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International Dairy Journal 19 (2009) 30–35

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International Dairy Journal

journal homepage: www.elsevier .com/locate/ idairy j

Effect of thermosonication, pulsed electric field and their combination oninactivation of Listeria innocua in milk

F. Noci*, M. Walkling-Ribeiro, D.A. Cronin, D.J. Morgan, J.G. LyngAgriculture and Food Science Centre, School of Agriculture, Food Science and Veterinary Medicine, UCD Dublin, Belfield, Dublin 4, Republic of Ireland

a r t i c l e i n f o

Article history:Received 11 December 2007Received in revised form 21 July 2008Accepted 24 July 2008

* Corresponding author. Tel.: þ353 1 7167603; fax:E-mail address: francesco.noci@ucd.ie (F. Noci).

0958-6946/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.idairyj.2008.07.002

a b s t r a c t

The impact of thermosonication (TS) and pulsed electric field (PEF), individually and combined, on thesurvival of Listeria innocua 11288 (NCTC) in milk was investigated. TS (400 W, 160 s) without pre-heatingreduced L. innocua by 1.2 log10 cfu mL�1, while shorter treatment times produced negligible inactivation,suggesting TS to be a hurdle rather than an effective standalone treatment. PEF (30 and 40 kV cm�1,50 ms) at 10 �C caused a reduction of L. innocua of 1.1 and 3.3 log cycles, respectively. The highest fieldstrength (40 kV cm�1) combined with TS (80 s) led to 6.8 log10 cfu mL�1 inactivation. Milk pre-heated to55 �C (over 60 s) prior to TS followed by PEF (30 and 40 kV cm�1) showed inactivation between 4.5 and6.9 log10 cfu mL�1, the latter being comparable (P> 0.05) with thermal pasteurisation. The data indicatethat TS followed by PEF represents a valid alternative for L. innocua inactivation in milk.

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1. Introduction

Treatment with pulsed electric field (PEF) is considered amongone of the most promising alternative technologies for the pres-ervation of liquid foods. However, the effectiveness of PEF pro-cessing has been limited so far to vegetative cells, as the technologyhas not been as successful in inactivating bacterial spores andenzymes (Raso & Barbosa-Canovas, 2003). Although the mecha-nism by which the application of PEF inactivates microorganisms isnot yet fully elucidated, a phenomenon called ‘electroporation’,which involves reversible or irreversible pore formation in the cellmembrane, as a result of the application of an electric field, is themost commonly accepted mechanism that causes cell death(Barbosa-Canovas & Sepulveda, 2005).

The results obtained from PEF application to food suggest it asan alternative to thermal pasteurisation of liquid foods, therebyreducing the heat-induced damage to product quality (Barbosa-Canovas & Sepulveda, 2005). A drawback to PEF is that its effec-tiveness varies depending on the technological parameters used,the nature of the product and the microorganisms being targeted(Wouters, Alvarez, & Raso, 2001). In this respect, PEF processing oflow pH foods has shown better results than those obtained inproducts closer to a neutral pH (Alvarez, Raso, Palop, & Sala, 2000).Furthermore, PEF processing of liquid foods of low conductivity hasshown higher levels of inactivation than those obtained in higherconductivity products under similar treatment conditions

þ353 1 7161147.

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(Wouters, Dutreux, Smelt, & Lelieveld, 1999). Studies conducted ondifferent liquid products showed an increased effect of PEF whenthe product undergoes pre-heating, possibly due to changes in thecellular membrane permeability caused by the effect of tempera-ture (Hulsheger, Potel, & Niemann, 1981; Pothakamury, Vega,Zhang, Barbosa-Canovas, & Swanson, 1996; Wouters et al., 1999).These studies indicate the potential of a mild pre-heating step as anadditional hurdle for microbial inactivation.

To date the use of ultrasound as a preservation technology, hasnot received the same level of interest as PEF or other technologies(e.g., high hydrostatic pressure), possibly due to the limited impactof this technology on microorganisms, when applied as a solemethod of inactivation. High intensity (>1 W cm�2) ultrasoundwith frequency ranging between 18 and 100 kHz has been reportedto inactivate a range of enzymes and microorganisms, in particular,when used in conjunction with heat, moderate pressure and anti-microbials (Lopez et al., 1994; Sala, Burgos, Condon, Lopez, & Raso,1995; Vercet, Sanchez, Burgos, Montanes, & Buesa, 2002; Villamiel& de Jong, 2000a,b). The mechanism of microbial inactivation is notyet fully elucidated, although mechanical shock due to cavitationhas often been suggested (Abee & Wouters, 1999; Butz & Tauscher,2002; Grahame, 1996). The inner cytoplasmic membrane seems tobe the location where the main damage occurs, since no inactiva-tion difference has been noted between Gram-positive and Gram-negative bacteria (Scherba, Weigel, & O’Brien, 1991).

The objective of this study was to investigate the inactivation ofListeria innocua in low-fat milk processed by thermosonication (TS),PEF or TS followed by PEF. In particular, because TS and PEF inac-tivate by different mechanisms, this study investigated possibleadditive or synergistic effects, as the cell damage induced by one

Fig. 1. Example of the square wave pulse produced within the pulsed electric fieldsystem (one square is equivalent to 1 ms on the x-axis and 4 kV on the y-axis).

F. Noci et al. / International Dairy Journal 19 (2009) 30–35 31

hurdle could enhance the effectiveness of the second hurdle.Thermal treatment, conducted in a tubular heat exchanger wasused as a control in all experiments.

2. Materials and methods

2.1. Bacterial strains and inoculation

L. innocua 11288 (NCTC) was obtained from the School ofBiomolecular and Biomedical Science at UCD Dublin. A culturegrown in Listeria enrichment broth base (Oxoid Ltd., Basingstoke,UK) was used as inoculum. Inocula were prepared by adding 15 mLof a frozen pre-culture to 5 mL of Listeria enrichment broth baseand by placing it in a shaking water bath at 37 �C and 70 rpm(Grant, Cambridge, UK) for 24 h. Then, 1 mL of the inoculated brothof growth was added to 100 mL of the same broth and placed at37 �C for 24 h in the shaking water bath. The culture thus produced,composed of cells in their stationary growth phase, was used as theinoculum for 1 L of milk. The complete 100 mL were used asinoculum and added to the milk in order to achieve an initialpopulation of approximately 109 cfu mL�1.

2.2. Milk

Low-fat UHT milk (Lakeland Dairies, Killeshandra, Ireland) wasused in all experiments to minimize the risk of contamination withother organisms. The electrical conductivity of the milk was6.5�103 ms at 23 �C and was determined with a portable conduc-tivity meter (Cyberscan, Model No. CON 400 Series, EutechInstruments, Singapore).

2.3. Bacterial enumeration

Samples of low-fat UHT milk were taken after inoculation inorder to measure the initial concentration of microorganisms andkept at 0 �C in icy water for up to 30 min until plating. Post-treat-ment samples were collected immediately after processing andkept at 0 �C in icy water prior to plating for up to 30 min. Sampleswere then serially diluted in sterile Ringers solution (pH 7.0) andplated (0.1 mL) onto Oxford Listeria selective agar supplementedwith Oxford Listeria selective supplement (Oxoid Ltd. Basingstoke,UK). Plates were then incubated at 37 �C for 48 h before counting.

2.4. Processing equipment

2.4.1. ThermosonicationAn ultrasonic processor (Model No. UP 400S, Hielscher, Berlin,

Germany), with a constant frequency of 24 kHz and a maximumamplitude of 117 mm was used. The sonotrode used (Model No.H22D, Hielscher) had a 22 mm diameter tip and delivered anacoustic power density of 85 W cm�2. The power output of theprocessor could be adjusted between 20 and 100% of maximumpower (400 W). The sonotrode was permanently inserted intoa continuous process chamber, with a volume of 21 mL. A circu-lating water bath (Model No. LTD20G, Grant) was used to maintaina constant outlet temperature from the ultrasonic cell by re-circulating a refrigerant in the outer jacket of the ultrasonicprocessing chamber.

2.4.2. Pulsed electric fieldThe experimental set-up consisted of a peristaltic pump (Model

No. SR25 S300, Esska GmbH, Hamburg, Germany). The PEF systemconsisted of a 40 kV high voltage generator (Model No. LT40P50,Glassman High Voltage, Inc., High Bridge, NJ, USA), a pulse-formingnetwork and the treatment chamber. A digital oscilloscope (ModelNo. TDS 2012, Tektronix, Beaverton, OR, USA) was connected to the

system to monitor the pulse and a sample of the waveformgenerated during the process in the PEF chamber is shown in Fig. 1.A spark gap switch was used to control the pulse rate. The processchamber consisted of two parallel stainless steel electrodes sepa-rated by a 2.0-mm gap, the length of the electrodes was 84 mm andthe electrode surface exposed was 5.2 mm wide for a total electrodearea of 4.4�10�4 m2. The flow rate (Q) used was 15.75 mL min�1,which ensured laminar flow through the tubing and the processchamber. The average residence time (Tr) in the process chamberwas 3.33 s, calculated from the formula below:

Tr ¼ Q�

mL s�1�.

vðmLÞ

The system generated a 1 ms square wave pulse at differentfrequencies ranging from 10 to 18 Hz to obtain the required numberof pulses (as described in the experimental design section, below).

The number of pulses was obtained from the followingexpression:

n ¼�

vf

�� F

where n represents the number of pulses, v the treatment chambervolume in mL, f the pump flow rate in mL s�1 and F the frequency ofthe pulse in s�1. The temperature of the milk was monitoredthroughout the process (at 8 locations) using type T thermocouples.The temperature at the inlet of the PEF cell was maintained below20 �C to limit the temperature rise within the PEF cell.

2.4.3. Thermal pasteurisationThe thermal pasteurisation conditions used in this experiment

were similar to those used in the dairy industry. The inoculatedmilk was passed through a tubular heat exchanger (Model No.FT74 T, Armfield, Ringwood, UK) and at a flow rate of 91 mL min�1.The temperature of the holding tube was set at 72 �C for the milkintended for storage at chilling temperature (HT72). The residencetime in the holding tube was 26 s. The time and temperatureprofiles were monitored using thermocouples at specific pointsalong the tubular heat exchanger.

All equipments was thoroughly washed by circulating a solutionof NaOH (5%, w/v) for 10 min, followed by water until the pH of thewater coming out of the system was restored to neutrality. Finally,the entire system was washed with a hypochlorite-based disinfec-tant, and thoroughly rinsed with de-ionised water prior to use.

Table 1Processing protocols (2� 2) applied to low-fat UHT milk in a fixed sequence ofthermosonication (TS) followed by pulsed electric field (PEF)

Protocol Fixed conditions Variable conditions

1 Pre-heatingtemperature (�C)

PEF treatmenttime (ms)

TS power (W) PEF strength(kV cm�1)

25 50 320 30400 40

2 PEF treatmenttime (ms)

TS power (W) Pre-heatingtemperature (�C)

PEF strength(kV cm�1)

50 400 25 3055 40

3 Pre-heatingtemperature (�C)

TS power (W) PEF strength(kV cm�1)

PEF energydensity (kJ L�1)

55 400 40 48050 576

F. Noci et al. / International Dairy Journal 19 (2009) 30–3532

2.5. Experimental design

2.5.1. Evaluation of TSA study was carried out on the TS unit to evaluate the effect of

power output and treatment time. The experiment was designed asa 2� 3 factorial arrangement, with 3 different power output levels(240, 320 or 400 W) and 3 different flow rates to give 3 treatmenttimes (80, 120 or 160 s).

2.5.2. Evaluation of PEFThe PEF treatment was designed as a 2� 2 factorial arrange-

ment, with electric field strength and total treatment time as mainfactors. The electric field strengths used were 30 or 40 kV cm�1, andthe total treatment times were 25 or 50 ms with 1 ms pulses. Theproduct entered the PEF process cell at ambient temperature andwas cooled immediately to 10 �C after the treatment.

2.5.3. TS followed by PEFThe description of the conditions used for the TS followed by PEF

is summarised in Table 1.In Protocol 1, two TS power outputs (320 or 400 W) combined

with 2 electric field strengths (30 or 40 kV cm�1) at a constanttreatment time (50 ms) were used . In combining the two technol-ogies, the product leaving the ultrasonic cell was entering the PEFprocess cell after passing through a cooling coil, to decrease thetemperature of the product to 10 �C prior to PEF treatment. Aschematic representation of the experimental set-up used for thedifferent protocols is shown in Fig. 2a and b.

In Protocol 2, two TS pre-heating regimes and 2 PEF strengthswere used. In the first heating regime, the milk entered the ultra-sonic chamber at ambient temperature and experienced a temper-ature rise to 55 �C during the residence time (80 s) in the ultrasonic

Fig. 2. Schematic representation of the processing system used for treatment of milk byfollowed by TS and PEF.

chamber. In the second regime, the temperature of the milk wasincreased from 25 to 55 �C prior to sonication by passing it througha heating coil for 55 s. The product exited the TS cell again at 55 �C.The residence time inside the ultrasonic chamber under bothregimes was 80 s and a constant power output of 400 W was used.The temperature was controlled by circulating liquid refrigerant inthe outer jacket of the TS cell. The PEF strengths were 30 or40 kV cm�1 and a constant number of pulses (50) was applied.

In Protocol 3, the heating regime described above (i.e., pre-heating to 55 �C prior to TS) was kept constant, followed byconstant TS conditions (400 W for 80 s). Two PEF strengths (40 or50 kV cm�1) were used combined with different numbers of pulses(50 or 60 for 40 kV cm�1, and 32 or 38 for 50 kV cm�1, respectively)to produce two energy density levels at each electric field strength(480 and 576 kJ L�1).

2.6. Statistical analysis

The statistical analysis of the treatments in Protocol 1 was per-formed according to a 2� 2 factorial arrangement where the mainfactors were the ultrasonic equipment power output and the PEFelectric field strength. Treatments in Protocol 2 were also analysedas a 2� 2 factorial arrangement with different TS heating regimesand PEF strength as main factors. Treatments in Protocol 3 wereanalysed as a 2� 2 factorial design, with the PEF strength and theenergy density level as main factors. All experiments were carriedout in triplicate batches, all consisting of 1 L of inoculated milk.Triplicate samples of 10 mL were collected for microbiologicalanalysis from each batch. Thermal pasteurisation was used asa control treatment. A two-way ANOVA was conducted and a LSDmultiple comparison test with a P< 0.05 was used to determinewhether the application of different experimental conditionscaused different degrees of bacterial inactivation or not. All statis-tical analysis were performed using Genstat (Version 8.1, VSNInternational, Hemel Hempstead, UK).

3. Results and discussion

3.1. Thermosonication

The effect of TS on the inactivation of L. innocua cells is shown inFig. 3. As expected there was a linear increase (P< 0.05) in themicrobial inactivation as the retention time in the process cellincreased from 80 to 120 and 160 s. However, the effect of TS wasnegligible when the treatment was applied for the shortest time atthe lowest power output (240 W). A retention time of 160 s wasobserved to be the most effective in inactivating L. innocua, sinceinactivation exceeded 1 log cycle regardless of the power output.Processing the milk for 120 s resulted in a significantly lower

(a) thermosonication (TS) followed by pulsed electric field (PEF) and (b) pre-heating

Fig. 3. Inactivation of Listeria innocua 11288 in milk treated by thermosonication asfunction of the treatment time at a power output of 240 W (A), 320 W (-) and 400 W(:). Error bars represent the standard deviation.

Table 3

F. Noci et al. / International Dairy Journal 19 (2009) 30–35 33

microbial inactivation (P< 0.05), with results ranging between 0.5and 1 log10 cfu mL�1. With the shortest retention time (80 s), onlythe 400 W treatment resulted in a significant microbial reduction(0.6 log10 cfu mL�1). An explanation for the low inactivationobserved could be the limited residence times used in the experi-ment. These results suggest that the successful application of TS asa sole method for food preservation is unlikely under these pro-cessing parameters. The main reason for the relatively low inacti-vation observed was the fact that the thermal load to which L.innocua was exposed was relatively limited when the residencetime in the process chamber is considered. Although D-values forListeria spp are largely species dependent, Tansey et al. (2005)reported that the D55 value of L. innocua 11288 on different foodmodels ranged from 6.9 to 16.5 min in Listeria selective agar,therefore, suggesting that the type of heat treatment Listeria cellswere subjected to in the TS chamber would have limited or negli-gible impact on the inactivation of the microorganisms.

To evaluate in isolation the effectiveness of pre-heating the milkto 55 �C prior to TS, samples were exposed to a treatment whichconsisted of sonication with 400 W power output for 80 s, but werenot exposed to any PEF treatment afterwards. The overall inacti-vation of L. innocua after being heated to 55 �C followed by TS for80 s was 3.3 log10 cfu mL�1, showing a substantial inactivationeffect of this pre-heating regime (data not shown). This result wasnot surprising considering the increased heat load and the longerresidence time at a temperature of 55 �C, coupled with the fact thatTS was carried out in milk already at 55 �C, which could increasethe effectiveness of sonication. In a study by Bermudez-Aguirre andBarbosa-Canovas (2008), 5 min of TS at 63 �C were necessary toinactivate almost 2 log10 cfu mL�1 of L. innocua ATCC 51742 incommercial milk with 1% fat content.

Overall, the results observed in this study were broadly similarto those obtained by Pagan, Manas, Raso, and Condon (1999) inwhich Listeria monocytogenes was exposed to ultrasound alone, orin combination with temperature and pressure (up to 70 �C and

Table 2Inactivation of Listeria innocua 11288 in milk exposed to pulsed electric field (PEF)

PEF treatment combinations L. innocuainactivation(log10 cfu mL�1)

Factorsa

Treatment time (ms) Field strength(kV cm�1)

TT FS TT� FS

25 30 0.2 (0.07)* (0.09)* (0.12)*25 40 2.550 30 1.150 40 3.3

*P< 0.05; standard error of difference given in parentheses.a TT, treatment time; FS, field strength.

400 kPa). While ultrasound processing at ambient temperature hadlittle or no effect on the inactivation of L. monocytogenes (Paganet al., 1999), temperatures in excess of 50 �C were reported togreatly enhance the effect of sonication, while the D-values forL. monocytogenes were reduced from 4.3 min (at atmosphericpressure) to 1.5 min (200 kPa above atmospheric pressure) and1.0 min (400 kPa above atmospheric pressure). In the currentexperiment, where no additional pressure was employed, the Dvalue was 2 min. Moreover, the temperature at which L. innocuawas grown (37 �C) in the current experiment may have contributedto the different results, since increased resistance to heat has beenshown by L. monocytogenes grown at 37 �C compared with strainsgrown at 4 �C.

3.2. PEF treatment

The results of PEF processing are shown in Table 2. In the currentexperiment, when the total treatment time was increased from 25to 50 ms (at a field strength of 40 kV cm�1) the inactivation ofL. innocua increased from 2.5 to 3.3 log10 cfu mL�1. Increasing theelectric field strength increased the effectiveness of inactivation ofL. innocua as exposing the milk to an electric field of 30 kV cm�1

resulted in just over 1 log cycle reduction with a total treatmenttime of 50 ms. In a similar study, Calderon-Miranda, Barbosa-Canovas, and Swanson (1999) showed an increase in inactivation ofL. innocua ranging from 1.2 to 2.1 log10 cfu mL�1 as the treatmenttime increased from 42 to 64 ms. In an experiment conducted withL. monocytogenes in a static chamber, Fleischman, Ravishankar, andBalasubramaniam (2004) achieved an inactivation of just over0.75 log10 cfu mL�1 with a field of 30 kV cm�1 and 10 pulses of3.25 ms, while in a continuous pilot scale system Calderon-Mirandaet al. (1999) observed a linear increase from 0.9 to 1.9 log10 cfu mL�1

at 30 kV cm�1 as the total treatment time increased from 42 to64 ms. The cells used in the present experiment, were in theirstationary phase, reported to be a status in which cells are more PEFresistant than cells in the logarithmic phase. L. monocytogenes, L.innocua, and Escherichia coli were reported to be more PEF sensitivein fact in studies by Pothakamury et al. (1996), Wouters et al. (1999)and Alvarez, Pagan, Raso, and Condon (2002). Additionally, amonga range of Gram-positive microorganisms L. monocytogenes wasfound to have the highest resistance to PEF, when the pH of theprocessing medium was maintained at 7.0 (Garcia, Gomez, Raso, &Pagan, 2005), which was similar to that used in the presentexperiment (pH 6.5) with L. innocua.

3.3. Treatment with TS followed by PEF

The results of Protocol 1 are reported in Table 3. Increasing thepower output of the sonotrode from 320 to 400 W led to anincrease in the L. innocua inactivation from 1.4 to 3.8 log10 cfu mL�1

when milk was PEF processed at 30 kV cm�1. Similarly, whenprocessing at 40 kV cm�1 the increase in inactivation for the two TSpower outputs was over 2.0 log10 cfu mL�1, from 4.7 to

Effect of a sequence of thermosonication (TS) and pulsed electric field (PEF)(Protocol 1) on Listeria innocua 11288 inactivation

PEF treatment combinations L. innocuainactivation(log10 cfu mL�1)

Factorsa

TS power (W) Field strength(kV cm�1)

TSP FS TSP� FS

320 30 1.4 (0.35)* (0.35)** NS (0.50)320 40 4.7400 30 3.8400 40 6.8

*P< 0.05; **P< 0.01; NS, not significant; standard error of difference given inparentheses.

a TSP, TS power; FS, field strength.

Fig. 4. Inactivation of Listeria innocua 11288 by: (,) thermosonication (TS) followed by pulsed electric field (PEF); ( ) pre-heating followed by TS (PHTS) and PEF; and (-)conventional pasteurisation (HE). Error bars represent the standard deviation.

Table 4Inactivation of Listeria innocua 11288 by pre-heating (55 �C over 60 s) followed bythermosonication (TS) and pulsed electric field (PEF) (Protocol 3): effect of fieldstrength and energy density

TS/PEF treatment combinations L. innocuainactivation(log10 cfu mL�1)

Factorsa

Energydensity (kJ L�1)

Field strength(kV cm�1)

ED FS ED� FS

480 40 5.9 NS (0.16) NS (0.16) NS (0.30)480 50 6.7576 40 6.5576 50 6.9

NS, not significant; standard error of difference given in parentheses.a ED, energy density; FS, field strength.

F. Noci et al. / International Dairy Journal 19 (2009) 30–3534

6.8 log10 cfu mL�1, respectively. The statistical analysis showeda non-significant P value for the interaction between the two maineffects (TS power output and PEF strength). As expected, there wasa significant difference between processing at the two electric fieldstrengths when averaging across TS power levels (3.1 vs.5.3 log10 cfu mL�1 reduction at 30 and 40 kV cm�1, respectively).The treatment combining 400 W of TS and a PEF of 40 kV cm�1

achieved a similar level of inactivation to that obtained with thetubular heat exchanger (6.8 vs.7.0 log10 cfu mL�1 [P� 0.05],respectively). Although an effect of the temperature rise within thePEF cell cannot be discounted, it should be noted that the milkentered the cell at 10 �C, and the residence time within the PEF cellwas extremely short (3.3 s) with the product passing througha cooling coil immediately after the cell outlet.

In Protocol 2, TS and PEF were combined as described in Table 1.In this procedure, the product was either TS treated as in Protocol 1or pre-heated to 55 �C, and then maintained at this temperatureduring TS. Combining PEF at a lower electric field strength(30 kV cm�1 for 50 ms) and TS without pre-heating resulted ina 3.1 log cycle reduction, while when milk was pre-heated prior toTS, the overall inactivation was 4.5 log10 cfu mL�1. However, whenPEF was increased to 40 kV cm�1 for 50 ms, the effect of PEF wasmore pronounced over the effect of TS as the total inactivationincreased to 5.9 and 6.2 log10 cfu mL�1 for TS with or without pre-heating, respectively (Fig. 4), which were not significantly different.While limited inactivation was achieved by applying TS withoutpre-heating, leaving a greater concentration of microorganism forPEF, when the pre-heating was applied, prior to TS, the more pro-longed heating may have contributed to a greater direct inactiva-tion by these hurdles, leaving a smaller number of microorganismsto be inactivated by the following application of PEF.

Data in relation to energy density and different field strength/pulse number combinations within Protocol 3 are shown in Table 4.In Protocol 3, the TS conditions were kept constant (pre-heatingfollowed by TS at 400 W for 80 s) while PEF settings were changedto achieve energy densities of 480 and 576 kJ L�1. The energydensity increase led to an inactivation of 6.3 and 6.7 log10 cfu mL�1,respectively (averaging across the two field strengths). The average

inactivation at 40 kV cm�1 was 6.2 log10 cfu mL�1, while at50 kV cm�1 it was 6.8 log10 cfu mL�1. These data suggest anadvantage in applying higher energy densities to liquid products formicrobial inactivation as documented in numerous studies(Rodrigo, Martinez, Harte, Barbosa-Canovas, & Rodrigo, 2001;Toepfl, Heinz, & Knorr, 2007). This is broadly in agreement witha study by Abram, Smelt, Bos, and Wouters (2003), showing thatthe dependency of Lactobacillus plantarum inactivation in a buffersolution exposed to PEF followed a linear pattern when treatmentconditions were not optimised. However, when in the same study,the solution was treated by PEF using an optimised set of conditionsthe decrease in the microbial population showed an improvementin the treatment energy efficiency (Abram et al., 2003). Increasingthe electric field strength to 50 kV cm�1, or extending the totaltreatment time to 60 ms led to an increase in the overall inactiva-tion. Another set of treatment conditions within this protocol were50 kV cm�1 for 38 ms. This set-up was used to achieve an energydensity matching the energy of 40 kV cm�1 and 60 ms. The inacti-vation observed was 6.9 log10 cfu mL�1 which was similar (P� 0.05)to the 7.0 log10 cfu mL�1 obtained by conventional pasteurisation.The data in this experiment suggest that there is a marginaladvantage in terms of microbial inactivation in applying strongerelectric fields for a shorter treatment time (while maintaining theenergy constant). In a study by Smith, Mittal, and Griffiths (2002)on unpasteurised skim milk, increasing the number of pulses from

F. Noci et al. / International Dairy Journal 19 (2009) 30–35 35

50 to 80, which increased the energy density of the treatment, didnot lead to any significant change in microbial inactivation, whilethere was a marked increase when the number of pulses wasincreased from 20 to 50.

4. Conclusions

Individually, the TS and PEF hurdles only achieved moderatereductions in the bacterial population in milk. However, TS of milk,when used in a combined approach with PEF, achieved a degree ofinactivation of L. innocua comparable with conventional pasteur-isation, while substantially reducing the severity of the time/temperature exposure. Further optimisation of this hurdleapproach could result in a lesser degree of thermal damage in thepasteurised product, thereby retaining more of the organolepticallyand nutritionally favourable characteristics of the fresh product,while potentially achieving similar or superior shelf life to theconventional treatment. Studies on the impact of such a sequenceof treatments on pathogenic and spoilage microorganisms ofconcern in milk processing and on the quality of the processed milkare required to establish the feasibility of processing unpasteurisedmilk using this approach.

Acknowledgements

The authors would like to thank and acknowledge Mr. B. Boutroyand Mr. M. Cooney for his technical support and University CollegeDublin Farm in Lyons Estate (Newcastle, Co. Kildare) for the rawmaterials supplied. This study was funded under the Food Institu-tional Research Measure which is administrated by the Departmentof Agriculture and Food, Ireland as a part of the National Devel-opment Plan 2000–2006.

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