Inactivation of Listeria innocua in skim milk by pulsed electric fields and nisin

International Journal of Food Microbiology 51 (1999) 19–30www.elsevier.nl / locate / ijfoodmicro

Inactivation of Listeria innocua in skim milk by pulsed electricfields and nisin

*´ ´ ´Marıa L. Calderon-Miranda, Gustavo V. Barbosa-Canovas , Barry G. Swanson

Washington State University, Biological Systems Engineering Department, Pullman, WA 99164-6120, USA

Received 6 April 1999; accepted 22 May 1999

Abstract

Pulsed electric fields (PEF) is an emerging nonthermal processing technology used to inactivate microorganisms in liquidfoods such as milk. PEF results in loss of cell membrane functionality that leads to inactivation of the microorganism. Thereare many processes that aid in the stability and safety of foods. The combination of different preservation factors, such asnisin and PEF, to control microorganisms should be explored. The objective of this research was to study the inactivation ofListeria innocua suspended in skim milk by PEF as well as the sensitization of PEF treated L. innocua to nisin. The selectedelectric field intensity was 30, 40 and 50 kV/cm and the number of pulses applied was 10.6, 21.3 and 32. The sensitizationexhibited by PEF treated L. innocua to nisin was assessed for 10 or 100 IU nisin /ml. A progressive decrease in the

1]population of L. innocua was observed for the selected field intensities, with the greatest reduction being 2 log cycles (U).2

The exposure of L. innocua to nisin after PEF had an additive effect on the inactivation of the microorganism as thatexhibited by the PEF alone. As the electric field, number of pulses and nisin concentration increased, synergism wasobserved in the inactivation of L. innocua as a result of exposure to nisin after PEF. The reduction of L. innocuaaccomplished by exposure to 10 IU nisin /ml after 32 pulsed electric fields was 2, 2.7, and 3.4 U for an electric field intensityof 30, 40, and 50 kV/cm, respectively. Population of L. innocua subjected to 100 IU nisin /ml after PEF was 2.8–3.8 U forthe selected electric field intensities and 32 pulses. The designed model for the inactivation of L. innocua as a result of thePEF followed by exposure to nisin proved to be accurate in the prediction of the inactivation of L. innocua in skim milkcontaining 1.2 or 37 IU nisin /ml. Inactivation of L. innocua in skim milk containing 37 IU nisin /ml resulted in a decrease inpopulation of 3.7 U. 1999 Elsevier Science B.V. All rights reserved.

Keywords: Pulsed electric fields; Listeria innocua; Nisin; Food preservation

1. Introduction

Milk preservation can be accomplished by the useof heat pasteurization. Although heating inactivates

*Corresponding author. Tel.: 11-509-335-6188; fax: 11-509-enzymes and microorganisms, it has a deleterious335-2722.effect on the organoleptic and nutritional propertiesE-mail address: [email protected] (G.V. Barbosa-

Ćanovas) of the milk. The most common organoleptic change

0168-1605/99/$ – see front matter 1999 Elsevier Science B.V. All rights reserved.PI I : S0168-1605( 99 )00069-0

´20 M.L. Calderon-Miranda et al. / International Journal of Food Microbiology 51 (1999) 19 –30

is the generation of ‘cooked flavor’ as a consequence milk resulting from infected animals or poor sanita-of the heat treatment. Nonthermal processes, such as tion practices presents a threat to public healthpulsed electric fields (PEF), maintain the fresh-like because L. monocytogenes causes listeriosis (Marth,qualities of foods desirable to consumers. 1989). The ability of Listeria spp. to reproduce at

Nonthermal processing of foods with PEF has refrigeration temperatures, survive a wide range ofexisted since the 1900’s and was first used in the pH values and survival after the production of Fetapasteurization of milk (Vega-Mercado et al., 1997). and Camembert cheese (Papageorgiou and Marth,PEF processing is conducted by introducing the food 1989; Ryser and Marth, 1987) affirm the importancein a chamber containing two electrodes. Once in the of the inactivation of Listeria spp. during foodchamber the food is exposed to a multiple, short processing.duration, high intensity pulsed electric fields to Nisin is used as an antimicrobial in processedinactivate microorganisms with no appreciable ther- cheese spreads at concentrations of 100–400 IU/gmal damage hence the original taste, color, and (Hurst and Hover, 1993). Nisin is a bacteriocinfunctional properties are retained. The PEF applied produced in milk by strains of lactic acid bacteriato the food originate irreversible loss of the cell such as Lactoccocus lactis subsp. lactis (Delves-membrane functionality that leads to inactivation of Broughton, 1990; Hurst and Hover, 1993; Martinis etthe microbial cell. This process is known as elec- al., 1997). A bacteriocin is a bacterial protein withtroporation and is effective in the inactivation of antimicrobial activity against bacteria of relatedyeast in various foods (Zhang et al., 1994; Castro et species as well as some pathogenic microorganismsal., 1993; Hamilton and Sale, 1967) and bacteria (Hill, 1995). The use of 32 or 250 IU nisin /mlsuch as Escherichia coli suspended in skim milk results in the inhibition of Listeria spp. (Mohamed et

´(Martın et al., 1997). al., 1984). Though nisin is active against gramA combination of factors or hurdles can be positive bacteria, it has no effect against gram

employed to accomplish food preservation (Wagner negative bacteria, yeast, or fungi. The nisin isand Moberg, 1989). Each of them contributes to the adsorbed by the cytoplasmic membrane of vegetativestability and safety of the food product (Leistner, cells (Delves-Broughton, 1990; Hurst and Hover,1992). PEF can be combined with moderately high 1993). Once in the cytoplasmic membrane, nisintemperatures ( , 608C) or other processing aids such inactivates sulphydryl groups originating membraneas antimicrobials to increase the extent of microbial disruption and leakage of cellular contents (Delves-

ïnactivation (Hulsheger et al., 1981; Jayaram and Broughton, 1990; Hurst and Hover, 1993; DaviesCastle, 1992; Kalchayanand et al., 1994; Liu et al., and Adams, 1994). Lysis of the cell can take place as1997). The use of several hurdles in combination or a result of the formation of ion channels or am-in a successive manner may act additively or syner- phiphilic pores on the cytoplasmic membrane (De-gistically and difficult the survival of spoilage and lves-Broughton, 1990; Davies and Adams, 1994).food poisoning microorganisms (Leistner, 1992). Since Listeria spp. develops resistance to nisinThe use of hurdle technology by the combination of (Ming and Daeschel, 1993; Davies and Adams,PEF and the antimicrobial nisin can aid in the 1994; Martinis et al., 1997) the combination of nisininactivation of Listeria spp. (Kalchayanand et al., with other food preservation technologies results in a1994). sensitization of the Listeria to the nisin (Kal-

Listeria spp. are able to grow in food products chayanand et al., 1994). Liu et al. (1997) andsuch as whole milk, skim milk, chocolate milk, Kalchayanand et al. (1994) reported that the micro-cheese, meat, cabbage, and liquid whole egg (Lovett bial inactivation as a result of the combination ofet al., 1987; Rosenow and Marth, 1987; Farber et al., PEF with antimicrobial agents was more effective.1989; Leasor and Foegeding, 1989). Listeria is able Thus, the use of nisin combined with other foodto grow under anaerobic to microaerophilic con- processing techniques following the ‘hurdle’ ap-ditions and survives over a wide range of pH values proach can be a suitable food preservation method.(5.0 to 9.6). Listeria reproduces in the temperature Besides, it is useful to study the possibility of arange of 3 to 458C, with an optimum growth sensitization of L. innocua to the nisin as a result oftemperature of 378C. Presence of Listeria spp. in PEF in skim milk.

´M.L. Calderon-Miranda et al. / International Journal of Food Microbiology 51 (1999) 19 –30 21

Though isolation of L. innocua from food is not glycerol and stored at 2 708C until used. Theconsidered hazardous, presence of L. innocua can following procedure was done to determine themask the existence of L. monocytogenes. For this growth curve and to culture the bacteria at the earlyresearch, L. innocua was selected because is closely stationary phase: Frozen vials of the initial culture ofrelated to L. monocytogenes, yet nonpathogenic L. innocua were thawed and the bacteria were grown(Giraffa et al., 1995; Kathariou et al., 1995). The in a 1 litre Erlenmeyer flask containing 500 ml ofpurpose of this work was to study the inactivation of TSBYE. The temperature and agitation used are theL. innocua by PEF, and the sensitization of L. same as used for the initial culture. The spectralinnocua to nisin as a result of exposure to PEF in absorbance of the solution containing the bacteriaskim milk (PEF-nisin). was observed every 30 min at 540 nm and the colony

forming units per ml (cfu /ml) was obtained by pourplating serial dilutions done in sterile 0.1% peptone(DIFCO Laboratories, Detroit, MI) into tryptic soy

2. Materials and methods agar (BIOPRO) enriched with 0.6% yeast extract(TSAYE) (Fig. 1). The early stationary phase was

1]2.1. Microbial preparation reached after approximately 8 h. L. innocua was2

harvested at the early stationary phase by centrifuga-Listeria innocua (ATCC 51742, Rockville MD) tion of the culture solution at 5500 rpm for 10 min at

was rehydrated in 10 ml of tryptic soy broth 108C.(DIFCO) enriched with 0.6% yeast extract L. innocua cells were washed and recovered in(TSBYE). After 30 min, the cell suspension was 120 ml of TSBYE. The suspension of L. innocuainoculated into 50 ml of TSBYE and incubated for was centrifuged at 5000 rpm and 108C for 5 min.18 h at 378C with continuous agitation at 190 rpm in The cells were once again suspended in 60 mlan orbital shaker (MSB-3322A-I, GS Blue Electric, TSBYE and were placed in vials containing 1 ml ofBlue Island, IL). The initial culture was dispensed 20% sterile glycerol. The pellets were stored atinto sterile vials containing 1 ml of 20% sterile 2 48C until inactivation studies.

Fig. 1. Growth curve of L. innocua (51742) in TSBYE.


2.2. Skim milk where n represents the number of pulses, v thevolume of the treatment chamber (ml), f the flow of

Skim milk purchased from a local supermarket the skim milk (ml /s) and F the pulsing frequencywas heated at 1008C for 10 min prior to PEF (Hz).treatment. The electrical conductivity of the skim Input voltages of 30, 35 and 40 kV were selected.

3milk was 5.5 3 10 mS/cm at 268C was determined The pulse waveform and input voltage to thewith a conductivity meter (Hydac, Cambridge Sci- chamber were recorded with a digital oscilloscopeentific Instruments, Cambridge, Maryland). (Hewlett-Packard 54530A, Colorado Springs, CO).

The approximate maximum electric field intensity inthe treatment chamber was calculated from data2.3. Microbial inoculation and countgathered with the oscilloscope. The electric fieldintensity was 30, 40 or 50 kV/cm according to theOne pellet was thawed and added to 4 litre of skimexpression used by Naidu and Kamaraju (1996) tomilk at 78C, 30 min prior to treatment with PEF tocalculate maximum electric field intensity in areanimate L. innocua cells. The initial microbialcoaxial electrode configuration with infinite length:concentration for the experiments was approximately

72.7 3 10 cfu /ml. To assess the cfu /ml and sensiti- V]]]]E 5zation of L. innocua to nisin after PEF, serial R

]F S DGr lndilutions were performed in sterile 0.1% peptone. rFor the effect of PEF in the sensitization of L.

where E the electric field intensity (kV/cm), V theinnocua to nisin, dilutions were plated on TSAYEinput voltage (kV), r the inner and R the outer radiiwith 10 IU of nisin /ml, and TSAYE with 100 IU ofin centimeters.nisin /ml. Plates were incubated at 278C for 48 h.

The flow rate of skim milk was 500 ml /min,Each PEF, PEF-nisin treatment was conducted twicecontrolled by a variable speed pump (Masterflexand duplicate microbial counts were recorded in each7654-00, Cole Palmer Instruments Co., Chicago, IL).experiment. Results for the inactivation of L. innocuaA cooling coil immersed in iced water was used toare the average of four separate counts and arecool the skim milk at the entrance and exit of theexpressed in logarithmic units where 1 unit (U)treatment chamber. The temperature at the entrancecorresponds to 1 log cycle.and exit of the treatment chamber was recorded witha digital thermometer (John Fluke Mfg. Co., Everett,2.4. PEF treatmentWA). The cleaning and disinfecting of the systemwas done with a 500 ppm chlorine solution rinsedThe PEF system used was a pilot plant highwith sterile distilled water.intensity electric field pulser manufactured by

Physics International (San Leandro, CA). A conce-2.5. Nisin treatmentntric cylindrical treatment chamber containing two

stainless steel electrodes with a gap of 0.6 cm and a63 Nisaplin with a nisin activity of 10 IU/g wastreatment volume of 25 cm was used. Exponentially

obtained from Aplin & Barret (Ventura, CA). Nisap-decaying electric field pulse waveforms and approxi-lin was suspended in sterile distilled water to obtainmate pulse duration of 2 ms were selected. Thethe desired concentrations of nisin. Preliminaryenergy storage capacitor was set at 0.2 mF and a gasstudies showed no significant difference (P#0.05)spark switch was used to control the pulse rate.on the activity of nisin against L. innocua in LWE orFor this study the frequency was 3.5 Hz and ain agar. Also no significant difference (P#0.05) wasstepwise process was used to apply a set of 10.6,found in the inactivation of L. innocua when nisin21.3 and 32 pulses to the skim milk. The number ofwas added before or after the PEF treatment. Thus,pulses given to the skim milk was obtained from thefor the sensitization study the nisaplin solution wasfollowing expression:added to the agar (TSAYE) prior to plating of the

v microorganisms subjected to the PEF treatment and]n 5 3 FS Df for the validation of the designed model the nisaplin


solution was added to the skim milk 1 min prior to (SPSS Inc., Chicago, IL). Error bars are given for athe PEF treatment. The nisin concentrations selected 95% confidence interval.for this study were similar to the nisin concentrationreported during the heat treatment of milk by Mais-nier-Patin et al. (1995). The concentrations of nisin 3. Results and discussionselected were determined not inhibitory to thegrowth of L. innocua on TSAYE. 3.1. PEF

The population of L. innocua suspended in skim2.6. Experimental design

milk decreased as the electric field intensity andnumber of pulses increased as proven for other

A three level factorial design was used to evaluate¨microorganisms (Hulsheger et al., 1981; Jayaram and

the effects of electric field intensity (30, 40 and 50Ćastle, 1992; Martın et al., 1997).

kV/cm), number of pulses (10.5, 21.3 and 32), andFor the electric field intensity of 30 kV/cm and 32

nisin concentration (0, 10, 100 IU/ml). Two trialspulses the reduction of the population of L. innocua

were performed for each variable combination. Thewas 1.9 U (Fig. 2). The inactivation of L. innocua

data were modeled in Design Expert for DOSaccomplished with an electric field intensity of 40

Version 3.0 (Stat-ease Inc., Minneapolis, MN) usingkV/cm and 10.6 pulses is equivalent to the reduction

a three level factorial as a response surface.observed when L. innocua is exposed only to 100 IUnisin /ml (Table 1). When PEF consisted of 32 pulses

2.7. Statistical analysis for an electric field intensity of 40 kV/cm a 2.1 Ureduction of L. innocua was observed while for the

Analysis of variance (ANOVA) of the effect of electric field intensity of 50 kV/cm a 2.5 U reductionpulsed electric fields and nisin treatment on the was attained (Fig. 2). Significant difference (P5

reduction of L. innocua was performed using the 0.05) was found for the inactivation of L. innocuageneral linear model procedure on the Statistics exposed to an electric field intensity of 30, 40, or 50Program for Social Science for Windows Version 8.0 kV/cm.

Fig. 2. Inactivation of L. innocua by PEF at different electric field intensities.


Table 1Reduction units (U) observed for L. innocua in LWE treated by PEF, nisin, or sensitization to nisin after PEF (PEF-nisin)

PEF PEF-nisin PEF-nisin Nisin Nisin(10 IU/ml) (100 IU/ml) (10 IU/ml) (100 IU/ml)

Electric field intensity (kV/cm)Pulse 30 40 50 30 40 50 30 40 50 – –10.6 0.36 0.75 1 0.5 0.95 1.4 1.2 1.8 2.5 0.24 0.6821.3 0.88 1.2 1.6 1.4 2.0 2.8 1.9 2.7 3.332 1.9 2.1 2.4 2.0 2.7 3.4 2.8 3.3 3.8

The mean exit temperature after PEF processing of inactivation of L. innocua was 2, 2.7, and 3.4 U,skim milk was 22, 28 and 348C for an electric field respectively (Fig. 3). For PEF with an electric fieldintensity of 30, 40, and 50 kV/cm, respectively. The intensity of 30 kV/cm followed by 10 or 100 IUprocessing temperatures are not lethal to L. innocua; nisin /ml compared to PEF alone, significant differ-in fact they are in the range of optimum growth ences were observed for 100 IU nisin /ml (Fig. 4).temperatures (Doyle, 1988). The inactivation of L. innocua by PEF or PEF

followed by 10 IU nisin /ml at electric field intensity3.2. Sensitization of L. innocua to nisin of 40 kV/cm showed significant differences (P5

0.05) for 21.3 and 32 pulses. However, when 100 IUIn general, PEF followed by exposure of L. nisin /ml were used, all values were significantly

innocua to nisin decreased the population compared different from PEF alone or PEF followed by 10 IUto the inactivation accomplished by PEF or nisin nisin /ml (Fig. 5). The extent of inactivation for PEFalone. For an electric field intensity of 30 or 40 conducted at an electric field intensity of 40 kV/cmkV/cm followed by exposure to 10 IU nisin /ml with 10.6, 21.3 and 32 pulses followed by exposuresignificant differences (P50.05) were observed only of L. innocua to 100 IU nisin /ml was 1.8, 2.7 andfor 32 pulses (Fig. 3). When the PEF consisted of an 3.3 U, respectively (Fig. 5). When an electric fieldelectric field intensity of 30, 40, or 50 kV/cm and 32 intensity of 50 kV/cm was applied, significant differ-pulses followed by exposure to 10 IU nisin /ml, the ences (P50.05) were observed between the PEF and

Fig. 3. Inactivation of L. innocua by exposure to 10 IU nisin /ml after PEF.


Fig. 4. Inactivation of L. innocua by PEF and exposure to 10 or 100 IU nisin /ml after PEF with an electric field intensity of 30 kV/cm.


PEF followed by exposure to 10 or 100 IU nisin /ml An analysis of variance using the general linear(Fig. 6). The inactivation of L. innocua accom- model procedure determined that the electric fieldplished for an electric field intensity of 50 kV/cm intensity, number of pulses and nisin concentrationfollowed by exposure to 100 IU nisin /ml was 2.5, exhibited a significant (P,0.0001) effect on the3.3 and 3.8 U (Fig. 6). inactivation of L. innocua. Exposure to nisin after

No significant differences (P50.05) were ob- PEF exhibited an additive effect on the inactivationserved in the inactivation of L. innocua at an electric of L. innocua for the electric field intensity of 30field intensity of 40 or 50 kV/cm followed by kV/cm for any number of pulses and nisin con-exposure to 100 IU nisin /ml. centration (Fig. 7). When the electric field intensity



Fig. 7. Additive effect of the nisin on the inactivation of L. innocua after PEF at an electric field intensity of 30 kV/cm and 32 pulses.

was 40 kV/cm an additive effect on the inactivation while synergism was observed for 21.3 and 32 pulsesof L. innocua as a result of PEF followed by nisin for 10 or 100 IU nisin /ml. The effect of the PEF andwas observed for 10.6 pulses and 10 or 100 IU nisin treatments was synergistic on the inactivationnisin /ml. Moreover, an additive effect was observed of L. innocua at electric field intensity of 50 kV/cmat electric field intensity of 40 kV/cm with 10 pulses, with 21.3 or 32 pulses for 10 or 100 IU nisin /ml


Fig. 8. Synergistic effect of nisin on the inactivation of L. innocua after PEF at an electric field intensity of 50 kV/cm and 32 pulses.

(Fig. 8). The inactivation of L. innocua can be sion for the inactivation of L. innocua by PEF andattributed to PEF and nisin activity on cell mem- nisin was:branes (Hamilton and Sale, 1967; Delves-Broughton,1990). Thus, the effect resulting from the two C

]log 5 2.26 2 0.05434 3 E 2 0.06732 3 nS Dtreatments will be additive according to Leistner C0(1992). Nonetheless, as the electric field intensity

2 0.59683 3 log(A)and number of pulses increased the effect of PEFfollowed by exposure to nisin was synergistic in theinactivation of L. innocua. Our findings accord with where C is the initial microbial count, C is the0

the reports of Kalchayanand et al. (1994) where L. microbial count after the PEF-nisin treatment, E ismonocytogenes treated with an electric field intensity the electric field intensity (kV/cm), n number ofof 12.5 kV/cm and 1 pulse in a media containing 50 pulses and A nisin concentration (IU/ml). The modelIU nisin /ml exhibited an increased sensitivity to the had an adjusted R squared of 0.8082 with a signifi-nisin. Synergism was also reported by Maisnier-Patin cance level of 0.0001.et al. (1995) on the inactivation of L. monocytogenes To test the accuracy of the designed model, twoduring the heat treatment of milk with addition of 25 nisin concentrations were selected arbitrarily and theor 50 IU nisin /ml. inactivation of L. innocua was predicted for an

¨Since both treatment media (Hulsheger et al., electric field intensity of 30, 40 or 50 kV/cm and1981) and nisin concentration (Mohamed et al., 10.6, 21.3, and 32 pulses. The results obtained from1984) are important factors in the extent of cellular the designed model were compared to experimentalinactivation in cells exposed either to PEF or nisin, data. The inactivation of L. innocua suspended inthe lack of sensitization or the additive effect ob- skim milk containing 1.2. IU nisin /ml was 1.5, 2.1,served in some of our data may be a consequence of and 2.6 for 32 pulses at an electric field intensity ofboth the PEF intensity and nisin concentration. 30, 40 and 50 kV/cm, respectively (Fig. 9). There

The data from the PEF and PEF-nisin were were no significant differences (P50.05) betweenmodeled with the Design Expert using a three level the obtained inactivation of the microorganism andfactorial as a response surface. The resulting expres- that accomplished by PEF alone.


Fig. 9. Predicted (PV) and experimental (EV) values for the inactivation of L. innocua by PEF in skim milk containing 1.2 IU nisin /ml.

Fig. 10. Predicted (PV) and experimental (EV) values for the inactivation of L. innocua by PEF in skim milk containing 37 IU nisin /ml.


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Hurst, A., Hover, E., 1993. Nisin. In: Davidson, P.M., Branen,A.L. (Eds.), Food Antimicrobials. 2nd ed. Marcel Dekker, Inc.,pp. 369–391.

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Inactivation of Listeria innocua in skim milk by pulsed electric fields and nisin

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