration of 10 10 CFUrml were placed in a 0.4-cm gap Gene Pulser 䊛 cuvette ŽBio-Rad Laboratories, Hercules, CA, USA. and exposed to 10 electric field pulses of 2500 V using an electroporation system ŽGeneZapper TM 2500 IBI, CT.. The average pulse width was 5.6 ms and the energy input was 65.6 Jrpulse. After the PEF treatment samples were subjected to PMF treatment. 2.4. High hydrostatic pressure treatment One milliliter of thawed culture was diluted 1:9 using 0.1% peptone solution ŽDIFCO, Sparks, MD, USA.. Ten milliliters of this suspension were placed in a sterile Whirl-Pack bags ŽNasco, Fort Atkinson, WI, USA.. A pressure of 207 MPa for 5 min at room temperature was applied in a pressure vessel ŽEngineered Pressure Systems, Inc., MA, USA.. After HHP treatment, the samples were exposed to the PMF treatment.
275
pour-plate method in nutrient agar, and violet red bile ŽVRB. agar in the high pressure treatment. The combination of each technology plus the magnetic field treatment constituted one experiment and was analyzed as a completely random design with three replicates. Confidence intervals were established at ␣ s 0.05.
3. Results and discussion Few reports exist on the ability of MF to cause microbial inactivation ŽHoffman, 1985.; however, in a previous work, Harte et al. Ž2000. reported that an 18-T PMF was not effective in inactivating E. coli or S. cere¨ isiae cells. As an attempt to study whether a PMF could inactivate previously stressed microorganisms, four treatments were selected and applied to E. coli cells as stressing factors to study if the combination of any of them and the pulsed magnetic fields treatment would cause any microbial inactivation or cell damage. 3.1. Ultrasound treatment
2.5. Treatment EDTA and lysozyme or nisin Two anti-microbials, nisin and lysozyme were selected based in their ability to damage cell membranes. One milliliter of thawed culture was diluted Ž1:9. in pH 7.0 phosphate buffer. The initial dilution was divided in three equal parts and the following treatments were applied: Ž1. EDTA Ž5 mgrml.; Ž2. EDTA Ž5 mgrml. q nisin Ž77.5 ppm.; and Ž3. EDTA Ž5 mgrml. q lysozyme Ž1 mgrml.. Bacteria were allowed to stand in contact with the EDTA, EDTA and nisin or lysozyme for 15 min prior to PMF treatment. 2.6. Pulsed magnetic field treatment Each of the pre-treated samples Ž0.4 ml. was transferred to a glass culture tube Ž6 mm OD= 50 mm length. and sealed and placed inside a hose that passed through the magnetic field shaper and was connected to a controlled temperature circulating bath at 42 ⬚C ŽModel 9501, Fisher scientific, Niles, IL, USA.. Another vial, used as control, was kept inside the water bath for the same time with the only difference being that it was not exposed to the PMF. The PMF was applied using a 7000 series Magneform ŽMaxwell Laboratories, San Diego, CA, USA. with two capacitors Ž60 F, 8 kJ.. The average pulse duration was 30 s and fundamental frequency was 10᎐15 kHz. Magnetic field intensity was 18 Tesla ŽT.. Microbial enumeration was, in all cases, done by serial dilution of samples in 0.1% peptone solution and
The ultrasound treatment by itself caused a 0.8 log cycle reduction in the concentration of microbial cells as compared to the control sample. However, subsequent treatment with 50 MF pulses did not have any significant effect in increasing the reduction achieved by the ultrasound treatment alone. These results are shown in Fig. 1. 3.2. Pulsed electric fields The results for treatment of E. coli by PEF and MF can be seen in Fig. 2. A PEF treatment that caused reversible poration of the cell membrane without inactivating the microorganism was selected, since the purpose of this work was to study if the use of a magnetic field could actually inactivate cells previously stressed by other factors. Schoenbach, Peterkin, Alden III and Beebe Ž1997. reported that for inactivation of E. coli a
Fig. 1. Effect of 5 US Ž20 kHz, 70 W. cycles and 50 PMF at 42 ⬚C on the inactivation of Escherichia coli ATCC 11775.
276
M. Fernanda San Martın ´ et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 273᎐277
Fig. 2. Effect of 10 PEF Ž6.25 kVrcm. and 50 PMF at 42 ⬚C on the inactivation of Escherichia coli ATCC 11775.
critical electric field of 4.9 kVrcm was needed when bacterial cells were suspended in tap water and when pulses in the order of milliseconds were applied. In our work, an electric field of 6.25 kVrcm was applied in the form of exponential decay pulses with an average duration of 5.6 ms. Our results showed that no inactivation due to the PEF treatment occurred after 10 pulses, and that the further application of 50 MF pulses did not have any effect on the inactivation of E. coli cells. 3.3. High hydrostatic pressure A mild HHP treatment was applied to induce mechanical stress in the cell membrane without significantly reducing the bacterial population since the objective of this work was to study whether a magnetic field treatment could cause inactivation in already damaged cells. Zhou Ž1997. reported that after a treatment of 135 MPa for 10 min, E. coli cells exhibited pores on the cell surface and clumped cytoplasm. In our study, a HHP treatment of 207 MPa for 5 min resulted in a 1.9 log cycle reduction in E. coli population when plated on VRB agar Žselective medium. as compared to a 0.7 log cycle reduction when plated in nutrient agar Žgeneral medium.. The difference in counts suggests a sublethal damage to the cells due to the high pressure treatment. However, as shown in Figs. 3 and 4, no additional inactivation or cell damage of 50 magnetic field pulses at 18 T was observed.
Fig. 3. Effect of HHP Ž207 MPar5 min. and 50 PMF at 42 ⬚C in VRB agar on the inactivation of Escherichia coli ATCC 11775.
further stress promoted by the action of EDTAq antimicrobial treatments were not increased by exposure to PMF Ždata not shown.. Our results for an 18-T PMF treatment on E. coli ATCC 11775 are similar to those reported by Caubet Ž1999. who observed that Listeria innocua, E. coli and Bacillus cereus exposed to 1᎐6 pulses of a 7-T MF did not affect significantly their growth parameters. On the other hand, he observed that exposure of Penicillum cyclopium spores to only one pulse modified the aspect of the colonies when compared to unexposed spores.
4. Conclusion A pulsed magnetic field of 18 T is not capable of causing inactivation of Escherichia coli when applied after ‘mild treatments’ Žtreatments that by themselves do not cause inactivation. of other technologies ŽHHP, PEF, US, anti-microbials . are used as stressing factors. However, higher magnetic field strengths should be
3.4. EDTA and nisin or lysozyme Since E. coli is a Gram-negative bacterium, EDTA was used to remove Ca2q and Mg 2q ions from the cell wall. The loss of these ions increases the permeability of the cell wall and allows anti-microbial agents to act on the cytoplasmic membrane ŽDelves-Broughton, Blackburn, Evans & Hugenholtz, 1996; Cutter & Siragusa, 1995.. Therefore, a bacterial suspension was pretreated by exposure to EDTA either alone, or in the presence of nisin or lysozyme, and then exposed to 50 MF pulses at 18 T. The damage induced by EDTA or
Fig. 4. Effect of HHP Ž207 MPar5 min. and 50 PMF at 42 ⬚C in nutrient agar on the inactivation of Escherichia coli ATCC 11775.
M. Fernanda San Martın ´ et al. r Inno¨ ati¨ e Food Science & Emerging Technologies 2 (2001) 273᎐277
tested to draw general conclusions on the ability of this technology to inactivate microorganisms. References Angersbach, A., Heinz, V., & Knorr, D. Ž2000.. Effects of pulsed electric fields on cell membranes in real food systems. Inno¨ ati¨ e Food Science and Emerging Technologies, 1, 135᎐149. Barbosa-Canovas, G. V., Gongora-Nieto, M. M., Pothakamury, U. R., ´ ´ & Swanson, B. G. Ž1999.. Preser¨ ation of Foods with Pulsed Electric Fields. San Diego, CA: Academic Press. Barbosa-Canovas, G. V., Pierson, M. D., Zhang, Q. H., & Schaffner, ´ D. W. Ž2000.. Pulsed electric fields. Journal of Food Science Special Supplement: Kinetics of Microbial Inacti¨ ation for Alternati¨ e Food Processing Technologies, 65, 65᎐79. Barsotti, L., Merle, P., & Cheftel, J. C. Ž1999.. Food processing by pulsed electric fields. 1. Physical aspects. Food Re¨ iews International, 15Ž2., 163᎐180. Blackman, C. F., Blanchard, J. P., Benane, S. G., & House, D. E. Ž1994.. Empirical test of an ion parametric resonance model for magnetic field interactions with PC-12 cells. Bioelectromagnetics, 15, 239᎐260. Berlin, D. L., Herson, D. S., Hicks, D. T., & Hoover, D. G. Ž1999.. Response of pathogenic Vibrio species to high hydrostatic pressure. Applied and En¨ ironmental Microbiology, 65Ž6., 2776᎐2780. Blank, M. Ž1993.. Biological effects of electromagnetic fields. Bioelectrochemistry and Bioenergetics, 32, 203᎐210. Byrne, M. Ž2000.. Food Processing: Emerging Technologies. Anuga Food Tec. Caubet, R. Ž1999.. Effets des champs magnetiques pulses ´ ´ `a haute densite ´ de flux sur les micro-organismes. Proceedings of the Technological Training in the Food Industry, conservation for tomorrow, 2nd Edition. Bordeaux Pessac, France. Crawford, Y. J., Murano, E. A., Olson, D. G., & Shenoy, K. Ž1996.. Use of high hydrostatic pressure and irradiation to eliminate Clostridium sporogenes spores in chicken breast. Journal of Food Protection, 59 Ž7., 711᎐715. Cutter, C. N., & Siragusa, G. R. Ž1995.. Population reduction of Gram-negative pathogens following treatments with nisin and chelators under various conditions. Journal of Food Protection, 58 Ž9., 977᎐983. Delves-Broughton, J., Blackburn, P., Evans, R. E., & Hugenholtz, J. Ž1996.. Applications of the bacteriocin nisin. Antonie Van Leeuwenhoeck, 69 Ž2., 193. Earnshaw, R. G. Ž1998.. Ultrasound: a new opportunity for food preservation. Ch. 10. In M. J. W. Povey, & T. J. Mason, Ultrasound in Food Processing. Lodnon, UK: Blackie Academic and Professional. Garcıa-Graells, D., Masschalck, B., & Michiels, C. W. Ž1999.. Inacti´ vation of Escherichia coli in milk by high-hydrostatic-pressure treatment in combination with antimicrobial peptides. Journal of Food Protection, 62 Ž11., 1248᎐1254. Gervilla, R., Felipe, X., Ferragut, V., & Guamis, B. Ž1997.. Effect of high hydrostatic pressure on Escherichia coli and Pseudomonas fluorescens strains in ovine milk. Journal of Dairy Science, 80, 2297᎐2303. Harte, F., San Martın, ´ M. F., Lacerda, A. H., Lelieveld, H. L. M., Swanson, B. G., & Barbosa-Canovas, G. V. Ž2001.. Inactivation ´ effect of 18 T static and pulsed magnetic fields on Escherichia coli and Saccharomyces cere¨ isiae. Journal of Food Processing and Preser¨ ation, 25Ž3., 223᎐235. Hoffman G. A. Ž1985.. Deactivation of microorganisms by an oscillating magnetic field. US Patent 4,524,079 Hoover, D. G., Metrick, C., Papineau, A. M., Farkas, D. F., & Knorr, D. Ž1989.. Biological effects of high hydrostatic pressure on food microorganisms. Food Technology, 43Ž3., 99᎐107.
277
Jeyamkondan, S., Jayas, D. S., & Holley, R. A. Ž1999.. Pulsed electric field processing of foods: a review. Journal of Food Protection, 62 Ž9., 1088᎐1096. Lednev, V. V. Ž1991.. Possible mechanism for the influence of weak magnetic fields on biological systems. Bioelectromagnetics, 12, 71᎐75. Leistner, L. Ž1994.. Further developments in the utilization of hurdle technology for food preservation. Journal of Food Engineering, 22 Ž1᎐4., 421᎐432. Liboff, A. R. Ž1985.. Cyclotron resonance in membrane transport. In A. Chiabrera, C. Nicolini, & H. P. Schwan, Interactions Between Electromagnetic Fields and Cells Žpp. 281᎐293.. NATO ASI Series. Series A: Life Sciences 97. Linton, M., McClements, J. M. J., & Patterson, M. F. Ž1999.. Inactivation of Escherichia coli O157:H7 in orange juice using a combination of high pressure and mild heat. Journal of Food Protection, 62 Ž3., 277᎐279. Malko, J. A., Constantinidis, I., Dillehay, D., & Fajman, W. A. Ž1994.. Search for influence of 1.5 Tesla magnetic field on growth of yeast cells. Bioelectromagnetics, 15, 495᎐501. Mertens, B., & Knorr, D. Ž1992.. Developments of nonthermal processes for food preservation. Food Technology, 46 Ž5., 124᎐133. Mittenzwey, R., Submuth, R., & Mei, W. Ž1996.. Effects of extremely ¨ low-frequency electromagnetic fields in bacteria ᎏ the question of a co-stressing factor. Bioelectromagnetics, 40, 21᎐27. Okuno, K., Tuchiya, K., Ano, T., & Shoda, M. Ž1993.. Effect of super high magnetic field on the growth of Escherichia coli under various medium compositions and temperatures. Journal of Fermentation and Bioengineering, 75Ž2., 103᎐106. Pandya, Y., Jewett, F. F., & Hoover, D. G. Ž1995.. Concurrent effects of high hydrostatic pressure, acidity and heat on the destruction and injury of cells. Journal of Food Protection, 58 Ž3., 301᎐304. Pothakamury, U. R., Barbosa-Canovas, G. V., & Swanson, B. G. ´ Ž1993.. Magnetic field inactivation of microorganisms and generation of biological changes. Food Technology, 47 Ž12., 85᎐93. Qin, B. L., Pothakamury, U. R., Barbosa-Canovas, G. V., & Swanson, ´ B. G. Ž1996.. Nonthermal Pasteurization of Liquid Foods Using High-Intensity Pulsed Electric Fields. Raso, J., Palop, A., Pagan, ´ R., & Condon, ´ S. Ž1998.. Inactivation of Bacillus subtilis spores by combining ultrasonic waves under pressure and mild heat treatment. Journal of Applied Microbiology, 85, 849᎐854. Schoenbach, K. H., Peterkin, F. E., Alden III, R. W., & Beebe, S. J. Ž1997.. The effect of pulsed electric fields on biological cells: experiments and applications. IEEE Transactions on Plasma Science, 25Ž2., 284᎐292. Styles, M. F., Hoover, D. G., & Farkas, D. F. Ž1991.. Response of Listeria monocytogenes and Vibrio parahaemolyticus to high hydrostatic pressure. Journal of Food Science, 56 Ž5., 1991. Van Nostrand, F. E., Reynolds, R. J., & Hedrick, H. G. Ž1967.. Effects of a high magnetic field at different osmotic pressures and temperatures on multiplication of Saccharomyces cere¨ isiae. Applied Microbiology, 15Ž3., 561᎐563. Vaughan, T. E., & Weaver, J. C. Ž1998.. Molecular change due to biomagnetic simulation and transient magnetic fields: mechanical interference constrains on possible effects by cell membrane pore creation via magnetic particles. Bioelectrochemistry and Bioenergetics, 46, 121᎐128. Wouters, P. C., & Smelt, J. P. P. M. Ž1997.. Inactivation of microorganisms with pulse electric fields: potential for food preservation. Food Biotechnology, 11 Ž3., 193᎐229. Zhou, J. Ž1997.. Inactivation, Recovery, Nucleic Acid Leakage and Cell Disruption of Escherichia coli by High Hydrostatic Pressure. MS Thesis. Washington State University. Zimmerman, U. Ž1986.. Electrical breakdown, electropermeabilization and electrofusion. Re¨ iews of Physiology, Biochemistry and Pharmacology, 105, 175᎐256.