Pulsed Electric Fields Controlling Microbial Growth in Milk

International Journal of FoodEngineering

Volume 4, Issue 7 2008 Article 1

Effectiveness of Pulsed Electric Fields inControlling Microbial Growth in Milk

Adedayo Otunola∗ Ayman El-Hag†

Shesha Jayaram‡ William A. Anderson∗∗

∗University of Waterloo, [email protected]†American University of Sharjah, [email protected]‡University of Waterloo, [email protected]

∗∗University of Waterloo, [email protected]

Copyright c©2008 The Berkeley Electronic Press. All rights reserved.

Effectiveness of Pulsed Electric Fields inControlling Microbial Growth in Milk

Adedayo Otunola, Ayman El-Hag, Shesha Jayaram, and William A. Anderson

Abstract

A study was conducted to assess the effectiveness of pulsed electric field (PEF) inactivation ofa heterogeneous community of microbes. The aim was to assess the impact of process parameterson an indigenous population of microbes present in milk, rather than pure cultures used in otherstudies. Tests over an electric field strength range of 10 – 40 kV/cm and 10 to 120 pulses permillilitre showed that high electric field strength and pulse number inactivated microbes by upto approximately 2 log. Inoculum size affected PEF effectiveness when only a few pulses wereapplied. A significant log-reduction was achieved against the indigenous microbes found in milkthat were apparently recalcitrant to commercial pasteurization. Microbial inactivation was moreextensive when E. coli was not added to the indigenous population, indicating that the added pureculture was more resistant than the indigenous microbes. The milk fat content had a significantnegative effect on the extent of log-reduction for indigenous microbes, when 2% and 18% levelswere compared.

KEYWORDS: E. coli, non-thermal processing, inactivation, pasteurization

Introduction

Food-borne diseases have major public health impact (Beuchat, 1996, Slutsker et al., 1998, Rowan, 1999, Rowan et al., 2000, Russell et al., 2000, Selma et al., 2003) hence controlling food-borne microbes is as much an issue of food safety as food quality.

Although thermal processes are widely used in the food industry (Steele, 2000) to meet pasteurization and sterilization requirements, the treatment not only kills pathogenic and spoilage microbes but it has undesirable effects on both nutritional and organoleptic quality of food products (Hansen, 1987, Oamen et al., 1989, Adams, 1991, Qin et al., 1995, Montenegro, 2000, Morales et al., 2000), while the natural antimicrobial and immuno-potency of milk are destroyed (Li-Chan et al., 1995).

Consumer’s requirements for foods are changing, they now demand foods that are safe, “fresh-like” and natural, and hence food processing technologies are now being designed to also preserve their natural quality among others (Jeyamkondan et al., 1999, Fernandez-Molina et al., 2005). Consequently, non-thermal food processing techniques are being developed for the preservation of foods (Barbosa-Canovas et al., 1999) as an alternative to traditional thermal methods, and pulsed electric field (PEF) is one of the promising non-thermal processes.

Early work on non-thermal food processing was mostly done on milk (Hite, 1899, Fetteman, 1928, Dutreux et al., 2000, Buffa et al., 2001) probably because it is the most complete single food (Bendicho, 2002), and a potential vehicle for transmission of disease (Dundee et al., 2001, Grant et al., 2000). But the lethal effects of pulsed electric fields on microorganisms were described first by Doevenspeck in the nineteen sixties (Grahl, 1996). It has since been a subject of intensive study mainly because of its potential for being a feasible alternative or supplement to thermal treatment (Peleg, 1995) for certain liquid food products (Yaqub et al., 2004, Heinz et al., 2001), and this electrotechnology may extend the shelf life of pasteurized products or be a valid second preservation treatment to thermal pasteurized milk without changing its sensory and nutritional attributes (Qin et al., 1995, Sepulveda et al., 2005).

Much has been reported on PEF treatment of food (Marquez et al., 1997, Ho and Mittal, 2000, Barbosa-Canovas and Zhang, 2001, Picart et al., 2002) within the last two decades, but the underlying mechanism(s) of inactivation of microorganisms is still not fully understood (Ho and Mittal, 1996, Simpson et al., 1999, Lebovka and Vorobiev, 2004, Aronsson and Ronner, 2005). The most studied possibilities are electroporation (Russell et al., 2000, Marquez et al., 1997, Dutreux et al., 2000, Chen and Lee, 1994, Knorr et al., 1994, Pothakamury et al., 1996, Alvarez et al., 2000, Hulsheger et al., 1983). While Hulsheger et al. (1983)

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Otunola et al.: Pulsed Electric Field Milk Preservation

Published by The Berkeley Electronic Press, 2008

did not observe any morphological destruction nor membrane rupture (Aronsson et al., 2001) of bacterial cells, Dutreux et al. (2000) noticed a rough cell surface and partial destruction of the cell membrane, similar to Hamilton and Sale (1967) and Sale and Hamilton (1967, 1968). Membrane destruction (electroporation), in the form of increased permeability of the membrane and/or membrane breakdown occurs when the induced membrane potential exceeds a critical value of electrical field (Tsong, 1990), and damage probability is maximal at membrane poles (Lebovka and Vorobiev, 2004).

The level of microbial inactivation by high Pulsed Electric Fields is a function of process parameters (Qin et al., 1995, Pothakamury et al., 1996, Grahl and Markl, 1996, Mizuno and Hori, 1988, Butz and Tauscher, 2002, Reyn et al., 2004) (electric field strength and electrode-type, total treatment time and temperature, pulse duration and waveform shapes), media factors (pH, antimicrobial and ionic compounds, conductivity and ionic strength) and microbial entity factors (species and strain, morphology, concentration, and growth phase of the microbes). Although PEF processing of food inactivates microbes and retains its qualities (Reyn et al., 2004) some microbes are recalcitrant to pulsed electric field inactivation treatments (Selma et al., 2003, Fleischman et al., 2004), and hence there is the need to elucidate parameters that may enhance microbial inactivation.

Curiously enough, the epidemiology of food illnesses is rapidly changing as newly recognized pathogens emerge and well recognised pathogens increase in prevalence and virulence or become associated with new food vehicles (Altekruse et al., 1997), and there is the need to develop food processing technology that will surpass the threat to life posed by food borne pathogens without altering its quality or taste. The effect of PEF treatment of food has been reported but with insufficient information on antimicrobial effectiveness and especially the kinetics of antimicrobial activity on indigenous milk-borne microbes. The objective of this work is to assess the potency of variable electric field strength and pulse numbers (of 1 µsecond duration) on a heterogeneous microbial community (E. coli plus milk-borne microbes) in a static chamber, and it is a step towards formulating optimal strategies for efficient PEF processing of milk.

Materials and Methods

Organisms and growth conditions

Escherichia coli (ATCC11229), an index of fecal pollution, was obtained from the American Type Culture Collection (ATCC, Rockville, MD), and trypticase soy broth was used as a batch cultivation medium. About 1 ml of thawed frozen culture was inoculated in 50 ml broth and continually agitated in a temperature

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International Journal of Food Engineering, Vol. 4 [2008], Iss. 7, Art. 1

http://www.bepress.com/ijfe/vol4/iss7/art110.2202/1556-3758.1494

controlled shaker at 37�C and 150 rpm for 15 hr as a pre-culture. The 5 ml of pre-culture was inoculated into 250 ml of growth medium for batch cultivation at 37�C for 18 hrs as described previously with modifications (Grahl and Markl, 1996, Garcia et al., 2005, Gupta et al., 2005). After 18 hrs, the broth was shaken to disperse the bacterial cells evenly before centrifugation at 3500 rpm for 15 minutes at 10�C (Aronsson et al., 2004). The supernatant liquid was removed and E. coli pellets washed with phosphate buffered saline solution. The bacterial suspension in saline was re-centrifuged repeatedly to remove traces of growth nutrient and other impurities. The clean bacterial cell suspension was serially diluted, and the optical density measured at 530 nm before plating out to determine the corresponding population. The cell suspension in saline was kept at ~0�C until needed for further use.

Milk-borne bacteria (mixed culture): the indigenous bacteria present in pasteurized milk were used after incubation for approximately 10 hr (see below).

Media

Lactose agar growth medium plates were prepared by dissolving (g/l) Lactose 5; Nutrient broth, 8; Agar 15, in water which was boiled before autoclaving to sterilize the media.

Trypticase Soy Broth Medium was prepared following Difco instruction on the medium container.

Phosphate buffer saline solution was prepared by dissolving (g/l) NaCl, 8.5; KH2PO4, 0.3; NaHPO4.2H 2O 0.72 in sterile distilled water and pH adjusted to7.0

Milk-sample preparation

Freshly pasteurized milk (Neilson milk, 2% partially skimmed, and 18% cream) purchased locally was incubated at 30�C for 10 hrs to allow the indigenous mixed-culture microbes to grow to the desired population (104 – 106 cfu/ml).

The E. coli contaminated milk sample was prepared by introducing a known concentration of the bacterium into a 10 hr old milk sample to give the desired bacterial population. Both the E. coli-contaminated and mixed culture 10 hr milk samples were kept at 4�C until needed for use.

Pulsed Electric Field Treatment Unit

A thyratron based pulse power supply was used in this study. The major components of the pulse power supply are: 30kV DC source, 6 M� limiting

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resistor, fast operating thyratron switch, 20 nF charging capacitor and the test cell. Figure 1 is a schematic diagram of the pulse power supply. The 20 nF capacitor is charged from the DC source via the 6 M� limiting resistor and when the thyratron switch closes it discharges through the test cell. The pulse repetition rate was fixed during the whole study at 1 pulse/second. Both the voltage and current waveform across the test cell were measured using a high voltage Tektronix probe with 1:1000 ratios and a current transformer with 0.1 V/A ratio and a 7 ns rise time, respectively. The test cell was a coaxial chamber with 3 mm distance between the high voltage and ground electrodes.

Figure 1: Schematic diagram of the pulse power supply.

Treatment Procedure in Static Chamber.

The chamber was cleaned with sterile distilled water, disinfected with 70% alcohol and was allowed to dry for a period of one hour. It was then filled gradually with 3 ml of sample (10hr old, E. coli-contaminated milk, or bacterial suspension in saline water) at 1~2�C without any air bubbles and pulse processing was initiated immediately after closing the chamber (Manas et al., 2001). The chamber contained two micro outlets that allowed for observation of bubbles if any, since air-spaces are undesirable in the system (Gupta et al., 2005, Manas et al., 2001).

The samples were subjected to selected pulsed electric field treatment with a field intensity ranging from 10 – 40 kV/cm and pulse numbers varying from 10 – 120/ml, but the pulse duration was kept at 1 µs. After each treatment, the sample (milk or saline) was removed from the chamber and kept on ice to keep the temperature below 10�C prior to plating. After removal of each processed sample, the chamber was washed with alcohol and dried with sterile paper before the next batch treatment. The width of the applied pulse was around 1 �s and hence the maximum treatment time when 120 pulses were applied was approximately 120 �s, with a maximum temperature rise of around 10oC.

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Bacterial count after PEF treatment of milk samples

PEF-treated milk samples were immediately returned to ice after treatment and moved to a laminar flow hood for plating. The total bacterial count before and after pulsed electric field processing was assayed using a modified standard plate count method described previously (Fernandez-Molina et al., 2005). One millilitre of sample was serially diluted (10-1 – 10-6) with sterile saline water and plated out in triplicate on the agar medium, which was then incubated at 30�C for 48 hr. The viability of the total microbes was determined by counting colony forming units (cfu) on each plate, multiplied by the dilution factor.

Results

a) Effect of Electric field strength The effect of electric field strength on microbial inactivation in 10 hr old (2% partially skimmed) milk inoculated with E.coli is presented in Figure 2.

The curve shows little microbial inactivation at field strengths of 10 – 20 kV/cm resulting in 0.03 log-reduction of the microbial population. The higher electric field strength of 30 kV/cm gave approximately 0.5 log reduction, and achieved 1.2 log reduction at 40 kV/cm field strength. Data from two different experimental runs are shown, indicating the reproducibility achieved in the experimentation.

Figure 2: Log inactivation (E. coli plus indigenous microbes) as a function of electric field at 120 pulses/ml, with starting concentrations of approximately 3.9x105 cfu/ml (5.6 log cfu/ml).

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b) Effect of microbial concentration and community composition Figure 3 shows the effect of initial concentration of E. coli plus indigenous microbes, while Figure 4 shows the effect of initial concentration of indigenous microbes alone on microbial inactivation over zero to 120 pulses per millilitre, at 40 kV/cm field strength.

There was 0.6 – 1.7 log reduction of organisms with the first 30 pulses when the concentration was greater than 5.1 log in both communities (Figures 3 and 4) but 0.5 log reduction at 4.2 log initial concentration. Microbial inactivation gradually increased to 1.5 for E. coli plus indigenous microbes, but increased sharply to 2.2 log for indigenous microbes alone, comparing results at 120 pulses/ml.

Figure 3: Log inactivation (E. coli plus indigenous microbes) as a function of number of pulses at 40 kV/cm, with starting concentrations of approximately 3.9x105 (�) and 1.5x105 (�) cfu/ml (5.6 and 5.2 log cfu/ml, respectively).

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Figure 4: Log inactivation (indigenous microbes alone) as a function of number of pulses at 40 kV/cm, with starting concentrations of 1.4x104 (�) and 1.0x107

(�) cfu/ml (4.1 and 7.0 log cfu/ml, respectively).

c) Effect of milk type at 40 kV/cm The effect of milk type (2% skimmed and 18% cream) on microbial inactivation of E.coli plus indigenous microbes in milk is shown in Figure 5.

At 30 pulses/ml there was a 0.2 log-reduction in 18% cream but 0.9 log in 2% skimmed milk. When the pulses were increased to 120 per millilitre, there was a 1.5 log-reduction in microbial population in 2% skimmed milk, but a 1.1 log reduction in 18% cream.

Figure 5: Log inactivation (E. coli plus indigenous microbes) as a function of number of pulses at 40 kV/cm for two different levels of milk fat, partly skimmed milk (2%) and cream (18%). Initial microbial concentrations were 3.9x105 and 6.6x106 cfu/ml for 2% and 18%, respectively.

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Discussion

It is evident from the above results that the antimicrobial potency of pulsed electric field is a function of electric field strength as previously reported (Aronsson et al., 2001, Raso et al., 2000, Evrendilek et al., 2004). Although microbial electroporation (dielectric rupture theory, Zimmermann et al., 1980) resulting in microbial log-reduction (cell inactivation) occurs when the induced membrane potential exceeds a critical value of electric strength (Tsong, 1990, Castro et al., 1993) a weak external field of 10 – 20 kV/cm did not do any appreciable damage to the microbial population in this investigation. However, irreversible membrane damage leading to membrane leakage at high field strength (Russell et al., 2000, Sale and Hamilton, 1967, Sale and Hamilton, 1968, Knorr et al., 2001) may account for the 1.5 – 2 log reduction observed in this report.

Although Alvarez et al. (2000) observed that microbial inactivation is not a function of initial cell concentration (inoculum size), Selma et al. (2003) claimed strain, inoculum size and product parameters affect the potency of PEF treatment. The results here help to explain the discrepancy, as follows. The antimicrobial potency of PEF may or may-not be affected by inoculum size, depending on the number of pulses and field strength applied. Microbial inactivation was affected by inoculum size when we applied few pulses (less than 30 pulses/mL, Figures 3-5) as observed by Selma et al. (2003), but it was not a factor at 120 pulses/mL. Similarly, Alvarez et al. (2000) reported no effect at 200 pulses/mL.

Pulsed electric field processing of milk have recorded impressive results by inactivating a single species of food pathogen in a homogenous system (Barbosa-Canovas et al., 1999, Bendicho et al. 2002, Picart et al., 2002, Evrendilek et al., 2004, Martin et al., 1997, Calderon-Miranda et al., 1999) but it is doubtful if such a situation is realistic. We therefore added the single species E.coli to indigenous milk-borne microbes to mimic a heterogeneous microbial community that might exist in practice. The milk-borne heterogeneous microbial community developed from the few colonies that were presumably recalcitrant to the commercial thermal pasteurization process. PEF treatment of the milk-borne community resulted in a greater than 2 log reduction (Figure 4) but the E. coliinoculated milk-borne community had only a 1.5 log reduction under similar conditions (Figure 3). These findings indicate that microbial community composition may also affect the potency of pulsed electric field inactivation of microbes in milk, and this appears to be one of the very few such reports to date.

Pulsed electric field processing of food shows promise for meeting the increasing demand for “fresh-like” nutritious and safe food, but the underlying mechanism(s) of microbial inactivation may be less an issue than the identification of possible parameters that may enhance optimization of the

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treatment. While this report may be a contribution towards this goal, additional research is clearly needed to elucidate the effects of initial microbial counts, strains, and growth phase and physiological status.

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Pulsed Electric Fields Controlling Microbial Growth in Milk