APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2004, p. 1116–1122 Vol. 70, No. 20099-2240/04/$08.00�0 DOI: 10.1128/AEM.70.2.1116–1122.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Evaluation of Structural Changes Induced by High HydrostaticPressure in Leuconostoc mesenteroides

Gonul Kaletunc,1* Jaesung Lee,1 Hami Alpas,2 and Faruk Bozoglu2

Department of Food, Agricultural and Biological Engineering, Ohio State University, Columbus, Ohio 43210,1

and Department of Food Engineering, Middle East Technical University, Ankara 06531, Turkey2

Received 24 July 2003/Accepted 7 November 2003

Scanning electron microcopy (SEM), transmission electron microscopy (TEM), and differential scanningcalorimetry (DSC) were used to evaluate structural changes in Leuconostoc mesenteroides cells as a function ofhigh-hydrostatic-pressure treatment. This bacterium usually grows in chains of cells, which were increasinglydechained at elevated pressures. High-pressure treatments at 250 and 500 MPa also caused changes in theexternal surface and internal structure of cells. Dechaining and blister formation on the surface of cellsincreased with pressure, as observed in SEM micrographs. TEM studies showed that cytoplasmic componentsof the cells were affected by high-pressure treatment. DSC studies of whole cells showed increasing denatur-ation of ribosomes with pressure, in keeping with dense compacted regions in the cytoplasm of pressure-treatedcells observed in TEM micrographs. Apparent reduction of intact ribosomes observed in DSC thermogramswas related to the reduction in number of viable cells. The results indicate that inactivation of L. mesenteroidescells is mainly due to ribosomal denaturation observed as a reduction of the corresponding peak in DSCthermograms and condensed interior regions of cytoplasm in TEM micrographs.

High-hydrostatic-pressure (HHP) processing is consideredfor food preservation as an alternative to conventional thermalpasteurization due to its potential for microbial inactivation.HHP can be used alone or in combination with thermal ornonthermal techniques for the production of a wide variety ofhigh-quality foods that are minimally processed, additive free,and microbiologically safe (7). Thus, HHP research has beenprimarily focused on the cellular targets and the mechanism ofthe HHP-induced inactivation of various food spoilage andfood-borne pathogenic bacteria with the ultimate goal of op-timizing the processing conditions. Resistance to HHP variesamong bacteria and is dependent on the physiological state ofthe organisms at the time of pressurization (1, 8, 24, 30).Although some studies (9, 26) suggest that the cell wall and cellmembrane lose their function as a result of pressure, the exactmechanism of inactivation caused by HHP is still not wellunderstood. Dissociation of ribosomes, thermotropic phasechanges in membrane lipids, and protein denaturation also areproposed as possible structural changes in the cell that causeinactivation of microorganisms subjected to high pressure (1, 9,16, 23). Electron microscopy has been employed to character-ize pressure-induced morphological changes in microorgan-isms in order to understand the events leading to cell inacti-vation (9, 16, 20, 29). Using scanning electron microscopy(SEM), Kalchayanand et al. (9) evaluated morphologicalchanges in Leuconostoc mesenteroides cells after pressure treat-ment at 345 MPa. These investigators reported that while thecell size, shape, and surface structure of inactivated cells im-mediately after pressure treatment were not different fromthose of living cells, cell lysis was observed after 2 h of storage

at 4°C. Transmission electron microscopy (TEM) studies byMackey et al. (16) of the cell structure of Salmonella entericaserovar Thompson and Listeria monocytogenes after pressuretreatments at 250 and 500 MPa showed structural changesspecific to each organism. While electron micrographs of pres-sure-treated L. monocytogenes cells showed formation of vac-uolar regions in the cytoplasm, vacuole formation was notreported for the pressure-treated cells of S. enterica serovarThompson. These investigators observed fewer ribosomes inpressure-treated S. enterica serovar Thompson cells than un-treated cells, suggesting apparent cell lysis. Using SEM,Tholozan et al. (33) and Ritz et al. (29) reported increasingirregularities referred to as “bud scars” on the surface of L.monocytogenes cells with increasing pressure. Although totalinactivation of the population was observed at 400 MPa, bac-terial cells retained their morphological characteristics withlimited cell disruptions. Tholozan et al. (33) observed increas-ing cell invaginations with increasing pressure for Salmonellaenterica serovar Typhimurium, but there was no lysis. SEMstudies by Malone et al. (20) revealed low-density intracellularregions in Lactococcus lactis subsp. cremoris cells after pressuretreatments at 300 and 800 MPa and cell envelope damage at800 MPa.

Differential scanning calorimetry (DSC) has been employedto monitor the conformational transitions specific to variouscellular components of intact cells as a function of temperaturein order to understand the sequence of events leading to in-activation of microorganisms (1, 2, 4, 12–14, 17–19, 22, 23, 34).The thermal stability of ribosomes has been shown to correlatewith growth temperature of the cells, and the denaturation ofribosomes has been proposed as a mechanism for cell injury ordeath (1, 4, 12–14, 17–19, 22, 31). In addition to thermaltreatment-induced changes, DSC has been used to evaluate theeffect of various physical and chemical factors on bacterialinactivation by comparing the thermograms before and after

* Corresponding author. Mailing address: Department of Food, Ag-ricultural and Biological Engineering, Ohio State University, Colum-bus, OH 43210. Phone: (614) 292-0419. Fax: (614) 292-9448. E-mail:kaletunc.1@osu.edu.

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treatment (1, 23). DSC analysis of pressure-treated bacteriaindicated a correlation between cell viability and a reduction inthe apparent enthalpy associated with ribosome denaturation,suggesting that cell inactivation and ribosomal denaturationare closely related (1, 23). While some interesting observationshave been reported, pressure-induced structural changes at thecellular and molecular levels and their implications on cellinactivation have not been characterized thoroughly.

The goal of this study was to investigate HHP-induced mor-phological changes and their relation to cell inactivation in L.mesenteroides. SEM and TEM were used to characterize chainarrangement and the surface and internal morphology of cellsas a function of HHP treatment. DSC was employed to detectand monitor changes in the thermal stabilities of DNA andribosomes as well as the apparent enthalpy of whole cells.

MATERIALS AND METHODS

Source and preparation of organisms. L. mesenteroides OSU553 (isolatedfrom a local source) was obtained from the Culture Collection, Department ofMicrobiology, The Ohio State University, Columbus. The culture was storedfrozen (�80°C) in 30% (vol/vol) sterile glycerol. A loopful of L. mesenteroideswas revived in 10 ml of MRS broth (Difco, Detroit, Mich.) and incubated at 30°Cfor 16 h.

L. mesenteroides culture was inoculated (1% [vol/vol]) into MRS broth andincubated at 30°C. Duplicate samples were taken every 1 h and pour plated withMRS agar (MRS broth plus Bacto-Agar; Difco). The plates were incubated at30°C for 36 h, and a growth curve for L. mesenteroides was constructed. Once thelate-exponential phase was reached, cells were harvested for HHP treatment,DSC analysis, viable count, and electron microscopy studies.

HHP treatment. Cells grown to a final concentration of 1.3 � 108 � 0.1 � 108

CFU ml�1 in MRS broth (200 ml) were placed in 2-mil-thick sterile polyethylenebags (3.8 by 15 cm) (Fisher Scientific, Inc., Pittsburgh, Pa.) for HHP treatment.Air was removed from the bags prior to heat sealing. The bags were placed insidea second polyethylene bag and heat-sealed under vacuum to prevent contami-nation of the high-pressure unit if the primary package were to fail. A hydrostaticpressurization unit (Quintus QFP-6; ABB Autoclave Systems, Inc., Columbus,Ohio) that was capable of operating up to 900 MPa was used to apply pressureto the L. mesenteroides cell suspensions. A water-propylene glycol (Houghton-Safe 620-TY; Houghton Intl., Inc., Valley Forge, Pa.) mixture (1:1 [vol/vol]) wasused as the pressure-transmitting fluid. Prior to pressurization, the fluid washeated to the desired temperature by an electrical heating system surroundingthe unit. The rate of pressure increase was approximately 400 MPa/min, and thepressure release time was less than 20 s. Pressurization times reported in thisstudy exclude the pressure increase and release times. The pressure level, time,and temperature of pressurization were set manually and were recorded as afunction of time during the treatment.

The polyethylene bags containing cell suspensions were pressurized for 5 minat pressures of 250 and 500 MPa at 35°C. Duplicate samples were prepared foreach treatment. Both pressure-treated and untreated cell suspensions were cen-trifuged (J2-21; Beckman, Palo Alto, Calif.) at 10,000 � g for 10 min at 4°C toform pellets prior to SEM, TEM, and DSC analysis.

Enumeration of cells. Pressure-treated and untreated cell suspensions wereserially diluted in 0.1% sterile peptone (Becton Dickinson, Cockeysville, Md.)solution. From the selected dilutions, 1-ml portions were pour plated in duplicateplates by using MRS agar media. The plates were incubated at 30°C for 36 h, andplates containing 25 to 250 CFU ml�1 were selected for counting.

Electron microscopy. The L. mesenteroides cell pellet was prepared fromuntreated and pressure-treated cell suspensions by centrifugation at 10,000 � gfor 10 min at 4°C and washed once with 150 ml of sterile distilled water. Cellpellets (1 mm3) were transferred to sterile vials and resuspended in 1 ml of 0.1M phosphate buffer at pH 7.4. Suspended bacteria were filtered (0.45-�m poresize) and fixed on the membrane with 10 ml of 3% glutaraldehyde in 0.1 Mphosphate buffer (pH 7.4). Fixative was left in contact with the cells overnight at4°C.

For SEM analysis, the fixed cells were washed with buffer and postfixed for 1 hin 1% osmium tetroxide in phosphate buffer. Filters were rinsed with buffer anddehydrated through a series of ethanol solutions with increasing concentrations(50, 70, 95, and 100% ethanol). Ethanol was replaced with liquid CO2, and thesamples were dried in a critical point dryer. Cells were sputter coated with

gold-palladium and examined in a Philips XL-30 scanning electron microscope at30 kV (FEI, Inc., Hillsboro, Oreg.).

For TEM analysis, fixed cells were rinsed with buffer and centrifuged, and thepellet was embedded in 2% agar. Agar was cut into 1-mm3 pieces and postfixedfor 1 h in 1% osmium tetroxide in phosphate buffer. Samples were rinsed indistilled water and stained en bloc for 1 h in 1% aqueous uranyl acetate. Afterdehydration through an ascending series of ethanol solutions (50, 70, 95, and100% ethanol), cells in agar were transferred to propylene oxide and infiltratedand embedded in Spurr’s resin (Ted Pella, Redding, Calif.). Sections (70 nm)were obtained with an ultramicrotome and stained with Reynolds’ lead citrate(27) prior to examination in a Philips CM-12 TEM at 60 kV (FEI, Inc.).

DSC analysis. A portion (�100 mg) of the L. mesenteroides pellet was trans-ferred into a tared (1.5 ml) polyethylene tube, weighed, freeze-dried (Freezone4.5; Labconco Freeze Dry System, Kansas City, Mo.), and reweighed to deter-mine the percentage of dry matter in the pellet. The amount of moisture in theL. mesenteroides pellet used in the DSC experiments was 83% � 0.3% (wt/wt).

A differential scanning calorimeter (DSC 111; Setaram, Lyon, France) wasused to collect thermograms of untreated control and pressure-treated L. mes-enteroides. A DSC thermogram with an empty stainless steel sample and refer-ence crucibles was collected to measure the empty crucible baseline. Tempera-ture calibration was confirmed with an indium sample in a stainless steel crucible.All thermograms were collected at a constant heating rate of 4°C min�1. Pelletsof cells were transferred into the sample crucible and weighed (70 � 0.3 mg wetweight). When the reference crucible was left empty, an artifact due to animbalance of heat capacity between crucibles was observed at the initiation oftemperature scanning. A known quantity of water, similar in mass to the mois-ture in the sample, was placed in the reference crucible to eliminate the artifact.The reference crucible was filled with 58 � 0.2 mg (83% of sample weight) ofdistilled water. Both crucibles were sealed with aluminum O-rings. The sealedcrucibles were refrigerated (4°C) until used for DSC. The sample and referencecrucibles were placed in the DSC and equilibrated at 1°C with liquid nitrogenand scanned to 140°C at 4°C min�1. Samples were reweighed after DSC mea-surements to check for loss of mass during heating. Thermograms of samplesshowing signs of leakage were not used.

Analysis of DSC data. An empty crucible thermogram was subtracted from asample thermogram to correct for differences in the empty crucibles. Total heatlevels corresponding to the area between the endothermic peaks and the baseline(apparent enthalpy in joules per gram) were determined by integrating thetemperature versus heat flow curve by using software provided by the instru-ment’s manufacturer. A curved baseline using three-temperature points wasutilized to calculate the apparent enthalpy of whole cells (12). The curvedbaseline was constructed between the segment of the thermogram prior to thefirst thermally induced transition (�36°C) and the segment of the thermogramafter the last peak (�115°C). The total peak area was determined for both thecontrol and pressure-treated samples. Peak temperatures for the thermally in-duced transitions were also determined.

RESULTS

Viability. The effect of HHP treatment on the viability of L.mesenteroides cells was determined by plate counting. The ini-tial number of cells prior to pressure treatment was found to be1.3 � 108 CFU ml�1. Pressure treatment at 250 MPa and 35°Cfor 5 min reduced the cell count to 2.1 � 106 CFU ml�1. Viablecell counts were not detected following pressure treatment at500 MPa and 35°C for 5 min. MRS medium is somewhatselective, and therefore severely injured cells may not havebeen recovered.

SEM. Pressure treatment at 250 and 500 MPa producedmorphological changes on the surface and in the internal struc-ture of the cells, as observed by SEM and TEM. When grownin rich media, the bacteria formed characteristic chains of up tofive coccidal lenticular cells with constrictions at the junctionsbetween cells (Fig. 1A). Electron micrographs of typical ar-rangements of untreated and pressure-treated cells are shownin Fig. 1. The number of long chains (three cells or more)decreased as the pressure increased relative to untreated sam-ples. More than 50% of untreated cells, counted in SEM mi-

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crographs, were in chains of three or more (Fig. 2). The com-bined percentage of single cells and cells in chains of twoincreased progressively as the pressure increased.

Untreated control cells exhibited a smooth surface structure(Fig. 3A). The surface appearance became rough and crackedwhen the cells were exposed to 500 MPa (Fig. 3B). Some cellsshowed even rougher surface structure and blister-like protru-sions after pressure treatment at 500 MPa (Fig. 3C). Pressuretreatment at 250 MPa and 35°C for 5 min produced cells witha surface morphology characteristic of both untreated cells andcells treated at 500 MPa (results not shown). Blister-like for-mations appeared to be in rows parallel to the division sites oflenticular cells.

TEM. Thin sections of untreated L. mesenteroides cells dis-played intact cell membrane, uniform cell cytoplasm, and elec-tron-transparent regions of nucleoid in electron micrographs(Fig. 4A). Application of pressure resulted in morphologicalchanges in the internal structures. The most notable changeswith application of pressure were a double-track bilayer struc-ture of the membrane instead of the single-thick-layer appear-ance and enlargement of electron-transparent areas in thecytoplasm. Application of pressure at 250 MPa and 35°C for 5min resulted in expanded nucleoid regions and compactedinterior regions (Fig. 4B). Most cells maintained a distinctmembrane. In some cells, breakdown of the peptidoglycanlayer was evident, because parts of the outer layer appeared tohave sloughed off after pressure treatment at 500 MPa (35°Cfor 5 min) (Fig. 4C). Aggregation of cytoplasmic material andenlarged electron-transparent regions of nucleoid with a fi-brous appearance were also observed (Fig. 4C). Blister forma-tions were notable on cell walls of some cells after pressuretreatment at 500 MPa (Fig. 4C).

DSC. DSC thermograms of control and pressure-treatedcells are shown in Fig. 5. An increase in pressure resulted in adecreased transition peak area (apparent enthalpy, �H injoules per gram). A large reduction was observed in the firstmajor transition over a temperature region of the thermogramof 50 to �85°C (peak a). In addition to the area of the peak,both the onset and peak temperatures of the transition de-

FIG. 1. SEM micrographs of L. mesenteroides cells. (A) Untreatedcells. (B) Cells treated with 250 MPa of pressure at 35°C for 5 min.(C) Cells treated with 500 MPa of pressure at 35°C for 5 min. Originalmagnification, �3,500.

FIG. 2. Effect of pressure on dechaining of L. mesenteroides cells.The total number of cells counted in each case was approximately1,000. A pressure of 0.1 MPa was used for untreated cells. Open bars,single cells and cells in chains of two; solid bars, cells in chains of threeor more.

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creased as the treatment pressure increased. While the en-thalpy of the DNA transition (peak b) remains unchanged as aresult of pressure treatment at 250 MPa, a 25% decrease in theenthalpy of the transition was observed after a 500-MPa pres-sure treatment. The thermal stability of DNA decreased pro-gressively due to pressure treatment. The peak temperaturesfor the DNA melting transition are 100°C for untreated controlcells, 95°C for cells treated at 250 MPa, and 91°C for cellstreated at 500 MPa.

The DSC thermograms exhibited differences in the apparentspecific heat capacity change associated with heating of the liveand inactivated cells. These changes in heat capacity wereobserved as deviations between the pre- and posttransitionbaselines. The change in heat capacity became progressivelysmaller as the pressure level increased due to the increase inspecific heat of cells inactivated by pressure treatment (Fig. 5).

DISCUSSION

Leuconostoc spp. are gram-positive chain-forming cocci thatoccur singly or in pairs (diplococci) and short chains (strepto-cocci) (25). Dechaining as a result of pressure treatment maybe due to a lower volume achieved by single cells and doublets,because a smaller volume is favored thermodynamically atincreased pressure (3). Dechaining has been proposed to bedue to enzymatic activity and is associated with autolysins inStreptococcus spp. (6, 15, 28, 32). McCarty (21) noted thatstreptococcal chains are difficult to disrupt without killing theorganisms, but the relationship between cell death anddechaining has not been established. The present work showsthat both viability and chain length decreased as the level ofpressure treatment increased. Chains containing more thanthree cells were present after a 500-MPa treatment, althoughthe cells were not viable.

Blister-like formations increased with pressure, and thehighest numbers of blisters appeared after a pressure treat-ment at 500 MPa. Similar surface formations have been re-ported for heat- and pressure-treated bacteria. Surface blistershave been observed on the cell envelope of Escherichia coliupon heating to 55°C for 15 s (11). Longer heating timesresulted in a decrease of cells with surface blisters. Deforma-tions on the surface of L. monocytogenes cells, described as budscars, pimples, and swellings, have been reported as a result ofpressure treatment at 400 MPa and 20°C for 10 min (29, 33).Both studies reveal an increase in the number of deformationsat pressures between 275 and 400 MPa. The blisters observedin our work intensified at treatment at 500 MPa and 35°C, andthus their presence on L. monocytogenes cells after pressuretreatment at 400 MPa and 20°C shows that blisters were in-duced by pressure treatment.

Tholozan et al. (33) reported cell membrane invaginations inS. enterica serovar Typhimurium without any blister formation.In our laboratory, blister formation has not been observed inhigh-pressure-treated (300 to 700 MPa) E. coli cells even at thehighest-pressure treatment (unpublished data). Heat-inducedblisters reported by Katsui et al. (11) consisted of outer mem-brane and had a multilayered structure and short life. Theblisters on L. mesenteroides cells observed here formed outsidethe cell wall and were composed of extracellular materials of

FIG. 3. SEM micrographs of L. mesenteroides cells. (A) Untreated.Original magnification, �25,000. (B) Cells treated with 500 MPa ofpressure at 35°C for 5 min. Original magnification, �25,000.(C) Higher magnification (�50,000) of some cells treated with 500MPa of pressure at 35°C for 5 min.

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gram-positive cells; no internal structures were evident in theseblisters.

DSC thermograms of microorganisms document endother-mic transitions indicating the denaturation of cellular compo-nents (2, 10, 14, 18, 22, 34). The main peaks observed inthermograms of untreated L. mesenteroides cells are identifiedas ribosomal subunits and DNA by comparison to the transi-tion temperatures of isolated cell components of E. coli (13,18). DSC data in the literature (1, 23) indicate that inactivationof bacteria by pressure correlates with the denaturation of themain ribosomal subunit. The thermograms in Fig. 5 are inagreement with the proposed denaturation of ribosomes by

high pressure within this temperature envelope. Reduction inthe area of the ribosomal peak as a function of pressure indi-cates irreversible changes with pressure and may be due todenaturation, with possible aggregation (an exothermic event)of ribosomes. The denatured ribosomes may manifest them-selves as the compacted interior regions of the cytoplasm ob-served in TEM micrographs.

The transition attributed to melting of cellular DNA exhib-ited progressive changes by pressure treatment. The decreasein thermal stability of the DNA peak may be due to partialdissociation of a DNA duplex during pressure treatment, fol-lowed by refolding to a thermally less stable state upon return

FIG. 4. TEM micrographs of L. mesenteroides cells. (A) Untreated. (B) Cells treated with 250 MPa of pressure at 35°C for 5 min. (C) Cellstreated with 500 MPa at 35°C for 5 min. cw, cell wall; cm, cell membrane; n, nucleoid; b, blisters. Scale bar, 0.5 �m.

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to atmospheric pressure. Given the small volume change (andconcomitant small pressure sensitivity) associated with DNAduplex disruption (5), the reduction in transition temperatureof the DNA peak may be due to pressure-induced changes inDNA packaging in the cell. The expansion of electron-trans-parent nucleiod regions correlates with the changes in theDNA peak in DSC thermograms with pressure.

This study demonstrates structural changes that occur dur-ing high-pressure treatment in the arrangement of chain-form-ing bacteria, blister formations on the external surface, andcondensation of nucleoid and cytoplasmic material in the cell

interior. The corresponding thermodynamic changes in cellu-lar components—specifically in ribosomes and DNA—are alsoshown in this paper. Calorimetric data showed increasing de-naturation of ribosomes with pressure, in keeping with thedense compacted regions in the cytoplasm of pressure-treatedcells observed in TEM micrographs. The data further our un-derstanding of complex events induced by pressure treatmentleading to cell injury and death. The results provide additionalcharacterization of HHP inactivation of cells. At lower pres-sures, the inactivation may be due to ribosomal denaturation,based on the DSC results. However, membrane damage can-

FIG. 4—Continued.

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not be eliminated as a potential source of lethality solely on thebasis of the electron micrographs. Once the events causingdeath have been identified and characterized, a rational selec-tion of optimal pressure and temperature treatment to preventspoilage or disease will be forthcoming.

ACKNOWLEDGMENTS

This study was supported by National Science Foundation grantINT-0096915 and The Scientific and Technical Research Council ofTurkey (TUBITAK; project no. TOGTAG-NSF-2001-1).

We thank Brian Kemmenoe and Kathy Wolken of the Microscopyand Imaging Facility at the Ohio State University for assistance withthe SEM and TEM studies.

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FIG. 5. DSC thermograms of L. mesenteroides cells. (A) Untreated.(B) Cells treated with 250 MPa of pressure at 35°C for 5 min. (C) Cellstreated with 500 MPa of pressure at 35°C for 5 min. Arrows mark thepeak temperatures of endotherms corresponding to ribosome dena-turation (peak a) and DNA melting (peak b).

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