International Journal of Food Microbiology 140 (2010) 49–56

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International Journal of Food Microbiology

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Inactivation of Salmonella Enteritidis strains by combination of high hydrostaticpressure and nisin

Jaesung Lee a, Gönül Kaletunç b,⁎a Department of Food Science and Technology, The Ohio State University, Columbus, OH, United Statesb Department of Food, Agricultural, and Biological Engineering, The Ohio State University, Columbus, OH, United States

⁎ Corresponding author. Department of Food, Agricultu590 Woody Hayes Drive, Columbus, OH 43210, Unitedfax: +1 614 292 9448.

E-mail address: kaletunc.1@osu.edu (G. Kaletunç).

0168-1605/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2010.02.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 November 2009Received in revised form 8 February 2010Accepted 10 February 2010

Keywords:High pressureDifferential scanning calorimeterBacteriaInactivation

The effects of high hydrostatic pressure (HHP) and nisin treatment alone and in combination on cellularcomponents and viability of two Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) strainswere evaluated by differential scanning calorimetry (DSC) and plate counting in order to evaluate therelative resistance and optimize the treatment conditions. S. Enteritidis FDA and OSU 799 strains weresubjected to HHP (0.1–550 MPa for 10 min at 25 °C) alone and in combination with nisin (200 IU/ml nisin) inculture broth. HHP (up to 200 MPa) or the nisin alone did not affect the viability and cellular components ofeither strain. An 8-log cfu/ml reduction was observed after a pressure treatment at 500 MPa for the FDAstrain and 450 MPa for the OSU 799 strain. When nisin was added, a similar reduction was obtained at400 MPa for FDA strain and 350 MPa for the OSU 799 strain. The decrease in apparent enthalpy appeared tobe mainly due to reduction in the ribosome denaturation peak for both the pressure alone and the pressure–nisin combination treatments. HHP facilitated penetration of nisin into the cell above 100 MPa pressure.Monitoring through DNA-binding probes the effect of pressure and nisin treatments on DNA in vivo showedthat nisin did not affect DNA at 200 IU/ml. The apparent enthalpy data obtained from DSC can be used foroptimization of pressure levels to reduce a microbial population in the presence of nisin.

ral and Biological Engineering,States. Te.: +1 614 292 0419;

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

High hydrostatic pressure (HHP) has been shown to inactivatespoilage and pathogenic bacteria while maintaining the qualityattributes of food products. HHP has been recognized as a promisingalternative to thermal processing among emerging food preservationtechnologies (Knorr, 1993; Mertens and Deplace, 1993; Roberts andHoover, 1996; Farkas and Hoover, 2000; Linton et al., 2001).Preservation of food by HHP processing requires pressure levelsabove 600 MPa for inactivation of pressure-resistant pathogens insome foods (Balasubramaniam et al., 2008). Such pressure levels mayadversely alter texture and color of many foods (Patterson et al, 1995;Porretta et al., 1995; Hauben et al. 1997; Mussa and Ramaswamy,1997; Trujillo et al., 1999) as well as increase initial and maintenancecosts, promote wear, and shorten the life of the equipment (Torresand Velazquez, 2005, Hoover et al., 1989;Mertens and Deplace, 1993).Application of moderately high pressure treatment (up to 400 MPa)may cause only sublethal injury compromising the safety of the foodproduct due to potential recovery of injured bacteria during storage(Earnshaw et al., 1995; Patterson et al., 1995). Therefore, a processing

protocol based on hurdle technology combining moderately highpressures with a chemical preservation method can be used tomanufacture safe food without high processing costs.

The concept of hurdle technology has been applied to inactivatepathogenic bacteria by combining HHP with low pH (Alpas et al.,2000), heat (Patterson and Kilpatrick, 1998; Benito et al., 1999),lysozyme (Masschalck et al., 2000), carbon dioxide (Hass et al., 1989)or antimicrobial peptides (Kalchayanand et al., 1998; Yuste et al.,1998; Garcia-Graells et al., 1999; Masschalck et al., 2000; Massachalcket al., 2001). Most antimicrobial peptides produced by gram-positivebacteria are bactericidal to Gram-positive bacteria while causingsublethal stress to Gram-negative bacteria (Ray et al., 2001).Kalchayanand et al. (1998) reported that the mixture of pediocinand nisin provided 1 to 5 log unit additional reduction above the effectof bacteriocin or HHP alone in Staphylococcus aureus, Listeriamonocytogenes, Salmonella enterica serovar Typhimurium (S. Typhi-murium) and Escherichia coli O157:H7. Nisin, produced by somestrains of Lactococcus lactis subsp. lactis, was shown to bind to thecytoplasmicmembrane of Gram-positive bacteria to form poreswhichleads to leakage of intracellular molecules and metabolites (Sahl andBierbaum, 1998). The impermeability of the outer membrane ofGram-negative bacteria does not allow nisin to reach the cytoplasmicmembrane and prevents its use as an antimicrobial agent (Kordel andSahl, 1986; Delves-Broughton, 1990). Studies in literature show thatHHP-treated Gram-negative bacterial cells are susceptible to nisin

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(Masschalck et al., 2000; Massachalck et al., 2001). Garcia-Graellset al. (1999) reported that pressurization in the presence of nisin(400 IU/ml) decreased the survival of E. coli by an additional 3 logunits in skim milk at 550 MPa. Similarly, 1 to 2 log unit additionalreductions in Pseudomonas fluorescens, E. coli O157:H7, and aSalmonella sp. cultures in phosphate buffer were observed withnisin addition (100 IU/ml) under moderate (b300 MPa) HHP treat-ment (Massachalck et al., 2001). Although the primary target for nisinand HHP in a bacterial cell is believed to be the cytoplasmicmembrane(Kalchayanand et al., 1998; Massachalck et al., 2001), the exactmechanism of the inactivation of bacteria by HHP and nisin is still notclearly known.

Differential scanning calorimetry has been applied to characterizechanges in cellular components of bacteria exposed to thermal ornon-thermal treatments. DSC thermograms of whole bacterial cellsdisplay endothermic transitions associated with the phase orconformational changes of cellular components of bacteria as afunction of temperature. The peak temperature corresponding to eachtransition represents the thermal stability of a cellular component ofbacteria (Miles et al., 1986; Mackey et al., 1991, 1993; Belliveau et al.,1992; Anderson et al., 1991; Mohacsi-Farkas et al., 1999; Kaletunç,2001; Lee and Kaletunç, 2002b, 2005). The change in the peak positionand the peak area in thermograms before and after exposure tothermal or non-thermal treatment can be quantitatively analyzed todetermine the impact of treatment (Niven et al., 1999; Alpas et al.,2003; Kaletunç et al., 2004; Lee and Kaletunç, 2005). Niven et al.(1999) reported reductions in ribosome-associated transitions for E.coli NCTC 8164 cells as a function of pressure between 50–250 MPa.Alpas et al. (2003) used apparent enthalpy data to evaluate therelative HHP resistance of two bacterial strains of E. coli O157:H7 andS. aureus. The cellular component associated with a peak in a complexwhole cell thermogram is in general identified by comparing thetransition temperature of an isolated cell component with thecorresponding transition temperature in whole cells (Mackey et al.,1991; Lee and Kaletunç, 2002b). A membrane-permeable compoundwhich can bind to a specific cellular component can be used for peakassignment in the complex DSC thermogram without the need forisolation of cellular components. There are DNA-binding probeswhich are used to distinguish between viable and non-viable cells ofbacteria using fluorescence spectroscopy. Bisbenzimide, a DNA-binding fluorescent probe used to assess the viability of cells, wasreported to increase the thermal stability of DNA in vitro studies(Canzonetta et al., 2002).

Development of an understanding for inactivation of Gram-negative bacteria by pressure–nisin treatment is necessary tooptimize HHP–nisin combinations that yield desired reduction ofGram-negative bacteria in food products. The objectives of this studyinclude i) evaluation of the effect of nisin or HHP, alone or incombination, on cellular components of two S. Enteritidis (Gram-negative foodborne pathogen) strains using DSC; ii) use of bisbenzi-mide as a probe to identify the impact of HHP–nisin treatment on thethermal stability of the cellular DNA transition in vivo; and iii)comparison of the calorimetric data to plate count data for evaluationof bacterial survival.

2. Materials and methods

2.1. Bacterial strains

S. Enteritidis FDA was obtained from the Food MicrobiologyLaboratory, University of Wyoming. S. Enteritidis OSU 799 wasobtained from the Culture Collection Center at the Ohio StateUniversity. A previous study has shown the FDA strain to be stronglyresistant to high pressure among S. Enteritidis strains (Alpas et al.,1999). An isolated pure colony of each organism grown on XLD agarplate was suspended in 10 ml Trypticase soy broth supplemented

with 0.6% (w/w) yeast extract (TSBY) and incubated at 37 °C for 18 h.Cultures were stored frozen (−80 °C) in 30% (v/v) sterile glycerol. Aloopful of stock culture was transferred to 10 ml Trypticase soy brothand incubated 10 h at 37 °C before use.

2.2. Preparation of organisms for pressure treatment

Each culture was inoculated (1% v/v) into a broth containing TSBYand incubated at 37 °C to early stationary growth phase. The growthcurve was obtained based on plate counts and used to determine theearly stationary phase for cells prior to pressure treatment. One-hundred fifty ml of cell culture at early stationary phase was placed ina 3-mm-thick sterile stomacher bag (Fisher scientific, Ottawa, Ontario,Canada) and heat-sealed. Sealed bag was placed inside a second bagfilled with chlorine solution (20% v/v) to kill the bacteria if theprimary package were to fail. Air was removed from all of the bags.The second bag was heat-sealed and the two bag system was placedinside a third bag and was heat-sealed under vacuum.

For antimicrobial-treated samples, commercially available Nisa-plin® (Aplin and Barrett, Milwaukee, WI, USA) was added to bacterialculture at early stationary phase prior to pressure treatment. Based ona preliminary study conducted by using nisin at 200, 400, and 600 IU/ml, nisin was added to the culture at concentration level of 200 IU/mlfor all pressure–antimicrobial combination studies.

2.3. High hydrostatic pressure processing

Pressurization of bacteria culture was carried out using a HighPressure Processing Unit (ABB Quintus Food Processor QFP-6 ColdIsostatic Press, Columbus, Ohio). The hydrostatic pressurization unit iscapable of operating up to 900 MPa. The pressure chamber was filledwith 50% (v/v) aqueous propylene glycol solution. The temperature ofthe liquid was controlled by an electrical heating system and watercirculation around the chamber. The rate of pressure increase wasapproximately 400 MPa/min. The pressure level and temperature ofpressurization were maintained during the 10 min of pressurizationcycle, excluding pressure come-up and pressure release times.

Cell cultures with or without nisin were pressurized at 150, 200,250, 300, 350, 400, 450, 500 or 550 MPa for 10 min at 25 °C. After HHPtreatment, 1 ml portion of the pressurized culture was serially dilutedin 0.1% sterile peptone solution and pour-plated into trypticase soyagar to determine viable cell counts. The remainder of the pressure-treated cell culture was centrifuged at 10,000×g for 10 min at 4 °C(Beckman J2-21 centrifuge) to obtain cell pellets for DSC analysis.Untreated cell cultures were prepared similarly to use as control forplate count and for calorimetry.

2.4. Calorimetry of whole cells

A differential scanning calorimeter (DSC 111, Setaram, Lyon,France) was used to record the thermograms of the control, pressure-treated, or pressure and nisin-treated cells. Cell pellets of 70±0.5 mgwet weight were placed in fluid-tight, stainless steel crucibles, sealedusing aluminum o-rings and were refrigerated at 4 °C prior to DSCruns. The dry material content of the pellets was determined byfreeze-drying (Freezone 4.5, Freeze dry system, Model 77510,Labconco, Missouri) as 18±0.2% on a wet basis. For each DSC run,reference crucible was filled with 57 mg of water approximately equalto the amount of moisture in the sample. Sample and referencecrucibles were heated from 1 °C to 150 °C at a 4 °C/min. Samples werereweighed after DSC measurements to check for the loss of massduring heating. Thermograms of samples showing signs of leakagewere discarded. A DSC run was performed with empty sample andreference crucibles to obtain a baseline thermogram. DSC thermo-grams of samples were corrected for differences in the emptycrucibles by subtracting baseline thermogram.

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2.5. Analysis of DNA transitions in vivo from whole cell DSC

Hoechst 33258 (bisbenzimide, (2-[2-(4-hydroxyphenyl)-6-benzi-midazolyl]-6-(1-methyl-4-piperazyl)-benzimidazole trihydrochlor-ide), Sigma-Aldrich, St. Louis, MO) is membrane permeable,fluorescent DNA stain. Hoechst 33258 binds in the minor groove atAT-rich regions of double-stranded DNA. S. Enteritidis OSU 799 strainwas used for Hoechst 33258-DNA-binding studies. Control, pressure-treated (at 250 MPa), or pressure and nisin-treated (250 MPa and200 IU/ml nisin) cell cultures were centrifuged at 10 000×g for10 min at 4 °C to prepare cell pellets. Cell pellet was re-suspended insterile distilled water at a concentration of 10 mg/ml. Hoechst 33258concentration in suspension was kept at 5 µg/ml based on thepreliminary studies. The suspension was incubated at 37 °C for30 min. The suspension was then centrifuged at 10,000×g for10 min at 4 °C to separate the cells as pellets. Pellets were transferredto DSC crucibles for thermal analysis.

2.6. Data analysis

Total apparent enthalpies in J/g of wet cell weight associated withdenaturation of cellular components between approximately 30 and130 °C were determined by integrating the temperature versus heatflow curve using software provided by the instrument manufacturer.A curved baseline, taking into account the variation in heat capacitybefore and after the denaturation of cellular components, was used tocalculate the total apparent enthalpy. Total apparent enthalpy isdefined as the area between the envelope of endothermic peaks andthe baseline as described by Lee and Kaletunç (2002a).

3. Results

3.1. Thermograms of S. Enteritidis whole cells

The DSC thermograms of S. Enteritidis OSU 799 (A) and FDA (B)strains are shown in Fig. 1. Several overlapping endothermic peaks (a,b, c and d) appear in the thermograms. The peak temperature (Tpeak)of first major transition, a, identified as denaturation of ribosome inbacteria (Mackey et al., 1991), appears at a higher temperature in theFDA strain thermogram (71.5 °C) in comparison to the OSU 799 strainthermogram (69.5 °C). The peaks b, Tpeak 94 °C and c, Tpeak 99 °Cdisplay similar thermal stabilities for the two strains; however, theendothermic transition reported earlier by Lee and Kaletunç (2002b)to be observed only in Gram-negative bacteria appears to exhibitdifferences both in shape and thermal stabilities for the two strains.While the thermogram of the FDA strain showed potentially twooverlapping transitions (d, Tpeak 125 and 132.3 °C), the OSU 799 strain

Fig. 1. DSC thermograms of whole cells of S. Enteritidis OSU 799 (A) and S. EnteritidisFDA (B) (1 to 150 °C with 4 °C/min heating rate).

showed one broad transition (Tpeak 119 °C). The apparent enthalpy ofboth control cells were 4.1±0.4 J/g wet weight.

3.2. Assignment of DNA transition in whole cell thermogram using dyebinding

Fig. 2a shows the whole cell thermogram for S. Enteritidis OSUstrain with (thermogram B) or without (thermogram A) Hoechst dye.Addition of Hoechst dye to DNA in vitro was reported to stabilize theDNA structure and to increase the thermal stability of DNA-meltingtransition (Canzonetta et al., 2002). Thermogram B exhibits atransition, b′, with a peak temperature 100 °C and a shoulder at106 °C. The transition, b′, can be attributed to the transition, b, inthermogram A with increased thermal stability due to addition ofHoechst dye.

Similar increased thermal stabilities for transition b are observedin the thermograms of HHP- or HHP–nisin-treated cells with addedHoechst dye (Fig. 2b). The comparison of thermograms A and B and

Fig. 2. (a) DSC thermograms for S. Enteritidis OSU 799 with (thermogram B) or without(thermogram A) Hoechst 33258 dye. (b) DSC thermograms of S. Enteritidis OSU 799pellets for HHP- or HHP–nisin-treated cells after with or without Hoechst 33258 dye.Cells treated by 250 MPa (A), cells treated by 250 MPa after Hoechst 33258 dye (B),cells treated by 250 MPa with 200 IU/ml nisin (C), and cells treated by 250 MPapressure with 200 IU/ml−1 nisin after Hoechst 33258 dye (D).

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thermograms C and D in Fig. 2b shows that the peak temperature ofendothermic transition, b, increased by 4 °C with the addition ofHoechst dye to the culture medium after HHP or HHP–nisintreatments at 250 MPa. Although changes in the shape and area oftransitions occur in thermograms, the peak temperature of transitionsa and d remain same in all of the thermograms. Comparison ofthermograms in Fig. 2a and b shows that the thermal stability increaseof transition b by addition of Hoechst dye is higher for untreated cellsthan HHP- or HHP–nisin-treated cells.

3.3. Evaluation of the effect of pressure or pressure–nisin treatment on S.Enteritidis by calorimetry

Thermograms of the OSU and FDA strains with or without addednisin at various pressure levels are shown in Figs. 3 and 4.Comparisons of thermogram A in Fig. 3a and b and similarlythermogram A in Fig. 4a and b show that addition of nisin to thecell culture medium at atmospheric pressure did not affect DSCprofiles of whole cells. As the intensity of the treatment increases, thefeatures on DSC profiles including shape and area of the transitions aswell as the temperatures of the transitions exhibit differences. Thetotal apparent enthalpy, ΔH, calculated from DSC thermogramscorresponds to the heat energy required for macromolecular phaseand conformational transitions in bacteria during temperaturescanning. The change of the total apparent enthalpy was observedas a function of pressure and nisin treatments. The total apparententhalpy reduction as a function of treatment was more pronouncedfor the pressure and nisin treatment than pressure treatment alone(Table 1).

The decrease in total apparent enthalpy was mainly due toreduction in the ribosome denaturation peak, a, for both pressure-and pressure and nisin-treated cells (Figs. 3 and 4). Interestingly, thearea of the peak a decreased but the peak temperature remained same

Fig. 3. DSC thermograms of S. Enteritidis FDA pellets. (a) After pressure alone treatment. Co(G), 450 MPa (H) and 500 MPa (I). (b) After nisin–pressure combination treatment. Control (Thermograms are stacked for clarity.

up to 450 MPa for FDA strain and up to 400 MPa for OSU strain with orwithout nisin treatment. On the other hand, the thermograms inFigs. 3 and 4 show that the response of peaks b and c to pressure orpressure and nisin treatment are different, although the control cellthermograms for both strains are similar exhibiting an overlapping b(Tpeak 94 °C) and c (Tpeak 99 °C) transitions. Thermograms of pressure-treated cells at and above 200 MPa display only one visibleendothermic transition with a peak temperature of 94 °C (Figs. 3aand 4a); however, for pressure and nisin-treated cells, a complexendothermic event potentially consists of multiple transitions wasobserved over the temperature range of 85 and 110 °C. The peak ddoes not appear to be affected neither by pressure nor pressure andnisin treatment.

The fractional viability based on calorimetric data, defined in termsof reduced apparent enthalpy, [(ΔH−ΔHf) / (ΔH0−ΔHf)], asexplained in detail elsewhere (Lee and Kaletunç, 2002a) wascalculated to compare the effect of pressure or pressure and nisintreatments for each strains (Figs. 5 and 6), and relative pressureresistances of each strain (Fig. 7) and relative pressure–nisinresistances of each strain (Fig. 8). A residual apparent enthalpy(ΔHf) was used in the calculation of reduced apparent enthalpy toadjust for the residual endothermic transitions present after noviability detected by plate count. Although ΔHf values were similar fordifferent treatment conditions (1.8–1.9 J/g), the pressure levels toreachΔHf were higher for only pressure-treated cells (500 MPa for theFDA strain and 450 MPa for the OSU strain) than for pressure andnisin-treated cells (400 MPa for the FDA strain and 350 MPa for theOSU strain) (Table 1).

3.4. Comparison of plate count and calorimetry data

Fractional viability based on plate count data were calculated as afunction of pressure with or without nisin treatment (Figs. 5 and 6).

ntrol (A), 200 MPa (B), 250 MPa (C), 275 MPa (D), 300 MPa (E), 350 MPa (F), 400 MPaA), 200 MPa (B), 250 MPa (C), 275 MPa (D), 300 MPa (E), 350 MPa (F) and 400 MPa (G).

Fig. 4. DSC thermograms of S. Enteritidis OSU 799 pellets. (a) After pressure alone treatment. Control (A), 150 MPa (B), 200 MPa (C), 250 MPa (D), 300 MPa (E), 350 MPa (F),400 MPa (G), 450 MPa (H) and 500 MPa (I). (b) After nisin–pressure combination treatment. Control (A), 150 MPa (B), 200 MPa (C), 250 MPa (D), 300 MPa (E), 350 MPa (F) and400 MPa (G). Thermograms are stacked for clarity.

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Fractional viability (N/N0) as a function of pressure was low until acritical pressure was reached followed by an exponential decrease(Figs. 5 and 6). For an 8-log reduction, the required pressure levelswere 500 MPa for the FDA strain and 450 MPa for the OSU strainwithout nisin while they were 400 MPa for the FDA strain and350 MPa for the OSU strain with nisin. The reduced apparententhalpy, [(ΔH−ΔHf) / (ΔH0−ΔHf)], as a function of pressure(Figs. 5 and 6) shows a similar trend observed for the viability data.Both plate count and calorimetry data show that resistance of bacteriato pressure is strain dependent (Fig. 7). The FDA strain is relativelymore pressure resistant than the OSU strain. Addition of nisin at200 IU/ml of culture medium prior to pressure treatment causedadditional viability loss at a given pressure in comparison to pressureonly treatment (Figs. 5 and 6). The relative resistance between thetwo strains became negligible when a combined pressure and nisintreatment was applied (Fig. 8).

Table 1Viability and apparent enthalpy values for cells of S. Enteritidis strains after HHP treatment

Pressure(MPa)

Viable counts (cfu/ml)

S. Enteritidis OSU S. Enteritidis FDA

0 nisin 200 IU/ml nisin 0 nisin 200 IU/ml

0.1 1.7×109 1.4×109 1.3×109 1.2×109

150 1.7×109 5.6×108

200 1.3×109 8.2×107 1.2×109 3.0×108

250 1.4×108 1.4×106 4.8×108 1.4×107

300 3.6×105 1.8×103 1.3×107 2.1×104

350 4.9×103 b1.0×101 6.5×104 1.2×102

400 5.0×101 1.5×104 b1.0×101

450 b1.0×101 1.9×103

500 b1.0×101

550

4. Discussion

Preliminary studies on S. Enteritidis FDA strain viability at severalnisin (200–600 IU/ml) – HHP (0.1–600 MPa) combinations wereconducted to determine the optimum nisin concentration. Theaddition of nisin at concentrations from 200 to 600 IU/ml to theculture without subsequent pressure treatment did not reduce theviability of the culture. Cultures with nisin showed additionalreduction of viability in comparison to the cultures without nisin asa result of pressure treatment (Figs 5 and 6); however, for pressure–nisin combination treatments, viability of cells was not significantlyaffected (PN0.05) by increasing nisin concentration from 200 IU/ml to600 IU/ml at all pressure levels (data not shown).

The S. Enteritidis FDA strain had a relatively higher resistance(Pb0.05) to pressure treatment in comparison to S. Enteritidis OSU799 strain (Fig. 7). Comparison of thermal stabilities (Tpeak) of

s.

Apparent enthalpy (J/g)

S. Enteritidis OSU S. Enteritidis FDA

nisin 0 nisin 200 IU/ml nisin 0 nisin 200 IU/ml nisin

4.1 4.0 4.1 4.03.9 3.53.5 3.0 3.8 3.13.2 2.7 3.7 2.92.7 2.4 3.1 2.42.2 2.0 2.9 2.21.9 1.9 2.6 1.91.8 2.31.8 1.81.8

Fig. 5. Pressure dependence of reduced enthalpy ((ΔH−ΔHf) / (ΔH0−ΔHf) andfractional viability (N/N0) of S. Enteritidis FDA strain. Fractional viability withoutnisin (●), with nisin (■), Reduced enthalpy without nisin (○), with nisin (□).

Fig. 7. Pressure dependence of reduced enthalpy ((ΔH−ΔHf) / (ΔH0−ΔHf) andfractional viability (N/N0) of S. Enteritidis FDA and OSU strains. Fractional viabilityOSU (●), FDA (■), reduced enthalpy OSU (○), FDA (□).

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ribosomal subunits for two control strains showed an approximately2 °C higher thermal stability for the FDA strain. Thermal stability ofribosomes has been shown to be related to thermal resistance ofbacterial cells (Mackey et al., 1991; Lee and Kaletunç, 2002b),however not necessarily being a determining factor for pressureresistance (Alpas et al., 2003). Thermal inactivation study on E. coliand Lactobacillus plantarum showed that the greater thermal stabilityof ribosome in E. coli coincided with the greater resistance of E. coli toheat treatment (Lee and Kaletunç, 2002b); however, a recent study byAlpas et al. (2003) on comparison of pressure sensitivities of twostrains of S. aureus by using DSC reports that while S. aureus 765 has ahigher thermal stability for ribosomal subunits than S. aureus 485, S.aureus 485 is more resistant to pressure than S. aureus 765, indicatingthe resistance of bacteria to pressure treatment cannot be predictedby its thermal resistance. Although the thermal stability cannot beused as a criterion to assess the pressure sensitivity of bacteria, Alpaset al. (2003) demonstrated that apparent enthalpy, a measurablephysical property from calorimetric data, can be utilized to determinethe relative pressure sensitivities of different species as well asdifferent strains of same species. Therefore, in the present study theapparent enthalpies were used to assess the relative sensitivities oftwo S. Enteritidis strains to HHP alone and HHP–nisin treatments. The

Fig. 6. Pressure dependence of reduced enthalpy ((ΔH−ΔHf) / (ΔH0−ΔHf) andfractional viability (N/N0) of S. Enteritidis OSU strain. Fractional viability withoutnisin (●), with nisin (■), reduced enthalpy without nisin (○), with nisin (□).

FDA strain was relatively more pressure resistant than the OSU strainbased on both calorimetry and plate count data (Fig. 7). Both strainsexhibited that above a critical pressure value both apparent enthalpyand viability decreased considerably; however, the critical pressureobserved by plate count data was somewhat higher than the onebased on enthalpy data. The lower critical pressure observed forenthalpy data can be attributed to the presence of injured micro-organisms at low pressure. While the plate counts may provideconditions for repair of the injury, the injured organisms are placed incalorimeter immediately after pressure treatment andwould not havethe time and necessary conditions for recovery. A similar observationwas reported for evaluation of viability after a thermal treatment byusing enthalpy and plate count data (Lee and Kaletunç, 2002b).

The apparent enthalpy of either strain did not change significantlyunder atmospheric pressure after nisin treatment for an hour, similarto nearly constant viabilities observed from plate count data. Both theplate count and apparent enthalpy data indicated that nisin alone didnot affect viability of S. Enteritidis; however, the cells becamesusceptible to nisin treatment as the applied pressure increases asassessed by additional cell viability reduction obtained above theeffect of the same HHP alone. It was proposed that HHP alters theouter membrane, thereby facilitating penetration of small water-

Fig. 8. Pressure dependence of reduced enthalpy ((ΔH−ΔHf) / (ΔH0−ΔHf) andfractional viability (N/N0) of pressure and nisin-treated S. Enteritidis FDA and OSUstrains. Fractional viability OSU (●), FDA (■), reduced enthalpy OSU (○), FDA (□).

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soluble peptides or proteins such as nisin and lysozyme into Gram-negative cells (Hauben et al., 1996; Kalchayanand et al., 1998). Thepressure-induced alteration of outer membrane components, wasconfirmed by the leakage of a periplasmic enzyme above a pressuretreatment of 110 MPa (Hauben et al., 1996). Hauben et al. (1997) alsoreported the dissociation of metal ions such as Ca2+ and Mg2+, whichare involved in stabilization of outermembrane, above 220 MPa basedon the studies of E. coli. Comparison of Figs. 7 and 8 indicates that,while the relative pressure resistances of the two strains weredifferent, that difference became negligible when pressure and nisintreatment were applied (Fig. 8). Also, the pressure level at which asignificant viability loss or a significant total enthalpy reduction firstoccurred was lower for pressure and nisin-treated cells than forpressure-treated cells. These observations suggest that membranealteration for allowing entrance of nisin into cell occurs at pressuresaround 100 MPa thereby leading to viability loss and enthalpyreduction. A similar observation was reported by Lee and Kaletunç(2005) for calorimetric assessment of chemical treatments on E. colithat chemical treatment increases the sensitivity of bacteria to heattreatment. In this study, the results indicate that the two hurdles,pressure and nisin have complementary effects in inactivation ofbacteria. Nisin on its own cannot penetrate into a Gram-negative cellto cause inactivation. Pressure facilitates the penetration of nisin intothe cell followed by the additive effect of two hurdles on inactivationof cell, thereby requiring lower levels of pressure to achieve thenecessary inactivation level.

Fluorescence spectra of water-soluble bisbenzimide (Hoechst33258) have been used to detect the DNA of eukaryotic cells(Baumstark-Khan et al., 2000). The purpose of the use of bisbenzimidein this calorimetric study was to detect DNA transition in vivo on thepremise of increased thermal stability of bacterial DNA transitionupon binding with the bisbenzimide. Fig. 2b shows the transitiontemperature peak b in the thermogram of pressure or nisin–pressurecombination treated bacterial cells was increased by 4 °C uponsuspension of these cells in solution containing bisbenzimidesuggesting the origin of peak b is double-stranded DNA transition.Among fluorescent dyes, Hoechst 33258 is known to bind in theminorgroove at AT-rich regions of double-stranded DNA. DNA–Hoechst33258 complex has a higher thermal stability than DNA alone in vitro.The results observed in Fig. 2a and b demonstrate that thermalstability increase can be used to identify the DNA transition in acomplex whole cell thermogram where the individual cellularcomponent transitions are difficult to identify unless compared toDSC transitions of isolated components.

Pressure levels less than 200 MPa enhance the stability of double-stranded DNA when the thermal stability of the DNA is higher than50 °C (Dubins et al., 2001). Since the Tm of bacterial DNA is generally95 °C, we do not expect any damage on DNA structures duringmoderate HHP treatment. Because little information is available inliterature, it is difficult to explain about the mechanism of nisin–pressure combination treatment which causes change in the shapeand area of DNA transitions (peaks b and c) in DSC thermograms after200-MPa treatment for both strains. Possible hypothesis would be theadditional reduction of internal cell pH by addition of nisin in the HHPtreatment. Wouters et al. (1998) reported that internal cell pH of L.plantarum dropped from 7 to 5.5 after the cells pressurized at 250 MPafor 10 min while external pH was maintained at pH 5.5 during thepressurization. HHP treatment was suggested to reduce the internalcell pH by damaging membrane bound ATPase which regulates theefflux of protons (Cheftel, 1995). Internal pH of L. monocytogenesdecreased from 7.9 to 5.5 as a result of nisin treatment (500 IU/ml, for12 min) when external pH was maintained at pH 5.5 (Budde andJakobsen, 2000). Nisin-induced pore formation in cytoplasmicmembrane was attributed to the collapse of proton motive forcedue to the dissipation of the membrane potential and the pH gradient(Bruno et al., 1992; Chung et al., 2000). The stability of DNA was

known to reduce below about pH 5 and above about 10 depending onthe ionic strength of medium and the GC content of DNA. Unfolding ofDNA structure might occur due to disruption of the hydrogen bondingwhen the bases are protonated or deprotonated (Cooper, 1997).Therefore, it is possible that the damage of cytoplasmic membrane,which leads to changes in DNA stability due to reduced internal cellpH, is greater when nisin is applied during pressure treatment.Further study on the measurement of internal pH of cells treated withnisin–pressure combination might be helpful to confirm abovehypothesis. On the other hand, because the melting temperature ofDNA remains same after the treatments, the shape and area changesobservedmay not be due to structural changes in DNA but may be dueto conformational and phase transitions of other cellular componentswhich might have endothermic transitions overlapping with meltingof DNA.

To this end, pressures 100–150 MPa increase the susceptibility ofboth relatively pressure-resistant and -sensitive S. Enteritidis strainsto nisin. The pressure might cause alterations in the outer membraneof the cells thereby facilitating penetration of nisin into the cell. DSCdata allowed us to compare various final states achieved underdifferent treatment conditions starting from the same initial state. Wecould thereby assess the effectiveness of using nisin in combinationwith pressure for inactivation of bacteria relative to pressuretreatment alone. Calorimetry data can be used to evaluate theeffectiveness of each hurdle on overall inactivation of bacterial cellas well as their effect on cellular components so that hurdle sequenceand intensities can be optimized to achieve necessary inactivationlevels.

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