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Inactivation of Escherichia Coli O157:H7 in Liquid Dialyzed Egg Using Pulsed Electric Fields
0960–3085/04/$30.00+0.00 # 2004 Institution of Chemical Engineers Trans IChemE, Part C, June 2004 Food and Bioproducts Processing, 82(C2): 151–156
www.ingentaselect.com=titles=09603085.htm
INACTIVATION OF ESCHERICHIA COLI O157:H7 IN LIQUID DIALYZED EGG USING PULSED ELECTRIC FIELDS M. AMIALI1, M. O. NGADI1,*, V. G. S. RAGHAVAN1 and J. P. SMITH2 2
1 Department of Bioresource Engineering, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada Department of Food science and Agricultural Chemistry, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada
P
ulsed electric field (PEF) pasteurization may be used either to replace or supplement conventional processing of heat-sensitive products such as liquid egg. The objective of this study was to investigate inactivation characteristics of Escherichia coli O157:H7 in liquid egg products at low temperature using square waveform pulsed electric fields. Dialyzed liquid egg products, namely whole egg, egg white and egg yolk, were exposed to an electric field of 15 kV cm1 at a low temperature of 0 C. The square voltage fields were generated across parallel plate treatment chambers. A pulse frequency of 1 Hz was used. Up to 500 pulses were applied. Product temperature during the PEF treatment was controlled using a water cooling system. About 1, 3 and 3.5 log reductions were obtained for the dialyzed egg white, egg yolk and whole egg products, respectively. The results showed that microbial inactivation rate increased with increasing number of pulses, especially for the egg yolk and whole egg products. The inactivation kinetics was exponential with some tailing. A new kinetic model for the bacteria inactivation was proposed. Keywords: dialyzed liquid egg products; liquid whole egg; egg yolk; egg white; Escherichia coli O157:H7; inactivation; pulsed electric field.
liquid egg products, namely 60 C and 3.5 min for liquid whole egg, 56.6 C and 3.5 min for egg white and, 61.1 C and 3.5 min for egg yolk (Anonymous, 1978). However, apart from bacteria inactivation, thermal processing also degrades product quality. Small variation in pasteurization temperatures significantly changes product quality and functionality. For instance, heating liquid egg products above 60 C for 3.5 min decreased their functionality due to insolubilization and denaturation of egg proteins (Cunningham, 1986; Herald and Smith, 1989). Woodward and Cotterill (1983) reported heat-induced aggregation of liquid egg proteins when heated at 57–87 C for 3 min. Thus, time–temperature control required for product safety is a major constraint in achieving high quality liquid egg products using thermal processing. Therefore, there is a need to develop alternative processing technology to achieve bacteria inactivation in liquid egg with minimal loss in functionality. Pulsed electric field (PEF) is a non-thermal technology with potential to replace or supplement reduced thermal treatment in a hurdle process. The technique has been widely used in the genetic engineering and biotechnology fields for cell hybridization in a process known as ‘electroporation’ (Chang et al., 1992). In this process, the applied PEF is controlled to maintain viability while cell contents are manipulated. However, in PEF treatment of foods, since the goal is to inactivate microbial cells, PEF is applied at sufficient intensity and duration to result in irreversible cell damage. Inactivation characteristics of various microorganisms including E. coli, Lactobacillus brevis and Saccharomyces cerevisiae in food
INTRODUCTION Egg is one of the most significant food products in North America. More than 5.4 billion eggs are produced annually in Canada, contributing approximately $500 million annually to the nation’s economy (Statistic Canada, 2002). Up to 71 billion eggs were consumed in the USA in 2000. Of the total number of eggs consumed, more than 30% were in the form of egg products (that is eggs removed from their shells), indicating the enormous size of the market. Escherichia coli O157:H7, one of over 100 different strains belonging to the group of Gram-negative facultative anaerobic bacteria Escherichia coli, is often an indicator of fecal contamination in food products. This bacterium is mostly found in soil and water, on plant, in the intestinal tract of animals, and in animal products as well as prepared foods handled by people. Liquid egg products may be contaminated during processing with Escherichia coli O157:H7 because of improper handling and unsanitary conditions (Banwart, 1989; Vanderzant and Splittstroesser, 2001). All raw egg products are required to be pasteurized by strict regulations in Canada and most other countries (Ma et al., 1993). Conventional thermal pasteurization protocols require different heating and holding times for different *Correspondence to: Dr M.O. Ngadi, Department of Bioresource Engineering, McGill University, MacDonald Campus, Ste-Anne-de-Bellevue, Quebec, Canada. E-mail: [email protected]
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media such as apple juice, skim milk and liquid whole egg have been reported (Hu¨lsheger et al., 1981; Jayaram et al., 1992; Castro et al., 1993; Qin et al., 1995; Martı´n et al., 1997; Martı´n-Belloso et al., 1997; Jeyamkondan et al., 1999). PEF processing of high electrical conductivity products such as liquid egg is technically more challenging than processing lower conductivity products. In general, foods with high electrical conductivities are difficult to treat since they generate low peak electric fields across the treatment chamber as a result of the high current they typically generate during PEF treatment (Barbosa-Ca´novas et al., 1999). In such case, pasteurization of high electrical conductivity food product will be accomplished with low efficiency. On the other hand, low conductivity products are generally more amenable to an effective PEF treatment. Electrical conductivity is related to the efficiency of energy transferred during PEF treatment. Consequently, it is suitable for lowering a food’s conductivity in order to obtain a greater microbial inactivation rate for the same applied electric field and with an application of equal input energy (Barbosa-Ca´novas et al., 1999). Different theories regarding the mechanism of microbial inactivation using PEF have been postulated in the literature (Jeyamkondan et al., 1999). The dielectric rupture theory postulates that electric field microbial inactivation is achieved as a result of pore formation and subsequent weakening and rupture of the cell membrane. A PEF system consists of an electric field pulse generator with appropriate discharge switch and a treatment chamber where the field is applied to the sample. Various pulse electric field generators and treatment chambers have been reported in the literature (Zhang et al., 1995; Grahl and Ma¨rkl, 1996). Successful application of PEF depends strongly on the type and characteristics of electric field waveform used, fluid medium and microorganisms of interest (Qin et al., 1994; Ho and Mittal, 2000). The objectives of this study were to investigate inactivation of E. coli O157:H7 suspended in dialyzed liquid egg products at low temperature, and to develop a kinetic model of inactivation during the PEF treatment. MATERIALS AND METHODS
where R (O), V (V), I (A), s (S m1), A (m2), and d (m) are, respectively, the resistance load, voltage, current cross the food, electrical conductivity of food, electrode surface area and the gap between electrodes. Because of the higher electrical conductivity of the samples (0.37, 0.34 and 0.17 S m1 for egg white, whole egg and egg yolk, respectively), the products were dialyzed to lower ionic strength in order to adjust their electrical conductivities. This was to match the impedance of the PEF generator used for this study and to reach higher electric field values. The dialysis processes were carried out using about 150 ml of liquid egg in ultrapure water at 4 C with gentle stirring. A cellulose membrane (Dialysis tubing, D-9402, Sigma-Aldrich, Canada) was used as a semi-permeable membrane to remove ions from the liquid egg products. The dialysis of whole egg and egg white was accomplished in about 8–10 h, whereas for egg yolk the process was concluded in about 26 h. Electrical conductivities of all the liquid egg products were reduced to the same electrical conductivity of 0.49 mS cm1 at 0 C after the dialysis process. The pH value of the samples did not change after the dialysis process. PEF System A pulse generator (Velonex Model 350, Velonex Division of Pulse Engineering Inc., Santa Clara, CA, USA) with a matched output impedance of 200 O was used in this experiment. The output voltage of the generator was 2 kV matched at maximum charge and the output voltage profile was negative square waveform with pulse duration varying up to 300 ms. Treatment Chamber A 0.23 ml static treatment chamber, consisting of two parallel stainless steel electrodes and polypropylene spacer with 0.15 cm gap and 1.53 cm2 surface area was used to treat the samples with PEF (Figure 1). The electric field intensity was set at 15 kV cm1. A cooling system was used to maintain constant temperature during the pulsed electric field treatment.
Preparation of Liquid Egg Products Three liquid egg products, namely 24.5% solid content whole egg ( pH 7.8), egg yolk ( pH 6.4) and egg white ( pH 8.3), were selected for the experiment. The whole egg and the egg white were obtained from a local egg processing company. However, the egg yolks samples were prepared in our laboratory by breaking eggs freshly purchased from the local grocery store. Egg yolk was carefully separated under aseptic conditions from egg white and chalaza, homogenized by gentle stirring and stored in a sterile 250 ml Erlenmeyer glass flask until 150 ml of liquid was collected. Electrical conductivities of the raw liquid egg products were estimated by measuring on-line the voltage and current across a treatment chamber at 0 C after each pulse from the PEF system. These electrical conductivity values were obtained using the following equations: V I d s¼ AR
R¼
(1) (2)
Treatment Procedure The chamber was filled with product conditioned at 0 C. The product was allowed to equilibrate in the test chamber for 5 min before each treatment. The process temperature was maintained at 0 C using a cooling system provided with the treatment chamber. The selected pulse numbers were 100, 200, 300 and 500. The source and load resistance were matched in series during the treatment process and the pulse frequency was set at 1 Hz with 200 ms pulse duration. The voltage and current across the treatment chamber were captured simultaneously using a two-channel digital oscilloscope (TDS3000, Tektronix, Wilsonville, OR, USA; see Figure 2). After treatment, the product was removed from the treatment chamber and the chamber was thoroughly washed with acetone solvent and rinsed twice with sterile distilled water. Energy input to the product during treatment was calculated as follows: Q¼
VIt v
(3)
Trans IChemE, Part C, Food and Bioproducts Processing, 2004, 82(C2): 151–156
INACTIVATION OF E. COLI O157:H7
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Figure 1. Dimensions of the treatment chamber.
where V is the voltage across the treatment chamber (V), I the applied current (A), t the treatment time (s), and v the volume of sample (m3). Microbial Analysis Culture of E. coli O157:H7 ATCC 43894 was grown to the early stationary growth phase in Brain Heart Infusion
Broth (BHIB, DIFCO, 0037-17-8) medium. The culture was spun at 10,000g and 4 C for 10 min using a centrifuge (Model 21000R, Needham Heights, MA, USA) to harvest the E. coli cells. The cell pellets were washed three times by re-suspension in distilled water. Washed pellets were finally re-suspended in 10 ml liquid egg product and further diluted in the product to achieve an initial cell concentration of about 106–107 cfu=ml. Viable cell count was evaluated
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Figure 2. Schematic of experimental setup.
before and after PEF treatment by plating on violet red bile agar (VRBA, DIFCO, 0012-17) and incubating at 37 C for 24 h. The mean count was reported for the two treated liquid egg samplings and the three plates used for each dilution. Analysis Statistical and regression analyses of data were conducted using Sigma-plot software (Sigmaplot 2000, Version 6.00, SPSS Inc.). Microsoft Excel software (Microsoft1 Excel 2002) was used to plot the curve of inactivation.
RESULTS AND DISCUSSIONS Survival fraction of E. coli decreased with increasing number of pulses as shown in Figure 3. About 1, 3 and 3.5 log reductions were obtained after 500 pulses at the 15 kV cm1 electric field intensity for egg white, egg yolk and whole egg, respectively. The pulse rate used for the treatment was 1 Hz and the temperature was maintained at 0 C using a cooling water system. Martı´n-Belloso et al. (1997) obtained 5–6 log reduction for E. coli in liquid whole egg in a continuous re-circulation PEF treatment set-up. The authors applied 26 kV cm1 pulses of 2 and 4 ms duration at the processing temperature of about 37 C. Ma et al. (2000) also
Figure 3. Inactivation rate of E. coli O157:H7 against number of pulses suspending in liquid egg products.
Trans IChemE, Part C, Food and Bioproducts Processing, 2004, 82(C2): 151–156
INACTIVATION OF E. COLI O157:H7 Table 1. Kinetic models for microbial inactivation by PEF. Model
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Table 3. Parameters for model Martı´n-Belloso et al. (1997). Parameter k
Standard error of estimate
r2
0.0548 0.0115 0.0396
0.0008 0.0666 0.0065
0.9999 0.9747 0.9998
Equations Model s ¼ ekt
s ¼ (t=tc )[(EEc )=k]
Hu¨lsheger et al. (1981)
Martı´n-Belloso et al. (1997) This work (model 1) This work (model 2)
s ¼ survival ratio t ¼ treatment time tc ¼ critical value of treatment time E ¼ field intensity Ec ¼ critical value of field intensity k ¼ independent kinetic constant s ¼ ekt s ¼ st þ (1 st)ekt st ¼ tailing survival ratio s ¼ sc ek1 t þ ð1 sc Þek2 t sc ¼ critical survival ratio
Whole egg Egg white Egg yolk
Table 4. Parameters for model 1 of this work. Model s ¼ st þ (1st)ekt Whole egg Egg white Egg yolk
Parameter st
Parameter k
Standard error of estimate
r2
0.0010 0.1151 0.0061
0.0576 0.0181 0.0436
0.0001 0.0301 0.0053
0.9999 0.9965 0.9999
Table 2. Parameters for model Hu¨lsheger et al. (1981). Model s ¼ (t=tc )[(EEc )=k] Whole egg Egg white Egg yolk
Parameter k
Parameter tc
Standard error of estimate
r2
1.64 0.76 1.31
3.5 16.17 4.94
— — —
0.989 0.961 0.863
obtained up to 6 log reduction with a continuous re-circulation PEF treatment of 48 kV cm1 at the processing temperature of about 41 C. The higher E. coli inactivation reported in these literature (Martı´n-Belloso et al., 1997; Ma et al., 2000) may be attributed to the higher electric field intensities and temperatures used in the studies. Microbial inactivation during PEF treatment typically increases with increasing electric field intensity and temperature (Martı´n-Belloso et al., 1997). The continuous re-circulation system used by the authors also may have improved agitation in the treatment chamber and thus improved inactivation. The egg products used in this study were dialyzed to reduce their electrical conductivities. Product characteristics and constituents such as protein influence microbial inactivation during PEF treatment. Fernandez-Diaz et al. (2000) reported that, although the gelling properties of egg white may change as result of dialysis, its protein solubility remained unchanged. Jeantet et al. (1999) determined that the protein content of ultrafiltered egg white was the same as fresh egg white. It is expected that E. coli inactivation obtained in this study with the dialyzed products should be in the same proportion for raw egg products at the same PEF parameters, considering that protein contents apparently remained unchanged with dialysis. This hypothesis could be verified with higher capacity generator. The result of the study demonstrates also that inactivation increases as the applied energy increases during PEF treatment. The energy density required to treat egg yolk was much lower than that needed to treat egg white and whole egg. The energy required to achieve 2.9 and 3.1 log microbial
reductions in egg yolk and whole egg, respectively, was only 3080 kJ l1 whereas for egg white, higher energy was required (5210 kJ l1) to obtain only 1.2 log reduction. However, the energy required to achieve the same 3 log reduction in egg yolk and whole egg was obtained after applying 500 and 300 pulses, respectively. The results show that much higher energy was required to process the higher electrical conductive egg white. For the same number of pulses, higher inactivation was achieved in whole egg than either in egg white or yolk. Martı`n-Belloso et al. (1997) reported energy input of about 505 kJ l1 (6000 kJ delivered into a chamber of 11.87 ml) to achieve about 4.5D reduction of E. coli (ATCC 11229) in whole egg at 37 C using 2 ms pulses. The authors also reported energy input of about 1011 kJ l1 (12,000 kJ in a chamber of 11.87 ml) for almost 6D reduction using a wider pulse width of 4 ms. Lower inactivation rates were obtained in this study for the similar energy input values reported by Martı`n-Belloso et al. (1997). This may be attributed to several factors including differences in the strains of E. coli, pulse widths and process temperatures used between the two studies. The energy density obtained in this study may be reduced considerably by using narrower pulse width. Results of this study reveal that different egg products have different energy requirements for similar bacterial inactivation. Accurate mathematical models describing the kinetics of inactivation of microbial population in real food systems are need to establish appropriate PEF process conditions to obtain known levels of microbial inactivation, which is necessary to achieve stable and safe products without over-processing (Wouter et al., 2001). Different kinetic models have been used to describe microbial inactivation kinetics during PEF treatment. These models include those of Hu¨lsheger et al. (1981) and Martı´n-Belloso et al. (1997). The model of Hu¨lsheger et al. (1981) accounts for a critical treatment time or field intensity below which there will be minimal inactivation. The Martı´n-Belloso et al. (1997) model is
Table 5. Parameters for model 2 of this work. Model s ¼ sc ek1 t þ ð1 sc Þek2 t Whole egg Egg white Egg yolk
Parameter Sc
Parameter k1
Parameter k2
Standard error of estimate
r2
0.9982 0.5989 0.9576
0.0589 18.5633 8.0066
0.0023 0.0046 0.0079
0.0000 0.0140 0.0043
1.0000 0.9996 0.9999
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basically a traditional exponential decay model. Observation of the numerous data on microbial inactivation using PEF suggests that these models might not be adequate in describing PEF inactivation (Wouter et al., 2001). Several authors have reported rapid inactivation within early pulses and subsequent tailing phenomena (Smelt et al., 2002). Therefore microbial inactivation during PEF appears to follow a two-phase kinetics. The first phase of inactivation consists of the rapid inactivation of bacteria due to increasing treatment time, whereas the second phase indicates the tailing effect which reflects the increasing resistance of the microorganisms to the treatment. These characteristics have not been represented by any available kinetic model. The suitability of four kinetic models as shown in Table 1 were examined. The model parameters obtained are shown in Tables 2–5. The results show that although the Hu¨lsheger et al. (1981) model yielded reasonable results, it did not adequately fit all the data as compared to the other models evaluated in our study. The Martı´n-Belloso et al. (1997) model yielded a better fit for the inactivation data with respect to treatment time. However, the models proposed in this study, namely model 1 and model 2, performed best. Considering the number of parameters involved, model 1 was selected as the best fit. It was able to predict the two-phase inactivation kinetics during PEF processing of liquid egg products. CONCLUSION From the results of this study, it can be concluded that PEF treatment can be successfully applied to obtain high levels of destruction with respect to the selected foodborne pathogen suspending in liquid egg products. The bacterial inactivation was a function of number of pulses, electrical conductivity and treatment time. PEF treatment with electric field of 15 kV cm1 and up to 500 pulses inactivated the bacteria with no risk of protein coagulation. The kinetic models used in this study showed that microbial inactivation with respect to treatment time follow exponential decay behavior with some tailing. A new model was developed to define the kinetics inactivation of E. coli O157:H7 inoculated in liquid egg products. NOMENCLATURE s A E Ec I k Q R s sc st t tc V v
electrical conductivity, S m1 surface area, m2 electric field intensity, kv cm1 critical electric field intensity, kv cm1 current, A kinetic constant energy input or density kJ L1 resistance, O survival ratio critical survival ratio tailing survival ratio treatment time, s critical treatment time, s voltage, kV volume, m3
REFERENCES
Banwart, G.J., 1989, Basic Food Microbiology, 2nd edn (Van Nostrand Reinhold, New York, USA). Barbosa-Ca´novas, G.V., Go´ngora-Nieto, M.M., Pothakamury, U.R. and Swanson, B.G., 1999, Preservation of Foods with Pulsed Electric Fields (Academic Press, San Diego, CA, USA). Castro, A., Barbosa-Ca´novas, G.V. and Swanson, B.G., 1993, Microbial inactivation of foods by pulsed electric fields, J Food Preserv, 17: 47–73. Chang, D.C., Chassy, B.M., Saunders, J.A. and Sowers, A.E., 1992, Guide to Electroporation and Electrofusion (Academic Press, San Diego, CA, USA). Cunningham, F.E., 1986, in Egg Science and Technology, 3rd edn, Stadelman, W.J. and Cotterhill, O.J. (eds) (AVI, Westport, CT, USA), p 243. Fernandez-Diaz, M.D., Barsotti, L., Dumay, E. and Cheftel, J.C., 2000, Effect of pulsed electric fields on ovalbumin solution and dialyzed egg white, J Agric Food Chem, 48: 2332–2339. Grahl, T. and Ma¨rkl, H., 1996, Killing of microorganisms by pulsed electric fields, App Microb Biotechnol, 45: 148–157. Herald, T.J. and Smith, D.M., 1989, Functional properties and composition of liquid whole egg proteins as influenced by pasteurization and frozen storage, Poult Sci, 68(11): 1461–1469. Ho, S. and Mittal, G.S., 2000, High voltage pulsed electrical field for liquid food pasteurization, Food Rev Int, 16(4): 395–434. Hu¨lsheger, H., Potel, J. and Niemann, E.G., 1981, Killing of bacteria with electric pulses of high field strength, Radiat Environ Biophys, 20: 53–65. Jayaram, S., Castle, G.S.P. and Margaritis, A., 1992, Kinetics of sterilization of lactobacillus brevis cells by the application of high voltage pulses, Biotechnol Bioeng, 40(11): 1412–1420. Jeantet, R., Baron, F., Nau, F., Roignant, M. and Brule´, G., 1999, High intensity pulsed electric fields applied to egg white: Effect on Salmonella enteritidis inactivation and protein denaturation, J Food Process, 62(12): 1381–1386. Jeyamkondan, S., Jayas, D.S. and Holley, R.A., 1999, Pulsed electric field processing of foods: A review, J Food Protec, 62(9): 1088–1096. Ma, C.Y., Harwalkar, V.R., Poste, L.M. and Sahasrabudhe, M.R., 1993, Effect of gamma irradiation on the physicochemical and functional properties of frozen liquid egg products, Food Res Int, 26: 247–254. Ma, L., Chang, F.J., Go´ngora-Nieto, M.M., Barbosa-Ca´novas, G.V. and Swanson, B.G., 2000, Food pasteurization using high-intensity pulsed electric fields: Promising new technology for non thermal pasteurization for eggs, Egg Nutr Biotechnol, pp 389–399. Martı´n, O., Qin, B.L., Chang, F.J., Barbosa-Ca´novas, G.V. and Swanson, B.G., 1997, Inactivation of Escherichia coli in skim milk by high intensity pulsed electric fields, J Food Process Eng, 20: 317–336. Martı´n-Belloso, O., Vega-Mercado, H., Qin, B.L., Chang, F.J., Barbosa-Ca´novas, G.V. and Swanson, B.G., 1997, Inactivation of Escherichia coli suspended in liquid egg using pulsed electric fields, J Food Process Preserv, 21: 193–208. Qin, B.L., Barbosa-Ca´novas, G.V., Swanson, B.G. and Pedrow, P.D., 1994, Inactivation of microorganisms by pulsed electric field of different voltage waveforms, IEEE Trans Dielec Elect Insul, 1(6): 1047–1057. Qin, B., Zhang, Q., Barbosa-Ca´novas, G.V., Swanson, B.G. and Pedrow, P.D., 1995, Pulsed electric field treatment chamber design for liquid food pasteurization using a finite element method, Trans ASAE, 38(2): 557–565. Smelt, J.P.P.M., Hellemons, J.C., Wouters, P.C. and van Gerwen, S.J.C., 2002, Physiological and mathematical aspects in setting criteria for decontamination of foods by physical means, Int J Food Microbiol, 78: 57–77. Statistic Canada, 2002, Production of Poultry and Eggs, Catalogue No. 23-202-XIB. Vanderzant, C. and Splittstroesser, D.F., 2001, Compendium of Methods for the Microbiological Examination of Foods, 4th edn (American Public Health Association, Washington, DC, USA). Woodward, S.A. and Cotterill, O.J., 1983, Electrophoresis and chromatography of heat treated plain, sugared and salted whole egg, J Food Sci, 48: 501–506. Wouter, P.C., Alvares, I. and Raso, J., 2001, Critical factors determining inactivation kinetics by pulsed electric field food processing, Trend Food Sci Technol, 12: 112–121. Zhang, Q.H., Chang, F.J., Barbosa-Ca´novas, G.V. and Swanson, B.G., 1995, Engineering aspects of pulsed electric field pasteurization, J Food Eng, 25: 261–291.
Anonymous, 1978, Eggs and egg products, in ASHRE Applications Handbook (American Society of Heating, Refrigeration and Air Conditioning Engineering, New York, USA).
Trans IChemE, Part C, Food and Bioproducts Processing, 2004, 82(C2): 151–156
www.ingentaselect.com=titles=09603085.htm
INACTIVATION OF ESCHERICHIA COLI O157:H7 IN LIQUID DIALYZED EGG USING PULSED ELECTRIC FIELDS M. AMIALI1, M. O. NGADI1,*, V. G. S. RAGHAVAN1 and J. P. SMITH2 2
1 Department of Bioresource Engineering, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada Department of Food science and Agricultural Chemistry, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada
P
ulsed electric field (PEF) pasteurization may be used either to replace or supplement conventional processing of heat-sensitive products such as liquid egg. The objective of this study was to investigate inactivation characteristics of Escherichia coli O157:H7 in liquid egg products at low temperature using square waveform pulsed electric fields. Dialyzed liquid egg products, namely whole egg, egg white and egg yolk, were exposed to an electric field of 15 kV cm1 at a low temperature of 0 C. The square voltage fields were generated across parallel plate treatment chambers. A pulse frequency of 1 Hz was used. Up to 500 pulses were applied. Product temperature during the PEF treatment was controlled using a water cooling system. About 1, 3 and 3.5 log reductions were obtained for the dialyzed egg white, egg yolk and whole egg products, respectively. The results showed that microbial inactivation rate increased with increasing number of pulses, especially for the egg yolk and whole egg products. The inactivation kinetics was exponential with some tailing. A new kinetic model for the bacteria inactivation was proposed. Keywords: dialyzed liquid egg products; liquid whole egg; egg yolk; egg white; Escherichia coli O157:H7; inactivation; pulsed electric field.
liquid egg products, namely 60 C and 3.5 min for liquid whole egg, 56.6 C and 3.5 min for egg white and, 61.1 C and 3.5 min for egg yolk (Anonymous, 1978). However, apart from bacteria inactivation, thermal processing also degrades product quality. Small variation in pasteurization temperatures significantly changes product quality and functionality. For instance, heating liquid egg products above 60 C for 3.5 min decreased their functionality due to insolubilization and denaturation of egg proteins (Cunningham, 1986; Herald and Smith, 1989). Woodward and Cotterill (1983) reported heat-induced aggregation of liquid egg proteins when heated at 57–87 C for 3 min. Thus, time–temperature control required for product safety is a major constraint in achieving high quality liquid egg products using thermal processing. Therefore, there is a need to develop alternative processing technology to achieve bacteria inactivation in liquid egg with minimal loss in functionality. Pulsed electric field (PEF) is a non-thermal technology with potential to replace or supplement reduced thermal treatment in a hurdle process. The technique has been widely used in the genetic engineering and biotechnology fields for cell hybridization in a process known as ‘electroporation’ (Chang et al., 1992). In this process, the applied PEF is controlled to maintain viability while cell contents are manipulated. However, in PEF treatment of foods, since the goal is to inactivate microbial cells, PEF is applied at sufficient intensity and duration to result in irreversible cell damage. Inactivation characteristics of various microorganisms including E. coli, Lactobacillus brevis and Saccharomyces cerevisiae in food
INTRODUCTION Egg is one of the most significant food products in North America. More than 5.4 billion eggs are produced annually in Canada, contributing approximately $500 million annually to the nation’s economy (Statistic Canada, 2002). Up to 71 billion eggs were consumed in the USA in 2000. Of the total number of eggs consumed, more than 30% were in the form of egg products (that is eggs removed from their shells), indicating the enormous size of the market. Escherichia coli O157:H7, one of over 100 different strains belonging to the group of Gram-negative facultative anaerobic bacteria Escherichia coli, is often an indicator of fecal contamination in food products. This bacterium is mostly found in soil and water, on plant, in the intestinal tract of animals, and in animal products as well as prepared foods handled by people. Liquid egg products may be contaminated during processing with Escherichia coli O157:H7 because of improper handling and unsanitary conditions (Banwart, 1989; Vanderzant and Splittstroesser, 2001). All raw egg products are required to be pasteurized by strict regulations in Canada and most other countries (Ma et al., 1993). Conventional thermal pasteurization protocols require different heating and holding times for different *Correspondence to: Dr M.O. Ngadi, Department of Bioresource Engineering, McGill University, MacDonald Campus, Ste-Anne-de-Bellevue, Quebec, Canada. E-mail: [email protected]
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media such as apple juice, skim milk and liquid whole egg have been reported (Hu¨lsheger et al., 1981; Jayaram et al., 1992; Castro et al., 1993; Qin et al., 1995; Martı´n et al., 1997; Martı´n-Belloso et al., 1997; Jeyamkondan et al., 1999). PEF processing of high electrical conductivity products such as liquid egg is technically more challenging than processing lower conductivity products. In general, foods with high electrical conductivities are difficult to treat since they generate low peak electric fields across the treatment chamber as a result of the high current they typically generate during PEF treatment (Barbosa-Ca´novas et al., 1999). In such case, pasteurization of high electrical conductivity food product will be accomplished with low efficiency. On the other hand, low conductivity products are generally more amenable to an effective PEF treatment. Electrical conductivity is related to the efficiency of energy transferred during PEF treatment. Consequently, it is suitable for lowering a food’s conductivity in order to obtain a greater microbial inactivation rate for the same applied electric field and with an application of equal input energy (Barbosa-Ca´novas et al., 1999). Different theories regarding the mechanism of microbial inactivation using PEF have been postulated in the literature (Jeyamkondan et al., 1999). The dielectric rupture theory postulates that electric field microbial inactivation is achieved as a result of pore formation and subsequent weakening and rupture of the cell membrane. A PEF system consists of an electric field pulse generator with appropriate discharge switch and a treatment chamber where the field is applied to the sample. Various pulse electric field generators and treatment chambers have been reported in the literature (Zhang et al., 1995; Grahl and Ma¨rkl, 1996). Successful application of PEF depends strongly on the type and characteristics of electric field waveform used, fluid medium and microorganisms of interest (Qin et al., 1994; Ho and Mittal, 2000). The objectives of this study were to investigate inactivation of E. coli O157:H7 suspended in dialyzed liquid egg products at low temperature, and to develop a kinetic model of inactivation during the PEF treatment. MATERIALS AND METHODS
where R (O), V (V), I (A), s (S m1), A (m2), and d (m) are, respectively, the resistance load, voltage, current cross the food, electrical conductivity of food, electrode surface area and the gap between electrodes. Because of the higher electrical conductivity of the samples (0.37, 0.34 and 0.17 S m1 for egg white, whole egg and egg yolk, respectively), the products were dialyzed to lower ionic strength in order to adjust their electrical conductivities. This was to match the impedance of the PEF generator used for this study and to reach higher electric field values. The dialysis processes were carried out using about 150 ml of liquid egg in ultrapure water at 4 C with gentle stirring. A cellulose membrane (Dialysis tubing, D-9402, Sigma-Aldrich, Canada) was used as a semi-permeable membrane to remove ions from the liquid egg products. The dialysis of whole egg and egg white was accomplished in about 8–10 h, whereas for egg yolk the process was concluded in about 26 h. Electrical conductivities of all the liquid egg products were reduced to the same electrical conductivity of 0.49 mS cm1 at 0 C after the dialysis process. The pH value of the samples did not change after the dialysis process. PEF System A pulse generator (Velonex Model 350, Velonex Division of Pulse Engineering Inc., Santa Clara, CA, USA) with a matched output impedance of 200 O was used in this experiment. The output voltage of the generator was 2 kV matched at maximum charge and the output voltage profile was negative square waveform with pulse duration varying up to 300 ms. Treatment Chamber A 0.23 ml static treatment chamber, consisting of two parallel stainless steel electrodes and polypropylene spacer with 0.15 cm gap and 1.53 cm2 surface area was used to treat the samples with PEF (Figure 1). The electric field intensity was set at 15 kV cm1. A cooling system was used to maintain constant temperature during the pulsed electric field treatment.
Preparation of Liquid Egg Products Three liquid egg products, namely 24.5% solid content whole egg ( pH 7.8), egg yolk ( pH 6.4) and egg white ( pH 8.3), were selected for the experiment. The whole egg and the egg white were obtained from a local egg processing company. However, the egg yolks samples were prepared in our laboratory by breaking eggs freshly purchased from the local grocery store. Egg yolk was carefully separated under aseptic conditions from egg white and chalaza, homogenized by gentle stirring and stored in a sterile 250 ml Erlenmeyer glass flask until 150 ml of liquid was collected. Electrical conductivities of the raw liquid egg products were estimated by measuring on-line the voltage and current across a treatment chamber at 0 C after each pulse from the PEF system. These electrical conductivity values were obtained using the following equations: V I d s¼ AR
R¼
(1) (2)
Treatment Procedure The chamber was filled with product conditioned at 0 C. The product was allowed to equilibrate in the test chamber for 5 min before each treatment. The process temperature was maintained at 0 C using a cooling system provided with the treatment chamber. The selected pulse numbers were 100, 200, 300 and 500. The source and load resistance were matched in series during the treatment process and the pulse frequency was set at 1 Hz with 200 ms pulse duration. The voltage and current across the treatment chamber were captured simultaneously using a two-channel digital oscilloscope (TDS3000, Tektronix, Wilsonville, OR, USA; see Figure 2). After treatment, the product was removed from the treatment chamber and the chamber was thoroughly washed with acetone solvent and rinsed twice with sterile distilled water. Energy input to the product during treatment was calculated as follows: Q¼
VIt v
(3)
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INACTIVATION OF E. COLI O157:H7
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Figure 1. Dimensions of the treatment chamber.
where V is the voltage across the treatment chamber (V), I the applied current (A), t the treatment time (s), and v the volume of sample (m3). Microbial Analysis Culture of E. coli O157:H7 ATCC 43894 was grown to the early stationary growth phase in Brain Heart Infusion
Broth (BHIB, DIFCO, 0037-17-8) medium. The culture was spun at 10,000g and 4 C for 10 min using a centrifuge (Model 21000R, Needham Heights, MA, USA) to harvest the E. coli cells. The cell pellets were washed three times by re-suspension in distilled water. Washed pellets were finally re-suspended in 10 ml liquid egg product and further diluted in the product to achieve an initial cell concentration of about 106–107 cfu=ml. Viable cell count was evaluated
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Figure 2. Schematic of experimental setup.
before and after PEF treatment by plating on violet red bile agar (VRBA, DIFCO, 0012-17) and incubating at 37 C for 24 h. The mean count was reported for the two treated liquid egg samplings and the three plates used for each dilution. Analysis Statistical and regression analyses of data were conducted using Sigma-plot software (Sigmaplot 2000, Version 6.00, SPSS Inc.). Microsoft Excel software (Microsoft1 Excel 2002) was used to plot the curve of inactivation.
RESULTS AND DISCUSSIONS Survival fraction of E. coli decreased with increasing number of pulses as shown in Figure 3. About 1, 3 and 3.5 log reductions were obtained after 500 pulses at the 15 kV cm1 electric field intensity for egg white, egg yolk and whole egg, respectively. The pulse rate used for the treatment was 1 Hz and the temperature was maintained at 0 C using a cooling water system. Martı´n-Belloso et al. (1997) obtained 5–6 log reduction for E. coli in liquid whole egg in a continuous re-circulation PEF treatment set-up. The authors applied 26 kV cm1 pulses of 2 and 4 ms duration at the processing temperature of about 37 C. Ma et al. (2000) also
Figure 3. Inactivation rate of E. coli O157:H7 against number of pulses suspending in liquid egg products.
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INACTIVATION OF E. COLI O157:H7 Table 1. Kinetic models for microbial inactivation by PEF. Model
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Table 3. Parameters for model Martı´n-Belloso et al. (1997). Parameter k
Standard error of estimate
r2
0.0548 0.0115 0.0396
0.0008 0.0666 0.0065
0.9999 0.9747 0.9998
Equations Model s ¼ ekt
s ¼ (t=tc )[(EEc )=k]
Hu¨lsheger et al. (1981)
Martı´n-Belloso et al. (1997) This work (model 1) This work (model 2)
s ¼ survival ratio t ¼ treatment time tc ¼ critical value of treatment time E ¼ field intensity Ec ¼ critical value of field intensity k ¼ independent kinetic constant s ¼ ekt s ¼ st þ (1 st)ekt st ¼ tailing survival ratio s ¼ sc ek1 t þ ð1 sc Þek2 t sc ¼ critical survival ratio
Whole egg Egg white Egg yolk
Table 4. Parameters for model 1 of this work. Model s ¼ st þ (1st)ekt Whole egg Egg white Egg yolk
Parameter st
Parameter k
Standard error of estimate
r2
0.0010 0.1151 0.0061
0.0576 0.0181 0.0436
0.0001 0.0301 0.0053
0.9999 0.9965 0.9999
Table 2. Parameters for model Hu¨lsheger et al. (1981). Model s ¼ (t=tc )[(EEc )=k] Whole egg Egg white Egg yolk
Parameter k
Parameter tc
Standard error of estimate
r2
1.64 0.76 1.31
3.5 16.17 4.94
— — —
0.989 0.961 0.863
obtained up to 6 log reduction with a continuous re-circulation PEF treatment of 48 kV cm1 at the processing temperature of about 41 C. The higher E. coli inactivation reported in these literature (Martı´n-Belloso et al., 1997; Ma et al., 2000) may be attributed to the higher electric field intensities and temperatures used in the studies. Microbial inactivation during PEF treatment typically increases with increasing electric field intensity and temperature (Martı´n-Belloso et al., 1997). The continuous re-circulation system used by the authors also may have improved agitation in the treatment chamber and thus improved inactivation. The egg products used in this study were dialyzed to reduce their electrical conductivities. Product characteristics and constituents such as protein influence microbial inactivation during PEF treatment. Fernandez-Diaz et al. (2000) reported that, although the gelling properties of egg white may change as result of dialysis, its protein solubility remained unchanged. Jeantet et al. (1999) determined that the protein content of ultrafiltered egg white was the same as fresh egg white. It is expected that E. coli inactivation obtained in this study with the dialyzed products should be in the same proportion for raw egg products at the same PEF parameters, considering that protein contents apparently remained unchanged with dialysis. This hypothesis could be verified with higher capacity generator. The result of the study demonstrates also that inactivation increases as the applied energy increases during PEF treatment. The energy density required to treat egg yolk was much lower than that needed to treat egg white and whole egg. The energy required to achieve 2.9 and 3.1 log microbial
reductions in egg yolk and whole egg, respectively, was only 3080 kJ l1 whereas for egg white, higher energy was required (5210 kJ l1) to obtain only 1.2 log reduction. However, the energy required to achieve the same 3 log reduction in egg yolk and whole egg was obtained after applying 500 and 300 pulses, respectively. The results show that much higher energy was required to process the higher electrical conductive egg white. For the same number of pulses, higher inactivation was achieved in whole egg than either in egg white or yolk. Martı`n-Belloso et al. (1997) reported energy input of about 505 kJ l1 (6000 kJ delivered into a chamber of 11.87 ml) to achieve about 4.5D reduction of E. coli (ATCC 11229) in whole egg at 37 C using 2 ms pulses. The authors also reported energy input of about 1011 kJ l1 (12,000 kJ in a chamber of 11.87 ml) for almost 6D reduction using a wider pulse width of 4 ms. Lower inactivation rates were obtained in this study for the similar energy input values reported by Martı`n-Belloso et al. (1997). This may be attributed to several factors including differences in the strains of E. coli, pulse widths and process temperatures used between the two studies. The energy density obtained in this study may be reduced considerably by using narrower pulse width. Results of this study reveal that different egg products have different energy requirements for similar bacterial inactivation. Accurate mathematical models describing the kinetics of inactivation of microbial population in real food systems are need to establish appropriate PEF process conditions to obtain known levels of microbial inactivation, which is necessary to achieve stable and safe products without over-processing (Wouter et al., 2001). Different kinetic models have been used to describe microbial inactivation kinetics during PEF treatment. These models include those of Hu¨lsheger et al. (1981) and Martı´n-Belloso et al. (1997). The model of Hu¨lsheger et al. (1981) accounts for a critical treatment time or field intensity below which there will be minimal inactivation. The Martı´n-Belloso et al. (1997) model is
Table 5. Parameters for model 2 of this work. Model s ¼ sc ek1 t þ ð1 sc Þek2 t Whole egg Egg white Egg yolk
Parameter Sc
Parameter k1
Parameter k2
Standard error of estimate
r2
0.9982 0.5989 0.9576
0.0589 18.5633 8.0066
0.0023 0.0046 0.0079
0.0000 0.0140 0.0043
1.0000 0.9996 0.9999
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basically a traditional exponential decay model. Observation of the numerous data on microbial inactivation using PEF suggests that these models might not be adequate in describing PEF inactivation (Wouter et al., 2001). Several authors have reported rapid inactivation within early pulses and subsequent tailing phenomena (Smelt et al., 2002). Therefore microbial inactivation during PEF appears to follow a two-phase kinetics. The first phase of inactivation consists of the rapid inactivation of bacteria due to increasing treatment time, whereas the second phase indicates the tailing effect which reflects the increasing resistance of the microorganisms to the treatment. These characteristics have not been represented by any available kinetic model. The suitability of four kinetic models as shown in Table 1 were examined. The model parameters obtained are shown in Tables 2–5. The results show that although the Hu¨lsheger et al. (1981) model yielded reasonable results, it did not adequately fit all the data as compared to the other models evaluated in our study. The Martı´n-Belloso et al. (1997) model yielded a better fit for the inactivation data with respect to treatment time. However, the models proposed in this study, namely model 1 and model 2, performed best. Considering the number of parameters involved, model 1 was selected as the best fit. It was able to predict the two-phase inactivation kinetics during PEF processing of liquid egg products. CONCLUSION From the results of this study, it can be concluded that PEF treatment can be successfully applied to obtain high levels of destruction with respect to the selected foodborne pathogen suspending in liquid egg products. The bacterial inactivation was a function of number of pulses, electrical conductivity and treatment time. PEF treatment with electric field of 15 kV cm1 and up to 500 pulses inactivated the bacteria with no risk of protein coagulation. The kinetic models used in this study showed that microbial inactivation with respect to treatment time follow exponential decay behavior with some tailing. A new model was developed to define the kinetics inactivation of E. coli O157:H7 inoculated in liquid egg products. NOMENCLATURE s A E Ec I k Q R s sc st t tc V v
electrical conductivity, S m1 surface area, m2 electric field intensity, kv cm1 critical electric field intensity, kv cm1 current, A kinetic constant energy input or density kJ L1 resistance, O survival ratio critical survival ratio tailing survival ratio treatment time, s critical treatment time, s voltage, kV volume, m3
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