1764 (2006) 1188ndash1197httpwwwelseviercomlocatebba

Biochimica et Biophysica Acta

Protection by sucrose against heat-induced lethal and sublethal injury ofLactococcus lactis An FT-IR study

Klaus V Kilimann ab Wolfgang Doster e Rudi F Vogel bChristoph Hartmann ad Michael G Gaumlnzle c

a Lehrstuhl fuumlr Fluidmechanik und Prozessautomation Technische Universitaumlt Muumlnchen Weihenstephaner Steig 23 D-85350 Freising Germanyb Lehrstuhl fuumlr Technische Mikrobiologie Technische Universitaumlt Muumlnchen Weihenstephaner Steig 16 D-85350 Freising Germany

c Department of Agricultural Food and Nutritional Science AgriculturalForestry Centre University of Alberta Edmonton AB Canada T6G 2P5d Department of Modelling and Simulation in Mechanics Faculty of Engineering and Material Science The German University in Cairo New Cairo City Egypt

e Physik Department E13 Technische Universitaumlt Muumlnchen James-Franck-Straβe 1 D-85747 Garching Germany

Received 2 December 2005 received in revised form 19 April 2006 accepted 25 April 2006Available online 16 May 2006

Abstract

The heat inactivation of Lactococcus lactis was studied by determination of cell counts and by FT-IR spectroscopy recording the averagestructure of cell proteins Cell counts were measured after incubation milk buffer or milk buffer with 1 5 M sucrose and FT-IR spectra wererecorded in 2H2O or 2H2O with 1 5 M sucrose in the range of 6ndash75 degC Sucrose protected L lactis against heat inactivation The cell countsdiffered by up to 6-log cycles after treatment in milk buffer as compared to milk buffer with sucrose The 1H2H exchange in proteins andsecondary structure elements were detected by the analysis of amide Iprime amide II and amide IIprime bands A reduced 1H2H exchange as well as alower content of disordered structural elements was observed when sucrose was present Conformational fluctuations of native proteins asindicated by the 1H2H exchange were apparent already at sublethal temperatures The loss of viability of L lactis occurred in the sametemperature range as the loss of the protein secondary structure These results demonstrate that sucrose protects L lactis against heat inactivationand that the increased heat stability of proteins in the presence of sucrose contributed to this enhanced heat resistancecopy 2006 Elsevier BV All rights reserved

Keywords FT-IR spectroscopy Bacterial inactivation Protein denaturation Heat resistance

1 Introduction

Heat treatment remains the most common processingtechnique to assure the microbiological safety of foodsMoreover combined osmotic and thermal treatments arefrequently applied in the stabilisation of bacterial cultures foruse as starter cultures in food fermentation or for use asprobiotics In both types of processes aiming to eliminate or tostabilise bacteria the bacterial heat resistance must be taken intoaccount The heat resistance of bacteria depends on extrinsicand intrinsic factors First stationary cells are more resistantthan cells in their exponential phase of growth due to the denovo synthesis of stress proteins in the early stationary phase[1ndash3] Second the medium composition with respect to the pH

Corresponding authorE-mail address mgaenzleualbertaca (MG Gaumlnzle)

1570-9639$ ndash see front matter copy 2006 Elsevier BV All rights reserveddoi101016jbbapap200604016

water activity (aw) and the concentration of osmolytes affectbacterial inactivation [4ndash6] Bacteria exhibit a maximalresistance against heat treatment at their optimal pH of growthFurthermore a reduction in water activity results in a higherresistance to thermal treatment [7]

Moderately elevated temperatures cause sublethal injuryfrom which the organisms recover only on non-selective media[89] Heat induced sublethal injury and cell death was linked toprotein denaturation and the inactivation of membrane boundenzymes [1011] Many foods are selective media due to theirparticular properties such as pH-value and presence of anti-microbial compounds In those foods the inactivation ofspecific resistance mechanisms required for growth or survivalin these products may be sufficient for preservation

Bacteria adapt to hyper-osmotic conditions by accumulatingorganic solutes such as sugars [12ndash14] Lactic acid bacteriaincluding Lactococcus lactisMG1363 react to osmotic upshock

1189KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

with sucrose by equilibration of the intracellular and extra-cellular sucrose concentrations [1215] This accumulation ofosmolytes furthermore provides protection towards other lethalstressors eg freeze-drying salt high pressure or heat[51516] FT-IR measurements demonstrated that the accumu-lation of osmolytes prevents membrane damage under stressconditions [151718]

The heat denaturation of proteins is caused by confor-mational transitions in the secondary structure Increasingtemperature enhances cooperative intra-molecular motionsuntil the melting temperature is reached At this temperaturenon-covalent forces that maintain the native structure nolonger prevail against entropic forces [1920] and the proteinunfolds The thermal stability of proteins can be changedintrinsically by modification of the primary sequence orextrinsically by the addition of suitable stabilising effectorseg peptides or osmolytes [2122] A general mechanismproposed to explain this stabilising effect of osmolytes istheir exclusion from the protein hydration shell orpreferential hydration [23]

Fourier transform infrared (FT-IR) spectroscopy is apowerful tool to study protein structure Changes in proteinconformation are recorded in the in the finger print regionof the amide bands at 1500 to 1600 cmminus1 of IR spectra[2425] For instance FT-IR records temperature inducedchanges of structural characteristics of proteins [26ndash28] Asa non-destructive technique it can be used for in-vivostudies to distinguish between the native and denatured oraggregated state of intracellular proteins FT-IR spectroscopywas previously applied to determine membrane phasetransitions in bacterial cells [151729] but its potential todetermine structural alterations of bacterial proteins duringsublethal and lethal heat treatments has not been exploited

It was the aim of this study to investigate the protectiveeffect of sucrose against heat inactivation in the range of40ndash75 degC of Lactococcus lactis ssp cremoris MG1363 bythe determination of lethal and sublethal injury In order todetermine the protection mechanisms of sucrose structuralchanges of the cytoplasmic membrane and cell proteinsinduced by heat were determined by recording FT-IR spectraduring heat application to the population Thermal inactiva-tion data of bacterial cell suspensions were compared totemperature-induced conformational changes of the proteinstructure

2 Material and methods

21 Microorganisms and media

Lactococcus lactis ssp cremorisMG 1363 was grown at 30 degC in M17 brothsupplemented with 1 glucose Cells of an overnight culture were harvested bycentrifugation washed and re-suspended in buffer to cell counts of about 109

cells mlminus1 This buffer was designated as ldquomilk bufferrdquo because it wasintentionally set up to resemble whey [30] It contained the followingcompounds (g lminus1) KCl 11 MgSO4times7H2O 07110 NaH2PO4times2H2O1874 CaSO4times2H2O 10 CaCl2times2H2O 099 citric acid 20 lactose 520The pH was adjusted to pH 65 When indicated 1 5 M sucrose (sucrose) wasadded to the buffer (milk buffer sucrose)

22 Determination of viable cell counts and stress resistant cell counts

Overnight cultures of L lactis were washed twice in milk buffer or milkbuffer sucrose as described above transferred to 1 ml plastic vials and exposedto temperatures ranging from 40 degC to 75 degC for 0 to 120 min Sampling timeswere chosen to obtain at least 4 cell counts during incubation times that reducedcell counts by 3ndash4 orders of magnitude This was not possible a very lowtemperatures without any inactivation and at high temperatures that reduced thecell counts by more than 6 log within few seconds After treatment cellsuspensions were cooled rapidly on ice Cells from each sample were diluted andplated on M17 agar supplemented with 1 of glucose or M17 agarsupplemented with 1 of glucose and 3 NaCl for determination of viableand stress resistant cell counts respectively [30] The plates were incubated for24 h at 30 degC under aerobic conditions to assess viable cell counts (CFU) andfor 48 h to assess stress resistant cell counts (CFUsub) All temperatureinactivation kinetics were performed in duplicate triplicate or quadruplicateindependent experiments The results are reported as meansplusmnstandard deviation

23 Data analysis

Thermal inactivation data was modelled assuming first order kinetics [31] toobtain a single parameter describing the kinetics of lethal and sub-lethal injury ofthe bacteria

logethN=N0THORN frac14 kt eth1THORNwith N=microbial population at time t N0= initial microbial population at t0 andk=the first-order reaction rate constant

24 FT-IR Spectroscopy

Conformational changes in proteins and internal vibrational modes oflipid acyl chains of the membrane of L lactis were measured by Fouriertransform infrared spectroscopy (FT-IR) Cells from 50 ml overnight culturewere harvested by centrifugation (5 min at 10000timesrelative centrifugal force)washed twice in milk buffer or milk buffer sucrose and incubated for 45 minin the buffer system in which they were subsequently analysed In thepresence of 1H2O the amide bands are obscured by the dominating spectra of1H2O Therefore the cells were harvested after incubation and washed intriplicate in deuterium oxide (2H2O) or 2H2O-sucrose to exchange easilyaccessible H+ ions by deuterium ions the resulting sample had an opticaldensity at 590 nm of about 10 corresponding to about 1010 cells per mlCells were poured into a 20-μm-thick infrared cell equipped with CaF2-windows the total volume of the cell was 20 μL CO2 was removed and theair moisture inside the chamber was reduced by flushing the chamber withnitrogen gas The FT-IR spectra were recorded with an Equinox 55spectrometer (Bruker Ettlingen Germany) equipped with a DTGs detectorSpectra were taken between 6 degC and 75 degC the temperature was adjusted byan external water thermostat The scans at a given temperature were startedwhen the average temperature measured across the diagonal of the CaF2-windows was within 0 5 degC of the desired temperature During each experimenttwenty scans were averaged for each temperature point and averaged data ofat least two independent experiments are presented The OPUS softwarepackage (Bruker Ettlingen Germany) was used for analysis of FT-IR spectraSecond derivative spectra were generated by using a 9-data point (9 cmminus1)function which is implemented in the OPUS software package

3 Results

31 Temperature induced lethal and sub-lethal injury ofL lactis

The heat inactivation of stationary cells L lactis suspendedin milk buffer or milk buffer sucrose was determined Theviability and sub-lethal injury of the cultures were measuredafter heat treatment A temperature range of 40ndash75 degC with 5 degC

1190 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

increments was chosen and process times ranged from 0 min to120 min In Fig 1 the thermal death time data of L lactis in milkbuffer are depicted Sub-lethal injury was detected afterincubation at 45 degC and lethal injury was apparent attemperature levels of 50 degC or higher After treatment at65 degC and 70 degC the cell counts were below the detection limitafter 4 min and less than 2 min respectively A processtemperature of 65 degC and a process time of 2 min resulted insub-lethal injury of all surviving cells

Thermal death time data of L lactis in milk buffer sucroseare shown in Fig 2 In general a protective effect for bothphysiological states was detected compared to the inactivationof L lactis in milk buffer For example a difference in the cellcounts of more than 6 log is apparent when treatments at 50 degCand 120 min in milk buffer and milk buffer with sucrose arecompared Nevertheless the cell counts of L lactis in milkbuffer sucrose were below the detection limit after 2 min at75 degC (Fig 2)

32 Analysis of infrared spectra

To assess the suitability of FT-IR spectroscopy for analysisof structural properties of cellular proteins spectra obtainedfrom microbial populations were compared with literature

Fig 1 Comparison of experimental and modelling results concerning lethal(CFU A) and sublethal (CFUsub B) injury of L lactis after a treatment in milkbuffer at various temperatures 40 degC () 45 degC () 50 degC () 55 degC ()60 degC () 65 degC () 70 degC (diams) and 75 degC (⋄) Lines represent the predictionof cell counts by first order kinetics Lines dropping below the x-axis indicatecell counts below the detection limit of 100 CFU mlminus1

Fig 2 Comparison of experimental and modelling results concerning lethal(CFU A) and sublethal (CFUsub B) injury of L lactis after a treatment in milkbuffer sucrose at various temperatures 40 degC () 45 degC () 50 degC () 55 degC() 60 degC () 65 degC () 70 degC (diams) and 75 degC (⋄) Lines represent theprediction of cell counts by first order kinetics Lines dropping below the x-axisindicate cell counts below the detection limit of 100 CFU mlminus1

spectra of purified proteins in aqueous solution An overviewof the bands in L lactis relevant to protein or membranestructure their assignment the location of their absorptionmaxima as well as their wave number range is given in Table1 the corresponding spectral range is shown in Fig 3 Thespectra measured with L lactis suspended in 2H2O comprisedthe same characteristics as the spectra measured in 2H2O-sucrose (Fig 3 and data not shown)

Temperature induced shifts of the spectra providedinformation about structural changes in the membrane andin proteins induced by increasing the temperature (Table 1)[1732] Membrane-phase transitions were detected by

Table 1Common range of the characteristic bands of the secondary structure of proteinsdetected by FT-IR in L lactis

Band Wave number (cmminus1) Width (cmminus1)

Amide A+HOD sim3400 3200ndash3800Symmetric CH2 sim2852 ndashAmide Iprime sim1652 1600ndash1700Amide II sim1550 ndashTyrosine sim1515 ndashSide chains ndash 1500ndash1600Amide IIprime sim1455 1350ndash1500

Fig 3 Segments of the FT-IR spectra of L lactis in 2H2O-sucrose used for the secondary structure analysis of proteins and the lipid acyl chains A temperature inducedshift of CH2 B temperature induced shift of amide A+HOD C temperature induced shift of amide IIprime and amide II D temperature induced shift of amide Iprime

1191KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

analysing the absorbance peak resulting from symmetric CH2

stretching vibrations at wave numbers near 2852 cmminus1 (Fig 3A)The internal vibrational modes of the lipid acyl chains wereassigned based on studies of polymethylenes and poly-methylene chain compounds [3334] In the region from2800 cmminus1 to 3100 cmminus1 there are infrared absorption bandsdue to symmetric and anti-symmetric modes of themethylene chain at about 2850 cmminus1 and 2920 cmminus1respectively The frequency of the absorption maximum ofthese bands is conformation sensitive and thus respond totemperature induced changes of the transgauche ratio in acylchains The vibrational mode (asymmetric stretch) of theterminal CH3 group has a characteristic frequency of2960 cmminus1 [15]

Fig 3B depicts the spectral information of the amide A andHOD-stretching bands Amide A is due to the NndashH-stretchingvibration band at frequencies ranging from 3300 cmminus1 to3400 cmminus1 [3235] In aqueous D2O solution the HOD-stretching band is masking the amide A band However themaximum frequency linearly increased with increasing temper-ature with a slope of 0716 (cmtimesdegC)minus1 in the case of 2H2O-sucrose and of 0702 (cmtimesdegC)minus 1 for 2H2O Therefore this peakserved as an internal thermometer of the sample (Fig 3B)

The amide II band (sim1550 cmminus1 NH) and its correspondingamide IIprime band (sim1455 cmminus1 ND) record the hydrogen-3-(1H2H) exchange in proteins (Fig 3C) [3637] The 1H2Hexchange provides information about the stability and flexibilityof proteins because the 1H2H exchange rates strongly depend

on the local environment of the protons The hydrogen ions ofan un-structured part of the protein exchange faster than thoseprotons protected from the aqueous environment [38] Thespectral range shown in Fig 3C is sensitive to conformationaltransitions in proteins as reflected by the HndashD exchange[173940] The intensity of the amide IIprime band at sim1455 cmminus1

grows with increasing HndashD exchange and a correspondingdecrease of the intensity of the amide II peak at sim1550 cmminus1 isgenerally observed Corresponding shifts in the amide II andamide IIprime bands with increasing temperature were also apparentin the IR spectra of L lactis (Fig 3C)

Fig 3D depicts the amide Iprime band (1600 cmminus1 to 1700 cmminus1)measured at different temperatures Spectral data in this rangeprovide information on the secondary structure of a protein TheC_O-stretching vibrations contribute 70ndash85 to the amide Iprimeintensity 10ndash20 involve the CndashN-stretching vibration [35]The frequency of amide Iprime decreases with the strength ofhydrogen bonding of the C_O acceptor [4142] Unfoldedproteins exhibit a broad amide Iprime band at approximately1645 cmminus1 In contrast spectra from aggregated proteinsdisplay a strong absorption band near 1620 to 1615 cmminus1 and aminor component at 1683 to 1680 cmminus1 reflecting anti-symmetric β-sheets Often protein unfolding at high tempera-ture will result in extensive aggregation accompanied by theformation intermolecular β-pleated sheets [43] Thus aggre-gated proteins can be distinguished from unfolded ones Withthe second derivative of the displayed absorbance spectra aclassification of the secondary structural changes in proteins

Fig 4 Vibrational frequencies for the CH2 symmetric stretch of membranelipids of L lactis cells in 2H2O () and 2H2O-sucrose () as a function oftemperature The solid line represents the fit using a sigmoidal function with 4parameters to describe the temperature shift of the membrane from the gel to theliquid crystalline phase

1192 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

is possible (Table 2) As a result of heat induced conformationalchanges ordered structures are shifted to unordered structuresby thermal treatment [42]

33 Sucrose effects on membrane phase transitions

FT-IR spectra of L lactis were recorded using stationaryphase cells re-suspended in 2H2O or 2H2O-sucrose Mem-brane phase transitions were detected by measuring thetemperature induced shift of the symmetric CH2 absorbancepeak maxima in the presence of 2H2O or 2H2O-sucrose (Fig4) If sucrose was added to 2H2O the peak was shifted tolower frequencies Therefore a difference in the frequencyresponse was observed between 2H2O and 2H2O-sucrose Theosmolyte induced a difference of 1 cmminus1 and 2 5 cmminus1 atlow and high temperatures respectively as was previouslyreported [1718] The temperature-induced shift of the peakmaxima to higher frequencies corresponds to ldquomeltingrdquo of theacyl chains of the phospholipids and the high conformationaldisorder of the phospho-lipids at high temperatures [44] ForL lactis suspended in 2H2O the phase transition was detectedbetween 7 degC (gel phase) and 35 degC (liquid crystalline phase)If sucrose was added to 2H2O the phase transition occurredbetween 8 degC and 30 degC (Fig 4)

34 Detection of the temperature-dependent conformationalchanges in the protein structure by the use of FT-IRspectroscopy

FT-IR spectra were recorded in the temperature range from30 degC to 65 degC and from 30 degC to 75 degC in 2H2O and 2H2O-sucrose respectively The temperature range was chosen toinclude the optimal temperature of growth of L lactis and thetemperatures that allowed a complete inactivation of thepopulation in less than 2 min (Figs 1 and 2)

35 Analysis of the amide IIprime- and the amide II band

The amide II band and its corresponding amide IIprime bandare sensitive indicators of hydrogen-deuterium (1H2H)exchange in proteins To highlight the temperature induced1H2H exchange in L lactis proteins difference spectra weregenerated by subtracting data obtained at 30 degC from thoseobtained at elevated temperatures (Fig 5) The centralfrequency corresponding to the amide II and amide IIprime

Table 2Protein secondary structure elements and the corresponding peak frequencies inabsorption FT-IR spectra

Structure element Wave number (cmminus1) Width (cmminus1)

α-helix sim1652 1649ndash1653β-sheet sim1681 1680ndash1683β-sheet sim1636 ndashβ-sheet sim1618 ndashTurn sim1687 ndashRandom coil sim1658 ndashUnordered sim1642 ndash

bands changed significantly with increase in temperature Theprotonated fraction (1550 cmminus1) decreased with increasingtemperature The amide IIprime band at 1450 cmminus1 representingthe deuterated fraction of proteins increased with increasingtemperature These spectral changes were observed in both

Fig 5 Difference FT-IR spectra of amide II and amide IIprime region (1360 cmminus1 to1600 cmminus1) generated by subtraction of the spectrum at 30 degC from the spectra atelevated temperatures A data generated in 2H2O B data generated in 2H2O-sucrose

1193KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

solvents 2H2O and 2H2O-sucrose however in the presenceof sucrose higher temperatures were needed to induce thehydrogen exchange To visualise the effect of sucrose on the1H2H exchange in L lactis proteins the area ratio of the amideII band (1510 cmminus1 to 1590 cmminus1) relative to the correspondingband areas at 30 degC is shown in Fig 6 The amide II peak area isdecreasing due to the increasing 1H2H exchange and acorresponding increase of the amide IIprime peak areas (1410 cmminus1

to 1490 cmminus1) was apparent (data not shown) The difference inthe relative peak areas suggests an enhanced 1H2H exchange in2H2O compared to 2H2O-sucrose For example in 2H2O adecrease of the protonated fraction of about 33 is observed at65degC whereas only a reduction of about 20 is seen with cellssuspended in 2H2O-sucrose

Fig 7 Difference FT-IR spectra of the amide Iprime region (1600 cmminus1 to 1700 cmminus1)generated by subtraction of the spectrum at 30 degC from the spectra at elevatedtemperatures A measurement data generated in 2H2O B measurement datagenerated in 2H2O-sucrose

36 Analysis of the amide Iprime band

The amide Iprime region detected in L lactis shows a broadand asymmetric band with the maximum around located near1645 cmminus1 (Figs 3D and 8) To emphasise temperaturedependent changes difference FT-IR spectra were calculatedby subtracting the spectrum measured at 30 degC from thespectra obtained at elevated temperatures (Fig 7) For Llactis suspended in 2H2O spectral changes were detected attemperatures above 45 degC whereas temperatures above 50 degCwere required to induce spectral changes with L lactis in2H2O-sucrose (Fig 7) This result suggests sucrose enhancesthe thermal stability of intra-cellular proteins

The peak at sim1645 cmminus1 derives from to α-helices andpolypeptide chains with random structure (Table 2) [3235414248] The temperature-induced changes of the spectrasuggest a decrease of the structural elements detected withinthis range of frequency with increase in temperature Theemerging new bands around 1620 cmminus1 and 1683 cmminus1 athigh temperatures reflect the formation of a hydrogen-bonded

Fig 6 Temperature effect on the amide II peak areas at elevated temperaturerelative to the amide II peak area at 30 degC Amide II peak areas were calculatedfor wave numbers ranging from 1510 cmminus1 to 1590 cmminus1 for data generated in2H2O () and 2H2O-sucrose ()

β-sheet structure leading to an irreversible re-association ofunfolded peptide segments (Table 2) [45ndash47]

A second-derivative-analysis of the amide Iprime regionprovided a more detailed decomposition of various second-ary structure elements in L lactis proteins (Table 2) Acomparison of the spectra of the amide Iprime region and thecorresponding second derivative spectra recorded at 32 degCand 74 degC is shown in Fig 8 Conformational changes thatwere induced by a temperature up-shift indicate theformation of β-sheet-structures at sim1683 cmminus1 (Fig 8)The new bands at 1658 cmminus1 and 1642 cmminus1 emerging athigh temperatures suggest a growing fraction of disorderedsegments Moreover the second derivative analysis reveals aloss in α-helical structures (1649 cmminus1) and a gain in (inter-molecular) β-sheet-structures (1618 cmminus1)

4 Discussion

The thermal stability of proteins can be modifiedintrinsically by altering the sequence amino acids orextrinsically by suitable stabilising effectors eg osmolytesThe general mechanism of protein stabilisation by solutesand carbohydrates in particular is the exclusion of solutefrom the protein domain [23] The ability of carbohydratesto stabilise proteins has been attributed to this preferentialhydration of proteins [212249] Bacteria are required to

Fig 8 Amide Iprime band of the infrared spectra obtained from L lactis at 32 degC and75 degC corresponding to viable and inactivated cells respectively The FT-IRspectra (A) and their second derivatives (B) were recorded in 2H2O-sucrose

Fig 9 Temperature effect on the different parameters describing thermalinactivation data of bacterial cell suspensions and temperature inducedconformational changes of the protein structure A intensity of the amide IIprimeband (1440 cmminus1) at elevated temperatures corrected for the peak intensity at30 degC B intensity of the amide Iprime band (1620 cmminus1) at elevated temperaturescorrected for the peak intensity at 30 degC C rate constants k describing lethal ()and sublethal () injury of the bacteria The filled symbols represent datagenerated in 2H2O-sucrose the empty symbols represent data generated in2H2O

1194 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

adapt to osmotic up-shift by accumulating compatible solutes[1213] This accumulation of solutes equilibrates theintracellular osmotic pressure to the environmental osmoticpressure and additionally provides protection to other stressconditions such as heat freezing drying and highhydrostatic pressure [56151718] The protective effect ofcompatible solutes on bacterial survival was previouslycorrelated with the protective effect of solutes on thebacterial membrane [151729] This study confirmed aprotective effect of sucrose on the bacterial survival andcytoplasmic membranes but the membrane phase transitionoccurred in the temperature range of 5ndash30 degC and thusappeared to be unrelated to the heat inactivation of L lactis

Conformational changes in the proteins of L lactis weredetermined with FT-IR spectroscopy The data obtained inthis work with L lactis are in excellent agreement with thespectra of purified proteins [32] and correspond to previousFT-IR measurements on bacterial cells [17]

The analysis of heat induced conformational changes ofthe proteins of L lactis enables a comparison of thetemperature effects on protein structure with bacterial survivaland sublethal injury (Fig 9) The difference in the intensitiesof the amide II and IIprime peaks (1550 cmminus1 and 1440 cmminus1

respectively) at elevated temperature compared to the peakintensity at 30 degC were used as an index for heat-induced1H2H exchange The buried residues of the protein structurebecame accessible to deuterium upon temperature up-shift andwere exchanged by the surrounding 2H2O (Figs 5 9A)[36373940] Modifications of the secondary structure of theproteins were detected by analysis of the amide Iprime band (Figs7 and 9B) To obtain a rate constant as an indicator for thetemperature effects on bacterial viability the experimentaldata on bacterial inactivation was fitted to a first orderequation (Fig 9C) Although first order models may beinadequate to describe inactivation curves with pronouncednon-linearities they have been widely used to describe

1195KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

thermal death time data and provided a reasonable agreementto our experimental data (correlation coefficient r2 gt087) Incontrast to more elaborate models that take into account non-linear survivor curves a simple first order model allows therepresentation of the inactivation kinetics by a singleparameter the rate constant k and thus greatly facilitatesthe comparison with the FT-IR data on protein structureHowever it should be stressed that the extensive washingsteps that were required to replace 1H2O by 2H2O for FT-IRspectroscopy of the cultures likely affected various aspects ofbacterial physiology

In the presence of sucrose the heat-induced structuralchanges in L lactis proteins detected by the amide Iprime and theamide IIprime bands were shifted to higher temperatures by about10 degC Inhibition of the 1H2H exchange by sucrose was moreprominent at lethal temperatures above 50 degC compared toambient temperatures of 30ndash45 degC (Figs 6 and 9) Thepresence of high (intracellular) sucrose concentration thusincreased the stability of L lactis proteins at denaturingtemperature Furthermore the bacterial viability was protectedby sucrose A temperature increase of about 10 degC wasrequired to achieve the same lethal effect in milk buffer sucrosecompared to milk buffer (Fig 9C)

The heat induced conformational changes of the L lactisproteins are related to the survival of this organism at lethaltemperatures No conformational changes in the L lactisproteins were detected by analysis of the amide Iprime band atsublethal temperatures The onset of conformational changescould be located between 50 and 60 degC in 2H2O and 2H2O-sucrose respectively These temperatures correspond to thelowest temperatures at which substantial inactivation of Llactis were detected in milk buffer and milk buffer withsucrose respectively (compare Fig 9B and C) Temperatureup-shift induced an increase of structural disorder andintermolecular β-sheet-structures in L lactis proteins a clearsign of irreversible protein aggregation and a decrease ofordered structural components mainly α-helices A moredetailed interpretation of the conformational changes ofprotein secondary structures is not feasible because thecombined spectra of the more than 2000 proteins expressedby L lactis [50] were determined and the structural changesof individual proteins overlap These results indicate heat-induced protein denaturation to an extent which is beyondthe cellular capacity of protein repair or de-novo synthesisand hence contributes to cell death The presence ofintracellular unordered or denatured proteins is attributableto heat denaturation of existing proteins or mis-translationoccurring at the ribosome Ribosome denaturation inEscherichia coli occurs in the range of 50ndash90 degC and waspreviously identified as a direct cause for heat-induced celldeath [51]

The 1H2H exchange was more susceptible to temperaturethan the spectral bands relating to protein secondarystructure Remarkably an increased 1H2H exchange wasobserved already in the range of 32ndash42 degC which is abovethe optimal temperature of growth but below the maximumgrowth temperature of L lactis This indicates that

conformational instabilities of proteins that may interferewith their respective functions occur already at temperaturespermitting growth of the organisms In keeping with thisinterpretation the primary response of L lactis to temper-ature upshift is the overexpression of heat shock proteinsincluding the DnaK-GrpE-DnaJ and the GroELS chaperonecomplexes [5253] In Lactobacillus sakei the induction ofheat shock proteins was apparent already at a temperature afew degrees above the optimum temperature of growth [54]The molecular chaperones are highly conserved families ofproteins that maintain or restore native protein conformationeven at high temperatures or assist in the degradation ofunfolded and aggregated proteins [55] As the ribosome hasbeen described as (heat) stress sensor and ribosome functionis coupled to protein quality control via trans-translation theexpression of chaperons is also a reaction to impairedtranslation by stalled ribosomes at sublethal stress tempera-tures [356]

In conclusion in this work demonstrated that molarconcentrations of sucrose increase the heat resistance of Llactis This finding extends previous findings on the role ofcompatible solutes for the bacterial resistance to freezingdrying and heat Additionally it demonstrates that highintracellular sucrose concentrations prevent the denaturationor misfolding of L lactis proteins Remarkably the use ofFT-IR spectroscopy enabled the detection of proteinconformational instabilities occurring already at elevatedtemperatures within the growth range of L lactis It isanticipated that these results will facilitate the process designfor thermal treatments to eliminate or stabilise bacterial cellsin food and biomedical applications

Acknowledgements

We gratefully acknowledge the support of DeutscheForschungsgemeinschaft Grant No FOR 3582 and SFB 533B11 and we wish to thank Gesa Groumlhnke for her excellentlaboratory work

References

[1] CE Dodd TG Aldsworth The importance of RpoS in the survival ofbacteria through food processing Int J Food Microbiol 74 (2002)189ndash194

[2] JD Taylor-Robinson M Child R Pickup P Strike C Edwards Cellndashcell interactions influence resistance and survival of Salmonella serotypeTyphimurium to environmental stress J Appl Microbiol 94 (2003)95ndash102

[3] M De Angelis R Di Cagno C Huet C Crecchio PF Fox M GobbettiHeat shock response in Lactobacillus plantarum Appl EnvironMicrobiol 70 (2004) 1336ndash1346

[4] CW Blackburn LM Curtis L Humpheson C Billon PJ McClureDevelopment of thermal inactivation models for Salmonella enteritidis andEscherichia coli O157H7 with temperature pH and NaCl as controllingfactors Int J Food Microbiol 38 (1997) 31ndash44

[5] KLMattick F Jorgensen JD Legan HM Lappin-Scott TJ HumphreyHabituation of Salmonella spp at reduced water activity and its effect onheat tolerance Appl Environ Microbiol 66 (2000) 4921ndash4925

1196 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

[6] L Coroller I Leguerinel P Mafart Effect of water activities of heatingand recovery media on apparent heat resistance of Bacillus cereus sporesAppl Environ Microbiol 67 (2001) 317ndash322

[7] C Laroche P Gervais Unexpected thermal destruction of dried glassbead-immobilized microorganisms as a function of water activity ApplEnviron Microbiol 69 (2003) 3015ndash3019

[8] EY Wuytack LD Phuong A Aertsen KM Reyns D Marquenie BDe Ketelaere B Masschalck I Van Opstal AM Diels CW MichielsComparison of sublethal injury induced in Salmonella enterica serovarTyphimurium by heat and by different nonthermal treatments J Food Prot66 (2003) 31ndash37

[9] RC Williams SS Sumner DA Golden Survival of Escherichiacoli O157H7 and salmonella in apple cider and orange juice asaffected by ozone and treatment temperature J Food Prot 67 (2004)2381ndash2386

[10] TB Hansen S Knochel Factors influencing resuscitation and growth ofheat injured Listeria monocytogenes 13-249 in sous vide cooked beef IntJ Food Microbiol 63 (2001) 135ndash147

[11] A Ikari M Nakano M Ishibashi K Kawano Y Suketa H Harada KTakagi Recovery from heat shock injury by activation of Na+ndashglucosecotransporter in renal epithelial cells Biochim Biophys Acta 1643 (2003)47ndash53

[12] E Glaasker FS Tjan PF Ter Steeg WN Konings B PoolmanPhysiological response of Lactobacillus plantarum to salt and nonelec-trolyte stress J Bacteriol 180 (1998) 4718ndash4723

[13] J Palmfeldt P Radstrom B Hahn-Hagerdal Optimisation of initial cellconcentration enhances freeze-drying tolerance of Pseudomonas chloro-raphis Cryobiology 47 (2003) 21ndash29

[14] MF Roberts Organic compatible solutes of halotolerant and halophilicmicroorganisms Saline Sys 1 (2005) 5

[15] AMolina-HoumlppnerWDosterRFVogelMGGaumlnzleProtectiveeffectofsucroseandsodiumchlorideforLactococcuslactisduringsublethalandlethalhigh-pressuretreatmentsApplEnvironMicrobiol70(2004)2013ndash2020

[16] G Zhao G Zhang Effect of protective agents freezing temperaturerehydration media on viability of malolactic bacteria subjected to freeze-drying J Appl Microbiol 99 (2005) 333ndash338

[17] LJM Linders WF Wolkers FA Hoekstra K Vant Riet Effect ofadded carbohydrates on membrane phase behavior and survival of driedLactobacillus plantarum Cryobiology 35 (1997) 31ndash40

[18] Y Mille L Beney P Gervais Viability of Escherichia coli after combinedosmotic and thermal treatment a plasma membrane implication BiochimBiophys Acta 1567 (2002) 41ndash48

[19] TJ Ahern AM Klibanov The mechanisms of reversible enzymeinactivation at 100 degC Science 228 (1985) 1280ndash1284

[20] GN Somero Proteins and temperature Annu Rev Physiol 57 (1995)43ndash68

[21] M Sola-Penna JR Meyer-Fernades Protective role of trehalose inthermal denaturation of yeast pyrophosphatase Z Naturforsch 49c (1994)327ndash330

[22] J Saad-Nehme AL Bezerra LA Fornells JL Silva JR Meyer-Fernandes A contribution of the mitochondrial adenosinetriphosphataseinhibitor protein to the thermal stability of the F0F1ndashATPase complexZ Naturforsch 52c (1997) 459ndash465

[23] SN Timasheff The control of protein stability and association by weak-interactions with watermdashHow do solvents affect these processes AnnuRev Biophys Biomol Struct 22 (1993) 67ndash97

[24] H-U Gremlich B Yan Infrared and raman spectroscopy of biologicalmaterials Marcel Dekker New York 2000

[25] H Fabian W Mantele Infrared spectroscopy of proteins Handbook ofVibrational Spectroscopy vol 5 John Wiley and Sons Chichester 2002pp 3424ndash3999

[26] K Murayama M Tomida Heat-induced secondary structure andconformation change of bovine serum albumin investigated by Fouriertransform infrared spectroscopy Biochemistry 43 (2004) 11526ndash11532

[27] SM Choi CY Ma Conformational study of globulin from commonbuckwheat (Fagopyrum esculentum Moench) by Fourier transforminfrared spectroscopy differential scanning calorimetry J Agric FoodChem 53 (2005) 8046ndash8053

[28] C Dirix T Duvetter A Van Loey M Hendrickx K Heremans The insitu observation of the temperature and pressure stability of recombinantAspergillus aculeatus pectin methylesterase with Fourier transforminfrared spectroscopy reveals an unusual pressure stability of beta-helicesBiochem J 392 (2005) 565ndash571

[29] SB Leslie E Israeli B Lighthart JH Crowe LM Crowe Trehaloseand sucrose protect both membranes and proteins in intact bacteria duringdrying Appl Environ Microbiol 61 (1995) 3592ndash3597

[30] A Molina-Houmlppner Physiological response of Lactococcus lactis to highpressure Doctoral thesis Technische Universitaumlt Muumlnchen Centre for Lifeand Food Sciences Weihenstephan 2002

[31] JM Jay MJ Loessner DA Golden (Eds) Modern food microbiologySpringer Berlin 2005

[32] A Barth C Zscherp What vibrations tell us about proteins Q RevBiophys 35 (2002) 369ndash430

[33] GR Snyder Vibrational spectra of crystalline n-paraffins J MolSpectrosc 7 (1961) 116ndash144

[34] O Reis R Winter TW Zerda The effect of high external pressure onDPPC-cholesterol multilamellar vesicles a pressure-tuning Fouriertransform infrared spectroscopy study Biochim Biophys Acta 1279(1996) 5ndash16

[35] Anonymous Determination of secondary structure in proteins by Fouriertransform infrared spectroscopy (FTIR) imb Jena without publishingDate download 27022005 URL httpwwwimb-jenadeImgLibDocftirIMAGE_FTIRhtml

[36] TM Raschke S Marqusee Hydrogen exchange studies of proteinstructure Curr Opin Biotechnol 9 (1998) 80ndash86

[37] E Goormaghtigh V Raussens JM Ruysschaert Attenuated totalreflection infrared spectroscopy of proteins and lipids in biologicalmembranes Biochim Biophys Acta 1422 (1999) 105ndash185

[38] H Susi The strength of hydrogen bonding infrared spectroscopyMethods A Compan Methods Enzymol 26 (Pt C) (1972) 381ndash391

[39] L Smeller P Rubens J Frank J Fidy K Heremans Two-dimensional Fourier-transform infrared correlation spectroscopy studyof the high-pressure tuning of proteins Vibr Spectrosc 22 (2000)119ndash125

[40] L Smeller F Meersman J Fidy K Heremans High-pressure FTIR studyof the stability of horseradish peroxidase Effect of heme substitutionligand binding Ca++ removal and reduction of the disulfide bondsBiochemistry 42 (2002) 553ndash561

[41] N Takeda M Kato Y Taniguchi Pressure-induced secondary structurechanges of ribonuclease A and ribonuclease S studied by FTIRspectroscopy Biospectroscopy 1 (1995) 207ndash216

[42] N Takeda M Kato Y Taniguchi Pressure and thermally-inducedreversible changes in the secondary structure of ribonuclease A studied byFTIR spectroscopy Biochemistry 34 (1995) 5980ndash5987

[43] M Jackson HH Mantsch Beware of proteins in DMSO BiochimBiophys Acta 1078 (1991) 231ndash235

[44] HM Ulmer H Herberhold S Fahsel MG Gaumlnzle R Winter RF VogelEffects of pressure-induced membrane phase transitions on inactivation ofHorA an ATP dependent multidrug resistance transporter in Lactobacillusplantarum Appl Environ Microbiol 68 (2002) 1088ndash1095

[45] M Jackson PI Haris D Chapman Fourier transform infraredspectroscopic studies of Ca(2+)-binding proteins Biochemistry 30(1991) 9681ndash9686

[46] S Luo CF Huang JF McClelland JD Graves A study of proteinsecondary structure by Fourier transform infraredphotoacoustic spectros-copy and its application for recombinant proteins Anal Biochem 216(1994) 67ndash76

[47] K Goosens L Smeller J Frank K Heremans Pressure-tuning theconformation of bovine pancreatic trypsin inhibitor studied by Fourier-transform infrared spectroscopy Eur J Biochem 236 (1996) 236ndash262

[48] R Gilmanshin M Gulotta RB Dyer RH Callender Structures ofapomyoglobins various acid-destabilized forms Biochemistry 40 (2001)5127ndash5136

[49] JH Crowe LM Crowe D Chapman Preservation of membranes inanhydrobiotic organismsmdashThe role of trehalose Science 223 (1984)701ndash703

1190 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

increments was chosen and process times ranged from 0 min to120 min In Fig 1 the thermal death time data of L lactis in milkbuffer are depicted Sub-lethal injury was detected afterincubation at 45 degC and lethal injury was apparent attemperature levels of 50 degC or higher After treatment at65 degC and 70 degC the cell counts were below the detection limitafter 4 min and less than 2 min respectively A processtemperature of 65 degC and a process time of 2 min resulted insub-lethal injury of all surviving cells

Thermal death time data of L lactis in milk buffer sucroseare shown in Fig 2 In general a protective effect for bothphysiological states was detected compared to the inactivationof L lactis in milk buffer For example a difference in the cellcounts of more than 6 log is apparent when treatments at 50 degCand 120 min in milk buffer and milk buffer with sucrose arecompared Nevertheless the cell counts of L lactis in milkbuffer sucrose were below the detection limit after 2 min at75 degC (Fig 2)

32 Analysis of infrared spectra

To assess the suitability of FT-IR spectroscopy for analysisof structural properties of cellular proteins spectra obtainedfrom microbial populations were compared with literature

Fig 1 Comparison of experimental and modelling results concerning lethal(CFU A) and sublethal (CFUsub B) injury of L lactis after a treatment in milkbuffer at various temperatures 40 degC () 45 degC () 50 degC () 55 degC ()60 degC () 65 degC () 70 degC (diams) and 75 degC (⋄) Lines represent the predictionof cell counts by first order kinetics Lines dropping below the x-axis indicatecell counts below the detection limit of 100 CFU mlminus1

Fig 2 Comparison of experimental and modelling results concerning lethal(CFU A) and sublethal (CFUsub B) injury of L lactis after a treatment in milkbuffer sucrose at various temperatures 40 degC () 45 degC () 50 degC () 55 degC() 60 degC () 65 degC () 70 degC (diams) and 75 degC (⋄) Lines represent theprediction of cell counts by first order kinetics Lines dropping below the x-axisindicate cell counts below the detection limit of 100 CFU mlminus1

spectra of purified proteins in aqueous solution An overviewof the bands in L lactis relevant to protein or membranestructure their assignment the location of their absorptionmaxima as well as their wave number range is given in Table1 the corresponding spectral range is shown in Fig 3 Thespectra measured with L lactis suspended in 2H2O comprisedthe same characteristics as the spectra measured in 2H2O-sucrose (Fig 3 and data not shown)

Temperature induced shifts of the spectra providedinformation about structural changes in the membrane andin proteins induced by increasing the temperature (Table 1)[1732] Membrane-phase transitions were detected by

Table 1Common range of the characteristic bands of the secondary structure of proteinsdetected by FT-IR in L lactis

Band Wave number (cmminus1) Width (cmminus1)

Amide A+HOD sim3400 3200ndash3800Symmetric CH2 sim2852 ndashAmide Iprime sim1652 1600ndash1700Amide II sim1550 ndashTyrosine sim1515 ndashSide chains ndash 1500ndash1600Amide IIprime sim1455 1350ndash1500

Fig 3 Segments of the FT-IR spectra of L lactis in 2H2O-sucrose used for the secondary structure analysis of proteins and the lipid acyl chains A temperature inducedshift of CH2 B temperature induced shift of amide A+HOD C temperature induced shift of amide IIprime and amide II D temperature induced shift of amide Iprime

1191KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

analysing the absorbance peak resulting from symmetric CH2

stretching vibrations at wave numbers near 2852 cmminus1 (Fig 3A)The internal vibrational modes of the lipid acyl chains wereassigned based on studies of polymethylenes and poly-methylene chain compounds [3334] In the region from2800 cmminus1 to 3100 cmminus1 there are infrared absorption bandsdue to symmetric and anti-symmetric modes of themethylene chain at about 2850 cmminus1 and 2920 cmminus1respectively The frequency of the absorption maximum ofthese bands is conformation sensitive and thus respond totemperature induced changes of the transgauche ratio in acylchains The vibrational mode (asymmetric stretch) of theterminal CH3 group has a characteristic frequency of2960 cmminus1 [15]

Fig 3B depicts the spectral information of the amide A andHOD-stretching bands Amide A is due to the NndashH-stretchingvibration band at frequencies ranging from 3300 cmminus1 to3400 cmminus1 [3235] In aqueous D2O solution the HOD-stretching band is masking the amide A band However themaximum frequency linearly increased with increasing temper-ature with a slope of 0716 (cmtimesdegC)minus1 in the case of 2H2O-sucrose and of 0702 (cmtimesdegC)minus 1 for 2H2O Therefore this peakserved as an internal thermometer of the sample (Fig 3B)

The amide II band (sim1550 cmminus1 NH) and its correspondingamide IIprime band (sim1455 cmminus1 ND) record the hydrogen-3-(1H2H) exchange in proteins (Fig 3C) [3637] The 1H2Hexchange provides information about the stability and flexibilityof proteins because the 1H2H exchange rates strongly depend

on the local environment of the protons The hydrogen ions ofan un-structured part of the protein exchange faster than thoseprotons protected from the aqueous environment [38] Thespectral range shown in Fig 3C is sensitive to conformationaltransitions in proteins as reflected by the HndashD exchange[173940] The intensity of the amide IIprime band at sim1455 cmminus1

grows with increasing HndashD exchange and a correspondingdecrease of the intensity of the amide II peak at sim1550 cmminus1 isgenerally observed Corresponding shifts in the amide II andamide IIprime bands with increasing temperature were also apparentin the IR spectra of L lactis (Fig 3C)

Fig 3D depicts the amide Iprime band (1600 cmminus1 to 1700 cmminus1)measured at different temperatures Spectral data in this rangeprovide information on the secondary structure of a protein TheC_O-stretching vibrations contribute 70ndash85 to the amide Iprimeintensity 10ndash20 involve the CndashN-stretching vibration [35]The frequency of amide Iprime decreases with the strength ofhydrogen bonding of the C_O acceptor [4142] Unfoldedproteins exhibit a broad amide Iprime band at approximately1645 cmminus1 In contrast spectra from aggregated proteinsdisplay a strong absorption band near 1620 to 1615 cmminus1 and aminor component at 1683 to 1680 cmminus1 reflecting anti-symmetric β-sheets Often protein unfolding at high tempera-ture will result in extensive aggregation accompanied by theformation intermolecular β-pleated sheets [43] Thus aggre-gated proteins can be distinguished from unfolded ones Withthe second derivative of the displayed absorbance spectra aclassification of the secondary structural changes in proteins

Fig 4 Vibrational frequencies for the CH2 symmetric stretch of membranelipids of L lactis cells in 2H2O () and 2H2O-sucrose () as a function oftemperature The solid line represents the fit using a sigmoidal function with 4parameters to describe the temperature shift of the membrane from the gel to theliquid crystalline phase

1192 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

is possible (Table 2) As a result of heat induced conformationalchanges ordered structures are shifted to unordered structuresby thermal treatment [42]

33 Sucrose effects on membrane phase transitions

FT-IR spectra of L lactis were recorded using stationaryphase cells re-suspended in 2H2O or 2H2O-sucrose Mem-brane phase transitions were detected by measuring thetemperature induced shift of the symmetric CH2 absorbancepeak maxima in the presence of 2H2O or 2H2O-sucrose (Fig4) If sucrose was added to 2H2O the peak was shifted tolower frequencies Therefore a difference in the frequencyresponse was observed between 2H2O and 2H2O-sucrose Theosmolyte induced a difference of 1 cmminus1 and 2 5 cmminus1 atlow and high temperatures respectively as was previouslyreported [1718] The temperature-induced shift of the peakmaxima to higher frequencies corresponds to ldquomeltingrdquo of theacyl chains of the phospholipids and the high conformationaldisorder of the phospho-lipids at high temperatures [44] ForL lactis suspended in 2H2O the phase transition was detectedbetween 7 degC (gel phase) and 35 degC (liquid crystalline phase)If sucrose was added to 2H2O the phase transition occurredbetween 8 degC and 30 degC (Fig 4)

34 Detection of the temperature-dependent conformationalchanges in the protein structure by the use of FT-IRspectroscopy

FT-IR spectra were recorded in the temperature range from30 degC to 65 degC and from 30 degC to 75 degC in 2H2O and 2H2O-sucrose respectively The temperature range was chosen toinclude the optimal temperature of growth of L lactis and thetemperatures that allowed a complete inactivation of thepopulation in less than 2 min (Figs 1 and 2)

35 Analysis of the amide IIprime- and the amide II band

The amide II band and its corresponding amide IIprime bandare sensitive indicators of hydrogen-deuterium (1H2H)exchange in proteins To highlight the temperature induced1H2H exchange in L lactis proteins difference spectra weregenerated by subtracting data obtained at 30 degC from thoseobtained at elevated temperatures (Fig 5) The centralfrequency corresponding to the amide II and amide IIprime

Table 2Protein secondary structure elements and the corresponding peak frequencies inabsorption FT-IR spectra

Structure element Wave number (cmminus1) Width (cmminus1)

α-helix sim1652 1649ndash1653β-sheet sim1681 1680ndash1683β-sheet sim1636 ndashβ-sheet sim1618 ndashTurn sim1687 ndashRandom coil sim1658 ndashUnordered sim1642 ndash

bands changed significantly with increase in temperature Theprotonated fraction (1550 cmminus1) decreased with increasingtemperature The amide IIprime band at 1450 cmminus1 representingthe deuterated fraction of proteins increased with increasingtemperature These spectral changes were observed in both

Fig 5 Difference FT-IR spectra of amide II and amide IIprime region (1360 cmminus1 to1600 cmminus1) generated by subtraction of the spectrum at 30 degC from the spectra atelevated temperatures A data generated in 2H2O B data generated in 2H2O-sucrose

1193KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

solvents 2H2O and 2H2O-sucrose however in the presenceof sucrose higher temperatures were needed to induce thehydrogen exchange To visualise the effect of sucrose on the1H2H exchange in L lactis proteins the area ratio of the amideII band (1510 cmminus1 to 1590 cmminus1) relative to the correspondingband areas at 30 degC is shown in Fig 6 The amide II peak area isdecreasing due to the increasing 1H2H exchange and acorresponding increase of the amide IIprime peak areas (1410 cmminus1

to 1490 cmminus1) was apparent (data not shown) The difference inthe relative peak areas suggests an enhanced 1H2H exchange in2H2O compared to 2H2O-sucrose For example in 2H2O adecrease of the protonated fraction of about 33 is observed at65degC whereas only a reduction of about 20 is seen with cellssuspended in 2H2O-sucrose

Fig 7 Difference FT-IR spectra of the amide Iprime region (1600 cmminus1 to 1700 cmminus1)generated by subtraction of the spectrum at 30 degC from the spectra at elevatedtemperatures A measurement data generated in 2H2O B measurement datagenerated in 2H2O-sucrose

36 Analysis of the amide Iprime band

The amide Iprime region detected in L lactis shows a broadand asymmetric band with the maximum around located near1645 cmminus1 (Figs 3D and 8) To emphasise temperaturedependent changes difference FT-IR spectra were calculatedby subtracting the spectrum measured at 30 degC from thespectra obtained at elevated temperatures (Fig 7) For Llactis suspended in 2H2O spectral changes were detected attemperatures above 45 degC whereas temperatures above 50 degCwere required to induce spectral changes with L lactis in2H2O-sucrose (Fig 7) This result suggests sucrose enhancesthe thermal stability of intra-cellular proteins

The peak at sim1645 cmminus1 derives from to α-helices andpolypeptide chains with random structure (Table 2) [3235414248] The temperature-induced changes of the spectrasuggest a decrease of the structural elements detected withinthis range of frequency with increase in temperature Theemerging new bands around 1620 cmminus1 and 1683 cmminus1 athigh temperatures reflect the formation of a hydrogen-bonded

Fig 6 Temperature effect on the amide II peak areas at elevated temperaturerelative to the amide II peak area at 30 degC Amide II peak areas were calculatedfor wave numbers ranging from 1510 cmminus1 to 1590 cmminus1 for data generated in2H2O () and 2H2O-sucrose ()

β-sheet structure leading to an irreversible re-association ofunfolded peptide segments (Table 2) [45ndash47]

A second-derivative-analysis of the amide Iprime regionprovided a more detailed decomposition of various second-ary structure elements in L lactis proteins (Table 2) Acomparison of the spectra of the amide Iprime region and thecorresponding second derivative spectra recorded at 32 degCand 74 degC is shown in Fig 8 Conformational changes thatwere induced by a temperature up-shift indicate theformation of β-sheet-structures at sim1683 cmminus1 (Fig 8)The new bands at 1658 cmminus1 and 1642 cmminus1 emerging athigh temperatures suggest a growing fraction of disorderedsegments Moreover the second derivative analysis reveals aloss in α-helical structures (1649 cmminus1) and a gain in (inter-molecular) β-sheet-structures (1618 cmminus1)

4 Discussion

The thermal stability of proteins can be modifiedintrinsically by altering the sequence amino acids orextrinsically by suitable stabilising effectors eg osmolytesThe general mechanism of protein stabilisation by solutesand carbohydrates in particular is the exclusion of solutefrom the protein domain [23] The ability of carbohydratesto stabilise proteins has been attributed to this preferentialhydration of proteins [212249] Bacteria are required to

Fig 8 Amide Iprime band of the infrared spectra obtained from L lactis at 32 degC and75 degC corresponding to viable and inactivated cells respectively The FT-IRspectra (A) and their second derivatives (B) were recorded in 2H2O-sucrose

Fig 9 Temperature effect on the different parameters describing thermalinactivation data of bacterial cell suspensions and temperature inducedconformational changes of the protein structure A intensity of the amide IIprimeband (1440 cmminus1) at elevated temperatures corrected for the peak intensity at30 degC B intensity of the amide Iprime band (1620 cmminus1) at elevated temperaturescorrected for the peak intensity at 30 degC C rate constants k describing lethal ()and sublethal () injury of the bacteria The filled symbols represent datagenerated in 2H2O-sucrose the empty symbols represent data generated in2H2O

1194 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

adapt to osmotic up-shift by accumulating compatible solutes[1213] This accumulation of solutes equilibrates theintracellular osmotic pressure to the environmental osmoticpressure and additionally provides protection to other stressconditions such as heat freezing drying and highhydrostatic pressure [56151718] The protective effect ofcompatible solutes on bacterial survival was previouslycorrelated with the protective effect of solutes on thebacterial membrane [151729] This study confirmed aprotective effect of sucrose on the bacterial survival andcytoplasmic membranes but the membrane phase transitionoccurred in the temperature range of 5ndash30 degC and thusappeared to be unrelated to the heat inactivation of L lactis

Conformational changes in the proteins of L lactis weredetermined with FT-IR spectroscopy The data obtained inthis work with L lactis are in excellent agreement with thespectra of purified proteins [32] and correspond to previousFT-IR measurements on bacterial cells [17]

The analysis of heat induced conformational changes ofthe proteins of L lactis enables a comparison of thetemperature effects on protein structure with bacterial survivaland sublethal injury (Fig 9) The difference in the intensitiesof the amide II and IIprime peaks (1550 cmminus1 and 1440 cmminus1

respectively) at elevated temperature compared to the peakintensity at 30 degC were used as an index for heat-induced1H2H exchange The buried residues of the protein structurebecame accessible to deuterium upon temperature up-shift andwere exchanged by the surrounding 2H2O (Figs 5 9A)[36373940] Modifications of the secondary structure of theproteins were detected by analysis of the amide Iprime band (Figs7 and 9B) To obtain a rate constant as an indicator for thetemperature effects on bacterial viability the experimentaldata on bacterial inactivation was fitted to a first orderequation (Fig 9C) Although first order models may beinadequate to describe inactivation curves with pronouncednon-linearities they have been widely used to describe

1195KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

thermal death time data and provided a reasonable agreementto our experimental data (correlation coefficient r2 gt087) Incontrast to more elaborate models that take into account non-linear survivor curves a simple first order model allows therepresentation of the inactivation kinetics by a singleparameter the rate constant k and thus greatly facilitatesthe comparison with the FT-IR data on protein structureHowever it should be stressed that the extensive washingsteps that were required to replace 1H2O by 2H2O for FT-IRspectroscopy of the cultures likely affected various aspects ofbacterial physiology

In the presence of sucrose the heat-induced structuralchanges in L lactis proteins detected by the amide Iprime and theamide IIprime bands were shifted to higher temperatures by about10 degC Inhibition of the 1H2H exchange by sucrose was moreprominent at lethal temperatures above 50 degC compared toambient temperatures of 30ndash45 degC (Figs 6 and 9) Thepresence of high (intracellular) sucrose concentration thusincreased the stability of L lactis proteins at denaturingtemperature Furthermore the bacterial viability was protectedby sucrose A temperature increase of about 10 degC wasrequired to achieve the same lethal effect in milk buffer sucrosecompared to milk buffer (Fig 9C)

The heat induced conformational changes of the L lactisproteins are related to the survival of this organism at lethaltemperatures No conformational changes in the L lactisproteins were detected by analysis of the amide Iprime band atsublethal temperatures The onset of conformational changescould be located between 50 and 60 degC in 2H2O and 2H2O-sucrose respectively These temperatures correspond to thelowest temperatures at which substantial inactivation of Llactis were detected in milk buffer and milk buffer withsucrose respectively (compare Fig 9B and C) Temperatureup-shift induced an increase of structural disorder andintermolecular β-sheet-structures in L lactis proteins a clearsign of irreversible protein aggregation and a decrease ofordered structural components mainly α-helices A moredetailed interpretation of the conformational changes ofprotein secondary structures is not feasible because thecombined spectra of the more than 2000 proteins expressedby L lactis [50] were determined and the structural changesof individual proteins overlap These results indicate heat-induced protein denaturation to an extent which is beyondthe cellular capacity of protein repair or de-novo synthesisand hence contributes to cell death The presence ofintracellular unordered or denatured proteins is attributableto heat denaturation of existing proteins or mis-translationoccurring at the ribosome Ribosome denaturation inEscherichia coli occurs in the range of 50ndash90 degC and waspreviously identified as a direct cause for heat-induced celldeath [51]

The 1H2H exchange was more susceptible to temperaturethan the spectral bands relating to protein secondarystructure Remarkably an increased 1H2H exchange wasobserved already in the range of 32ndash42 degC which is abovethe optimal temperature of growth but below the maximumgrowth temperature of L lactis This indicates that

conformational instabilities of proteins that may interferewith their respective functions occur already at temperaturespermitting growth of the organisms In keeping with thisinterpretation the primary response of L lactis to temper-ature upshift is the overexpression of heat shock proteinsincluding the DnaK-GrpE-DnaJ and the GroELS chaperonecomplexes [5253] In Lactobacillus sakei the induction ofheat shock proteins was apparent already at a temperature afew degrees above the optimum temperature of growth [54]The molecular chaperones are highly conserved families ofproteins that maintain or restore native protein conformationeven at high temperatures or assist in the degradation ofunfolded and aggregated proteins [55] As the ribosome hasbeen described as (heat) stress sensor and ribosome functionis coupled to protein quality control via trans-translation theexpression of chaperons is also a reaction to impairedtranslation by stalled ribosomes at sublethal stress tempera-tures [356]

In conclusion in this work demonstrated that molarconcentrations of sucrose increase the heat resistance of Llactis This finding extends previous findings on the role ofcompatible solutes for the bacterial resistance to freezingdrying and heat Additionally it demonstrates that highintracellular sucrose concentrations prevent the denaturationor misfolding of L lactis proteins Remarkably the use ofFT-IR spectroscopy enabled the detection of proteinconformational instabilities occurring already at elevatedtemperatures within the growth range of L lactis It isanticipated that these results will facilitate the process designfor thermal treatments to eliminate or stabilise bacterial cellsin food and biomedical applications

Acknowledgements

We gratefully acknowledge the support of DeutscheForschungsgemeinschaft Grant No FOR 3582 and SFB 533B11 and we wish to thank Gesa Groumlhnke for her excellentlaboratory work

References

[1] CE Dodd TG Aldsworth The importance of RpoS in the survival ofbacteria through food processing Int J Food Microbiol 74 (2002)189ndash194

[2] JD Taylor-Robinson M Child R Pickup P Strike C Edwards Cellndashcell interactions influence resistance and survival of Salmonella serotypeTyphimurium to environmental stress J Appl Microbiol 94 (2003)95ndash102

[3] M De Angelis R Di Cagno C Huet C Crecchio PF Fox M GobbettiHeat shock response in Lactobacillus plantarum Appl EnvironMicrobiol 70 (2004) 1336ndash1346

[4] CW Blackburn LM Curtis L Humpheson C Billon PJ McClureDevelopment of thermal inactivation models for Salmonella enteritidis andEscherichia coli O157H7 with temperature pH and NaCl as controllingfactors Int J Food Microbiol 38 (1997) 31ndash44

[5] KLMattick F Jorgensen JD Legan HM Lappin-Scott TJ HumphreyHabituation of Salmonella spp at reduced water activity and its effect onheat tolerance Appl Environ Microbiol 66 (2000) 4921ndash4925

1196 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

[6] L Coroller I Leguerinel P Mafart Effect of water activities of heatingand recovery media on apparent heat resistance of Bacillus cereus sporesAppl Environ Microbiol 67 (2001) 317ndash322

[7] C Laroche P Gervais Unexpected thermal destruction of dried glassbead-immobilized microorganisms as a function of water activity ApplEnviron Microbiol 69 (2003) 3015ndash3019

[8] EY Wuytack LD Phuong A Aertsen KM Reyns D Marquenie BDe Ketelaere B Masschalck I Van Opstal AM Diels CW MichielsComparison of sublethal injury induced in Salmonella enterica serovarTyphimurium by heat and by different nonthermal treatments J Food Prot66 (2003) 31ndash37

[9] RC Williams SS Sumner DA Golden Survival of Escherichiacoli O157H7 and salmonella in apple cider and orange juice asaffected by ozone and treatment temperature J Food Prot 67 (2004)2381ndash2386

[10] TB Hansen S Knochel Factors influencing resuscitation and growth ofheat injured Listeria monocytogenes 13-249 in sous vide cooked beef IntJ Food Microbiol 63 (2001) 135ndash147

[11] A Ikari M Nakano M Ishibashi K Kawano Y Suketa H Harada KTakagi Recovery from heat shock injury by activation of Na+ndashglucosecotransporter in renal epithelial cells Biochim Biophys Acta 1643 (2003)47ndash53

[12] E Glaasker FS Tjan PF Ter Steeg WN Konings B PoolmanPhysiological response of Lactobacillus plantarum to salt and nonelec-trolyte stress J Bacteriol 180 (1998) 4718ndash4723

[13] J Palmfeldt P Radstrom B Hahn-Hagerdal Optimisation of initial cellconcentration enhances freeze-drying tolerance of Pseudomonas chloro-raphis Cryobiology 47 (2003) 21ndash29

[14] MF Roberts Organic compatible solutes of halotolerant and halophilicmicroorganisms Saline Sys 1 (2005) 5

[15] AMolina-HoumlppnerWDosterRFVogelMGGaumlnzleProtectiveeffectofsucroseandsodiumchlorideforLactococcuslactisduringsublethalandlethalhigh-pressuretreatmentsApplEnvironMicrobiol70(2004)2013ndash2020

[16] G Zhao G Zhang Effect of protective agents freezing temperaturerehydration media on viability of malolactic bacteria subjected to freeze-drying J Appl Microbiol 99 (2005) 333ndash338

[17] LJM Linders WF Wolkers FA Hoekstra K Vant Riet Effect ofadded carbohydrates on membrane phase behavior and survival of driedLactobacillus plantarum Cryobiology 35 (1997) 31ndash40

[18] Y Mille L Beney P Gervais Viability of Escherichia coli after combinedosmotic and thermal treatment a plasma membrane implication BiochimBiophys Acta 1567 (2002) 41ndash48

[19] TJ Ahern AM Klibanov The mechanisms of reversible enzymeinactivation at 100 degC Science 228 (1985) 1280ndash1284

[20] GN Somero Proteins and temperature Annu Rev Physiol 57 (1995)43ndash68

[21] M Sola-Penna JR Meyer-Fernades Protective role of trehalose inthermal denaturation of yeast pyrophosphatase Z Naturforsch 49c (1994)327ndash330

[22] J Saad-Nehme AL Bezerra LA Fornells JL Silva JR Meyer-Fernandes A contribution of the mitochondrial adenosinetriphosphataseinhibitor protein to the thermal stability of the F0F1ndashATPase complexZ Naturforsch 52c (1997) 459ndash465

[23] SN Timasheff The control of protein stability and association by weak-interactions with watermdashHow do solvents affect these processes AnnuRev Biophys Biomol Struct 22 (1993) 67ndash97

[24] H-U Gremlich B Yan Infrared and raman spectroscopy of biologicalmaterials Marcel Dekker New York 2000

[25] H Fabian W Mantele Infrared spectroscopy of proteins Handbook ofVibrational Spectroscopy vol 5 John Wiley and Sons Chichester 2002pp 3424ndash3999

[26] K Murayama M Tomida Heat-induced secondary structure andconformation change of bovine serum albumin investigated by Fouriertransform infrared spectroscopy Biochemistry 43 (2004) 11526ndash11532

[27] SM Choi CY Ma Conformational study of globulin from commonbuckwheat (Fagopyrum esculentum Moench) by Fourier transforminfrared spectroscopy differential scanning calorimetry J Agric FoodChem 53 (2005) 8046ndash8053

[28] C Dirix T Duvetter A Van Loey M Hendrickx K Heremans The insitu observation of the temperature and pressure stability of recombinantAspergillus aculeatus pectin methylesterase with Fourier transforminfrared spectroscopy reveals an unusual pressure stability of beta-helicesBiochem J 392 (2005) 565ndash571

[29] SB Leslie E Israeli B Lighthart JH Crowe LM Crowe Trehaloseand sucrose protect both membranes and proteins in intact bacteria duringdrying Appl Environ Microbiol 61 (1995) 3592ndash3597

[30] A Molina-Houmlppner Physiological response of Lactococcus lactis to highpressure Doctoral thesis Technische Universitaumlt Muumlnchen Centre for Lifeand Food Sciences Weihenstephan 2002

[31] JM Jay MJ Loessner DA Golden (Eds) Modern food microbiologySpringer Berlin 2005

[32] A Barth C Zscherp What vibrations tell us about proteins Q RevBiophys 35 (2002) 369ndash430

[33] GR Snyder Vibrational spectra of crystalline n-paraffins J MolSpectrosc 7 (1961) 116ndash144

[34] O Reis R Winter TW Zerda The effect of high external pressure onDPPC-cholesterol multilamellar vesicles a pressure-tuning Fouriertransform infrared spectroscopy study Biochim Biophys Acta 1279(1996) 5ndash16

[35] Anonymous Determination of secondary structure in proteins by Fouriertransform infrared spectroscopy (FTIR) imb Jena without publishingDate download 27022005 URL httpwwwimb-jenadeImgLibDocftirIMAGE_FTIRhtml

[36] TM Raschke S Marqusee Hydrogen exchange studies of proteinstructure Curr Opin Biotechnol 9 (1998) 80ndash86

[37] E Goormaghtigh V Raussens JM Ruysschaert Attenuated totalreflection infrared spectroscopy of proteins and lipids in biologicalmembranes Biochim Biophys Acta 1422 (1999) 105ndash185

[38] H Susi The strength of hydrogen bonding infrared spectroscopyMethods A Compan Methods Enzymol 26 (Pt C) (1972) 381ndash391

[39] L Smeller P Rubens J Frank J Fidy K Heremans Two-dimensional Fourier-transform infrared correlation spectroscopy studyof the high-pressure tuning of proteins Vibr Spectrosc 22 (2000)119ndash125

[40] L Smeller F Meersman J Fidy K Heremans High-pressure FTIR studyof the stability of horseradish peroxidase Effect of heme substitutionligand binding Ca++ removal and reduction of the disulfide bondsBiochemistry 42 (2002) 553ndash561

[41] N Takeda M Kato Y Taniguchi Pressure-induced secondary structurechanges of ribonuclease A and ribonuclease S studied by FTIRspectroscopy Biospectroscopy 1 (1995) 207ndash216

[42] N Takeda M Kato Y Taniguchi Pressure and thermally-inducedreversible changes in the secondary structure of ribonuclease A studied byFTIR spectroscopy Biochemistry 34 (1995) 5980ndash5987

[43] M Jackson HH Mantsch Beware of proteins in DMSO BiochimBiophys Acta 1078 (1991) 231ndash235

[44] HM Ulmer H Herberhold S Fahsel MG Gaumlnzle R Winter RF VogelEffects of pressure-induced membrane phase transitions on inactivation ofHorA an ATP dependent multidrug resistance transporter in Lactobacillusplantarum Appl Environ Microbiol 68 (2002) 1088ndash1095

[45] M Jackson PI Haris D Chapman Fourier transform infraredspectroscopic studies of Ca(2+)-binding proteins Biochemistry 30(1991) 9681ndash9686

[46] S Luo CF Huang JF McClelland JD Graves A study of proteinsecondary structure by Fourier transform infraredphotoacoustic spectros-copy and its application for recombinant proteins Anal Biochem 216(1994) 67ndash76

[47] K Goosens L Smeller J Frank K Heremans Pressure-tuning theconformation of bovine pancreatic trypsin inhibitor studied by Fourier-transform infrared spectroscopy Eur J Biochem 236 (1996) 236ndash262

[48] R Gilmanshin M Gulotta RB Dyer RH Callender Structures ofapomyoglobins various acid-destabilized forms Biochemistry 40 (2001)5127ndash5136

[49] JH Crowe LM Crowe D Chapman Preservation of membranes inanhydrobiotic organismsmdashThe role of trehalose Science 223 (1984)701ndash703

Fig 3 Segments of the FT-IR spectra of L lactis in 2H2O-sucrose used for the secondary structure analysis of proteins and the lipid acyl chains A temperature inducedshift of CH2 B temperature induced shift of amide A+HOD C temperature induced shift of amide IIprime and amide II D temperature induced shift of amide Iprime

1191KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

analysing the absorbance peak resulting from symmetric CH2

stretching vibrations at wave numbers near 2852 cmminus1 (Fig 3A)The internal vibrational modes of the lipid acyl chains wereassigned based on studies of polymethylenes and poly-methylene chain compounds [3334] In the region from2800 cmminus1 to 3100 cmminus1 there are infrared absorption bandsdue to symmetric and anti-symmetric modes of themethylene chain at about 2850 cmminus1 and 2920 cmminus1respectively The frequency of the absorption maximum ofthese bands is conformation sensitive and thus respond totemperature induced changes of the transgauche ratio in acylchains The vibrational mode (asymmetric stretch) of theterminal CH3 group has a characteristic frequency of2960 cmminus1 [15]

Fig 3B depicts the spectral information of the amide A andHOD-stretching bands Amide A is due to the NndashH-stretchingvibration band at frequencies ranging from 3300 cmminus1 to3400 cmminus1 [3235] In aqueous D2O solution the HOD-stretching band is masking the amide A band However themaximum frequency linearly increased with increasing temper-ature with a slope of 0716 (cmtimesdegC)minus1 in the case of 2H2O-sucrose and of 0702 (cmtimesdegC)minus 1 for 2H2O Therefore this peakserved as an internal thermometer of the sample (Fig 3B)

The amide II band (sim1550 cmminus1 NH) and its correspondingamide IIprime band (sim1455 cmminus1 ND) record the hydrogen-3-(1H2H) exchange in proteins (Fig 3C) [3637] The 1H2Hexchange provides information about the stability and flexibilityof proteins because the 1H2H exchange rates strongly depend

on the local environment of the protons The hydrogen ions ofan un-structured part of the protein exchange faster than thoseprotons protected from the aqueous environment [38] Thespectral range shown in Fig 3C is sensitive to conformationaltransitions in proteins as reflected by the HndashD exchange[173940] The intensity of the amide IIprime band at sim1455 cmminus1

grows with increasing HndashD exchange and a correspondingdecrease of the intensity of the amide II peak at sim1550 cmminus1 isgenerally observed Corresponding shifts in the amide II andamide IIprime bands with increasing temperature were also apparentin the IR spectra of L lactis (Fig 3C)

Fig 3D depicts the amide Iprime band (1600 cmminus1 to 1700 cmminus1)measured at different temperatures Spectral data in this rangeprovide information on the secondary structure of a protein TheC_O-stretching vibrations contribute 70ndash85 to the amide Iprimeintensity 10ndash20 involve the CndashN-stretching vibration [35]The frequency of amide Iprime decreases with the strength ofhydrogen bonding of the C_O acceptor [4142] Unfoldedproteins exhibit a broad amide Iprime band at approximately1645 cmminus1 In contrast spectra from aggregated proteinsdisplay a strong absorption band near 1620 to 1615 cmminus1 and aminor component at 1683 to 1680 cmminus1 reflecting anti-symmetric β-sheets Often protein unfolding at high tempera-ture will result in extensive aggregation accompanied by theformation intermolecular β-pleated sheets [43] Thus aggre-gated proteins can be distinguished from unfolded ones Withthe second derivative of the displayed absorbance spectra aclassification of the secondary structural changes in proteins

Fig 4 Vibrational frequencies for the CH2 symmetric stretch of membranelipids of L lactis cells in 2H2O () and 2H2O-sucrose () as a function oftemperature The solid line represents the fit using a sigmoidal function with 4parameters to describe the temperature shift of the membrane from the gel to theliquid crystalline phase

1192 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

is possible (Table 2) As a result of heat induced conformationalchanges ordered structures are shifted to unordered structuresby thermal treatment [42]

33 Sucrose effects on membrane phase transitions

FT-IR spectra of L lactis were recorded using stationaryphase cells re-suspended in 2H2O or 2H2O-sucrose Mem-brane phase transitions were detected by measuring thetemperature induced shift of the symmetric CH2 absorbancepeak maxima in the presence of 2H2O or 2H2O-sucrose (Fig4) If sucrose was added to 2H2O the peak was shifted tolower frequencies Therefore a difference in the frequencyresponse was observed between 2H2O and 2H2O-sucrose Theosmolyte induced a difference of 1 cmminus1 and 2 5 cmminus1 atlow and high temperatures respectively as was previouslyreported [1718] The temperature-induced shift of the peakmaxima to higher frequencies corresponds to ldquomeltingrdquo of theacyl chains of the phospholipids and the high conformationaldisorder of the phospho-lipids at high temperatures [44] ForL lactis suspended in 2H2O the phase transition was detectedbetween 7 degC (gel phase) and 35 degC (liquid crystalline phase)If sucrose was added to 2H2O the phase transition occurredbetween 8 degC and 30 degC (Fig 4)

34 Detection of the temperature-dependent conformationalchanges in the protein structure by the use of FT-IRspectroscopy

FT-IR spectra were recorded in the temperature range from30 degC to 65 degC and from 30 degC to 75 degC in 2H2O and 2H2O-sucrose respectively The temperature range was chosen toinclude the optimal temperature of growth of L lactis and thetemperatures that allowed a complete inactivation of thepopulation in less than 2 min (Figs 1 and 2)

35 Analysis of the amide IIprime- and the amide II band

The amide II band and its corresponding amide IIprime bandare sensitive indicators of hydrogen-deuterium (1H2H)exchange in proteins To highlight the temperature induced1H2H exchange in L lactis proteins difference spectra weregenerated by subtracting data obtained at 30 degC from thoseobtained at elevated temperatures (Fig 5) The centralfrequency corresponding to the amide II and amide IIprime

Table 2Protein secondary structure elements and the corresponding peak frequencies inabsorption FT-IR spectra

Structure element Wave number (cmminus1) Width (cmminus1)

α-helix sim1652 1649ndash1653β-sheet sim1681 1680ndash1683β-sheet sim1636 ndashβ-sheet sim1618 ndashTurn sim1687 ndashRandom coil sim1658 ndashUnordered sim1642 ndash

bands changed significantly with increase in temperature Theprotonated fraction (1550 cmminus1) decreased with increasingtemperature The amide IIprime band at 1450 cmminus1 representingthe deuterated fraction of proteins increased with increasingtemperature These spectral changes were observed in both

Fig 5 Difference FT-IR spectra of amide II and amide IIprime region (1360 cmminus1 to1600 cmminus1) generated by subtraction of the spectrum at 30 degC from the spectra atelevated temperatures A data generated in 2H2O B data generated in 2H2O-sucrose

1193KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

solvents 2H2O and 2H2O-sucrose however in the presenceof sucrose higher temperatures were needed to induce thehydrogen exchange To visualise the effect of sucrose on the1H2H exchange in L lactis proteins the area ratio of the amideII band (1510 cmminus1 to 1590 cmminus1) relative to the correspondingband areas at 30 degC is shown in Fig 6 The amide II peak area isdecreasing due to the increasing 1H2H exchange and acorresponding increase of the amide IIprime peak areas (1410 cmminus1

to 1490 cmminus1) was apparent (data not shown) The difference inthe relative peak areas suggests an enhanced 1H2H exchange in2H2O compared to 2H2O-sucrose For example in 2H2O adecrease of the protonated fraction of about 33 is observed at65degC whereas only a reduction of about 20 is seen with cellssuspended in 2H2O-sucrose

Fig 7 Difference FT-IR spectra of the amide Iprime region (1600 cmminus1 to 1700 cmminus1)generated by subtraction of the spectrum at 30 degC from the spectra at elevatedtemperatures A measurement data generated in 2H2O B measurement datagenerated in 2H2O-sucrose

36 Analysis of the amide Iprime band

The amide Iprime region detected in L lactis shows a broadand asymmetric band with the maximum around located near1645 cmminus1 (Figs 3D and 8) To emphasise temperaturedependent changes difference FT-IR spectra were calculatedby subtracting the spectrum measured at 30 degC from thespectra obtained at elevated temperatures (Fig 7) For Llactis suspended in 2H2O spectral changes were detected attemperatures above 45 degC whereas temperatures above 50 degCwere required to induce spectral changes with L lactis in2H2O-sucrose (Fig 7) This result suggests sucrose enhancesthe thermal stability of intra-cellular proteins

The peak at sim1645 cmminus1 derives from to α-helices andpolypeptide chains with random structure (Table 2) [3235414248] The temperature-induced changes of the spectrasuggest a decrease of the structural elements detected withinthis range of frequency with increase in temperature Theemerging new bands around 1620 cmminus1 and 1683 cmminus1 athigh temperatures reflect the formation of a hydrogen-bonded

Fig 6 Temperature effect on the amide II peak areas at elevated temperaturerelative to the amide II peak area at 30 degC Amide II peak areas were calculatedfor wave numbers ranging from 1510 cmminus1 to 1590 cmminus1 for data generated in2H2O () and 2H2O-sucrose ()

β-sheet structure leading to an irreversible re-association ofunfolded peptide segments (Table 2) [45ndash47]

A second-derivative-analysis of the amide Iprime regionprovided a more detailed decomposition of various second-ary structure elements in L lactis proteins (Table 2) Acomparison of the spectra of the amide Iprime region and thecorresponding second derivative spectra recorded at 32 degCand 74 degC is shown in Fig 8 Conformational changes thatwere induced by a temperature up-shift indicate theformation of β-sheet-structures at sim1683 cmminus1 (Fig 8)The new bands at 1658 cmminus1 and 1642 cmminus1 emerging athigh temperatures suggest a growing fraction of disorderedsegments Moreover the second derivative analysis reveals aloss in α-helical structures (1649 cmminus1) and a gain in (inter-molecular) β-sheet-structures (1618 cmminus1)

4 Discussion

The thermal stability of proteins can be modifiedintrinsically by altering the sequence amino acids orextrinsically by suitable stabilising effectors eg osmolytesThe general mechanism of protein stabilisation by solutesand carbohydrates in particular is the exclusion of solutefrom the protein domain [23] The ability of carbohydratesto stabilise proteins has been attributed to this preferentialhydration of proteins [212249] Bacteria are required to

Fig 8 Amide Iprime band of the infrared spectra obtained from L lactis at 32 degC and75 degC corresponding to viable and inactivated cells respectively The FT-IRspectra (A) and their second derivatives (B) were recorded in 2H2O-sucrose

Fig 9 Temperature effect on the different parameters describing thermalinactivation data of bacterial cell suspensions and temperature inducedconformational changes of the protein structure A intensity of the amide IIprimeband (1440 cmminus1) at elevated temperatures corrected for the peak intensity at30 degC B intensity of the amide Iprime band (1620 cmminus1) at elevated temperaturescorrected for the peak intensity at 30 degC C rate constants k describing lethal ()and sublethal () injury of the bacteria The filled symbols represent datagenerated in 2H2O-sucrose the empty symbols represent data generated in2H2O

1194 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

adapt to osmotic up-shift by accumulating compatible solutes[1213] This accumulation of solutes equilibrates theintracellular osmotic pressure to the environmental osmoticpressure and additionally provides protection to other stressconditions such as heat freezing drying and highhydrostatic pressure [56151718] The protective effect ofcompatible solutes on bacterial survival was previouslycorrelated with the protective effect of solutes on thebacterial membrane [151729] This study confirmed aprotective effect of sucrose on the bacterial survival andcytoplasmic membranes but the membrane phase transitionoccurred in the temperature range of 5ndash30 degC and thusappeared to be unrelated to the heat inactivation of L lactis

Conformational changes in the proteins of L lactis weredetermined with FT-IR spectroscopy The data obtained inthis work with L lactis are in excellent agreement with thespectra of purified proteins [32] and correspond to previousFT-IR measurements on bacterial cells [17]

The analysis of heat induced conformational changes ofthe proteins of L lactis enables a comparison of thetemperature effects on protein structure with bacterial survivaland sublethal injury (Fig 9) The difference in the intensitiesof the amide II and IIprime peaks (1550 cmminus1 and 1440 cmminus1

respectively) at elevated temperature compared to the peakintensity at 30 degC were used as an index for heat-induced1H2H exchange The buried residues of the protein structurebecame accessible to deuterium upon temperature up-shift andwere exchanged by the surrounding 2H2O (Figs 5 9A)[36373940] Modifications of the secondary structure of theproteins were detected by analysis of the amide Iprime band (Figs7 and 9B) To obtain a rate constant as an indicator for thetemperature effects on bacterial viability the experimentaldata on bacterial inactivation was fitted to a first orderequation (Fig 9C) Although first order models may beinadequate to describe inactivation curves with pronouncednon-linearities they have been widely used to describe

1195KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

thermal death time data and provided a reasonable agreementto our experimental data (correlation coefficient r2 gt087) Incontrast to more elaborate models that take into account non-linear survivor curves a simple first order model allows therepresentation of the inactivation kinetics by a singleparameter the rate constant k and thus greatly facilitatesthe comparison with the FT-IR data on protein structureHowever it should be stressed that the extensive washingsteps that were required to replace 1H2O by 2H2O for FT-IRspectroscopy of the cultures likely affected various aspects ofbacterial physiology

In the presence of sucrose the heat-induced structuralchanges in L lactis proteins detected by the amide Iprime and theamide IIprime bands were shifted to higher temperatures by about10 degC Inhibition of the 1H2H exchange by sucrose was moreprominent at lethal temperatures above 50 degC compared toambient temperatures of 30ndash45 degC (Figs 6 and 9) Thepresence of high (intracellular) sucrose concentration thusincreased the stability of L lactis proteins at denaturingtemperature Furthermore the bacterial viability was protectedby sucrose A temperature increase of about 10 degC wasrequired to achieve the same lethal effect in milk buffer sucrosecompared to milk buffer (Fig 9C)

The heat induced conformational changes of the L lactisproteins are related to the survival of this organism at lethaltemperatures No conformational changes in the L lactisproteins were detected by analysis of the amide Iprime band atsublethal temperatures The onset of conformational changescould be located between 50 and 60 degC in 2H2O and 2H2O-sucrose respectively These temperatures correspond to thelowest temperatures at which substantial inactivation of Llactis were detected in milk buffer and milk buffer withsucrose respectively (compare Fig 9B and C) Temperatureup-shift induced an increase of structural disorder andintermolecular β-sheet-structures in L lactis proteins a clearsign of irreversible protein aggregation and a decrease ofordered structural components mainly α-helices A moredetailed interpretation of the conformational changes ofprotein secondary structures is not feasible because thecombined spectra of the more than 2000 proteins expressedby L lactis [50] were determined and the structural changesof individual proteins overlap These results indicate heat-induced protein denaturation to an extent which is beyondthe cellular capacity of protein repair or de-novo synthesisand hence contributes to cell death The presence ofintracellular unordered or denatured proteins is attributableto heat denaturation of existing proteins or mis-translationoccurring at the ribosome Ribosome denaturation inEscherichia coli occurs in the range of 50ndash90 degC and waspreviously identified as a direct cause for heat-induced celldeath [51]

The 1H2H exchange was more susceptible to temperaturethan the spectral bands relating to protein secondarystructure Remarkably an increased 1H2H exchange wasobserved already in the range of 32ndash42 degC which is abovethe optimal temperature of growth but below the maximumgrowth temperature of L lactis This indicates that

conformational instabilities of proteins that may interferewith their respective functions occur already at temperaturespermitting growth of the organisms In keeping with thisinterpretation the primary response of L lactis to temper-ature upshift is the overexpression of heat shock proteinsincluding the DnaK-GrpE-DnaJ and the GroELS chaperonecomplexes [5253] In Lactobacillus sakei the induction ofheat shock proteins was apparent already at a temperature afew degrees above the optimum temperature of growth [54]The molecular chaperones are highly conserved families ofproteins that maintain or restore native protein conformationeven at high temperatures or assist in the degradation ofunfolded and aggregated proteins [55] As the ribosome hasbeen described as (heat) stress sensor and ribosome functionis coupled to protein quality control via trans-translation theexpression of chaperons is also a reaction to impairedtranslation by stalled ribosomes at sublethal stress tempera-tures [356]

In conclusion in this work demonstrated that molarconcentrations of sucrose increase the heat resistance of Llactis This finding extends previous findings on the role ofcompatible solutes for the bacterial resistance to freezingdrying and heat Additionally it demonstrates that highintracellular sucrose concentrations prevent the denaturationor misfolding of L lactis proteins Remarkably the use ofFT-IR spectroscopy enabled the detection of proteinconformational instabilities occurring already at elevatedtemperatures within the growth range of L lactis It isanticipated that these results will facilitate the process designfor thermal treatments to eliminate or stabilise bacterial cellsin food and biomedical applications

Acknowledgements

We gratefully acknowledge the support of DeutscheForschungsgemeinschaft Grant No FOR 3582 and SFB 533B11 and we wish to thank Gesa Groumlhnke for her excellentlaboratory work

References

[1] CE Dodd TG Aldsworth The importance of RpoS in the survival ofbacteria through food processing Int J Food Microbiol 74 (2002)189ndash194

[2] JD Taylor-Robinson M Child R Pickup P Strike C Edwards Cellndashcell interactions influence resistance and survival of Salmonella serotypeTyphimurium to environmental stress J Appl Microbiol 94 (2003)95ndash102

[3] M De Angelis R Di Cagno C Huet C Crecchio PF Fox M GobbettiHeat shock response in Lactobacillus plantarum Appl EnvironMicrobiol 70 (2004) 1336ndash1346

[4] CW Blackburn LM Curtis L Humpheson C Billon PJ McClureDevelopment of thermal inactivation models for Salmonella enteritidis andEscherichia coli O157H7 with temperature pH and NaCl as controllingfactors Int J Food Microbiol 38 (1997) 31ndash44

[5] KLMattick F Jorgensen JD Legan HM Lappin-Scott TJ HumphreyHabituation of Salmonella spp at reduced water activity and its effect onheat tolerance Appl Environ Microbiol 66 (2000) 4921ndash4925

1196 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

[6] L Coroller I Leguerinel P Mafart Effect of water activities of heatingand recovery media on apparent heat resistance of Bacillus cereus sporesAppl Environ Microbiol 67 (2001) 317ndash322

[7] C Laroche P Gervais Unexpected thermal destruction of dried glassbead-immobilized microorganisms as a function of water activity ApplEnviron Microbiol 69 (2003) 3015ndash3019

[8] EY Wuytack LD Phuong A Aertsen KM Reyns D Marquenie BDe Ketelaere B Masschalck I Van Opstal AM Diels CW MichielsComparison of sublethal injury induced in Salmonella enterica serovarTyphimurium by heat and by different nonthermal treatments J Food Prot66 (2003) 31ndash37

[9] RC Williams SS Sumner DA Golden Survival of Escherichiacoli O157H7 and salmonella in apple cider and orange juice asaffected by ozone and treatment temperature J Food Prot 67 (2004)2381ndash2386

[10] TB Hansen S Knochel Factors influencing resuscitation and growth ofheat injured Listeria monocytogenes 13-249 in sous vide cooked beef IntJ Food Microbiol 63 (2001) 135ndash147

[11] A Ikari M Nakano M Ishibashi K Kawano Y Suketa H Harada KTakagi Recovery from heat shock injury by activation of Na+ndashglucosecotransporter in renal epithelial cells Biochim Biophys Acta 1643 (2003)47ndash53

[12] E Glaasker FS Tjan PF Ter Steeg WN Konings B PoolmanPhysiological response of Lactobacillus plantarum to salt and nonelec-trolyte stress J Bacteriol 180 (1998) 4718ndash4723

[13] J Palmfeldt P Radstrom B Hahn-Hagerdal Optimisation of initial cellconcentration enhances freeze-drying tolerance of Pseudomonas chloro-raphis Cryobiology 47 (2003) 21ndash29

[14] MF Roberts Organic compatible solutes of halotolerant and halophilicmicroorganisms Saline Sys 1 (2005) 5

[15] AMolina-HoumlppnerWDosterRFVogelMGGaumlnzleProtectiveeffectofsucroseandsodiumchlorideforLactococcuslactisduringsublethalandlethalhigh-pressuretreatmentsApplEnvironMicrobiol70(2004)2013ndash2020

[16] G Zhao G Zhang Effect of protective agents freezing temperaturerehydration media on viability of malolactic bacteria subjected to freeze-drying J Appl Microbiol 99 (2005) 333ndash338

[17] LJM Linders WF Wolkers FA Hoekstra K Vant Riet Effect ofadded carbohydrates on membrane phase behavior and survival of driedLactobacillus plantarum Cryobiology 35 (1997) 31ndash40

[18] Y Mille L Beney P Gervais Viability of Escherichia coli after combinedosmotic and thermal treatment a plasma membrane implication BiochimBiophys Acta 1567 (2002) 41ndash48

[19] TJ Ahern AM Klibanov The mechanisms of reversible enzymeinactivation at 100 degC Science 228 (1985) 1280ndash1284

[20] GN Somero Proteins and temperature Annu Rev Physiol 57 (1995)43ndash68

[21] M Sola-Penna JR Meyer-Fernades Protective role of trehalose inthermal denaturation of yeast pyrophosphatase Z Naturforsch 49c (1994)327ndash330

[22] J Saad-Nehme AL Bezerra LA Fornells JL Silva JR Meyer-Fernandes A contribution of the mitochondrial adenosinetriphosphataseinhibitor protein to the thermal stability of the F0F1ndashATPase complexZ Naturforsch 52c (1997) 459ndash465

[23] SN Timasheff The control of protein stability and association by weak-interactions with watermdashHow do solvents affect these processes AnnuRev Biophys Biomol Struct 22 (1993) 67ndash97

[24] H-U Gremlich B Yan Infrared and raman spectroscopy of biologicalmaterials Marcel Dekker New York 2000

[25] H Fabian W Mantele Infrared spectroscopy of proteins Handbook ofVibrational Spectroscopy vol 5 John Wiley and Sons Chichester 2002pp 3424ndash3999

[26] K Murayama M Tomida Heat-induced secondary structure andconformation change of bovine serum albumin investigated by Fouriertransform infrared spectroscopy Biochemistry 43 (2004) 11526ndash11532

[27] SM Choi CY Ma Conformational study of globulin from commonbuckwheat (Fagopyrum esculentum Moench) by Fourier transforminfrared spectroscopy differential scanning calorimetry J Agric FoodChem 53 (2005) 8046ndash8053

[28] C Dirix T Duvetter A Van Loey M Hendrickx K Heremans The insitu observation of the temperature and pressure stability of recombinantAspergillus aculeatus pectin methylesterase with Fourier transforminfrared spectroscopy reveals an unusual pressure stability of beta-helicesBiochem J 392 (2005) 565ndash571

[29] SB Leslie E Israeli B Lighthart JH Crowe LM Crowe Trehaloseand sucrose protect both membranes and proteins in intact bacteria duringdrying Appl Environ Microbiol 61 (1995) 3592ndash3597

[30] A Molina-Houmlppner Physiological response of Lactococcus lactis to highpressure Doctoral thesis Technische Universitaumlt Muumlnchen Centre for Lifeand Food Sciences Weihenstephan 2002

[31] JM Jay MJ Loessner DA Golden (Eds) Modern food microbiologySpringer Berlin 2005

[32] A Barth C Zscherp What vibrations tell us about proteins Q RevBiophys 35 (2002) 369ndash430

[33] GR Snyder Vibrational spectra of crystalline n-paraffins J MolSpectrosc 7 (1961) 116ndash144

[34] O Reis R Winter TW Zerda The effect of high external pressure onDPPC-cholesterol multilamellar vesicles a pressure-tuning Fouriertransform infrared spectroscopy study Biochim Biophys Acta 1279(1996) 5ndash16

[35] Anonymous Determination of secondary structure in proteins by Fouriertransform infrared spectroscopy (FTIR) imb Jena without publishingDate download 27022005 URL httpwwwimb-jenadeImgLibDocftirIMAGE_FTIRhtml

[36] TM Raschke S Marqusee Hydrogen exchange studies of proteinstructure Curr Opin Biotechnol 9 (1998) 80ndash86

[37] E Goormaghtigh V Raussens JM Ruysschaert Attenuated totalreflection infrared spectroscopy of proteins and lipids in biologicalmembranes Biochim Biophys Acta 1422 (1999) 105ndash185

[38] H Susi The strength of hydrogen bonding infrared spectroscopyMethods A Compan Methods Enzymol 26 (Pt C) (1972) 381ndash391

[39] L Smeller P Rubens J Frank J Fidy K Heremans Two-dimensional Fourier-transform infrared correlation spectroscopy studyof the high-pressure tuning of proteins Vibr Spectrosc 22 (2000)119ndash125

[40] L Smeller F Meersman J Fidy K Heremans High-pressure FTIR studyof the stability of horseradish peroxidase Effect of heme substitutionligand binding Ca++ removal and reduction of the disulfide bondsBiochemistry 42 (2002) 553ndash561

[41] N Takeda M Kato Y Taniguchi Pressure-induced secondary structurechanges of ribonuclease A and ribonuclease S studied by FTIRspectroscopy Biospectroscopy 1 (1995) 207ndash216

[42] N Takeda M Kato Y Taniguchi Pressure and thermally-inducedreversible changes in the secondary structure of ribonuclease A studied byFTIR spectroscopy Biochemistry 34 (1995) 5980ndash5987

[43] M Jackson HH Mantsch Beware of proteins in DMSO BiochimBiophys Acta 1078 (1991) 231ndash235

[44] HM Ulmer H Herberhold S Fahsel MG Gaumlnzle R Winter RF VogelEffects of pressure-induced membrane phase transitions on inactivation ofHorA an ATP dependent multidrug resistance transporter in Lactobacillusplantarum Appl Environ Microbiol 68 (2002) 1088ndash1095

[45] M Jackson PI Haris D Chapman Fourier transform infraredspectroscopic studies of Ca(2+)-binding proteins Biochemistry 30(1991) 9681ndash9686

[46] S Luo CF Huang JF McClelland JD Graves A study of proteinsecondary structure by Fourier transform infraredphotoacoustic spectros-copy and its application for recombinant proteins Anal Biochem 216(1994) 67ndash76

[47] K Goosens L Smeller J Frank K Heremans Pressure-tuning theconformation of bovine pancreatic trypsin inhibitor studied by Fourier-transform infrared spectroscopy Eur J Biochem 236 (1996) 236ndash262

[48] R Gilmanshin M Gulotta RB Dyer RH Callender Structures ofapomyoglobins various acid-destabilized forms Biochemistry 40 (2001)5127ndash5136

[49] JH Crowe LM Crowe D Chapman Preservation of membranes inanhydrobiotic organismsmdashThe role of trehalose Science 223 (1984)701ndash703

Fig 4 Vibrational frequencies for the CH2 symmetric stretch of membranelipids of L lactis cells in 2H2O () and 2H2O-sucrose () as a function oftemperature The solid line represents the fit using a sigmoidal function with 4parameters to describe the temperature shift of the membrane from the gel to theliquid crystalline phase

1192 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

is possible (Table 2) As a result of heat induced conformationalchanges ordered structures are shifted to unordered structuresby thermal treatment [42]

33 Sucrose effects on membrane phase transitions

FT-IR spectra of L lactis were recorded using stationaryphase cells re-suspended in 2H2O or 2H2O-sucrose Mem-brane phase transitions were detected by measuring thetemperature induced shift of the symmetric CH2 absorbancepeak maxima in the presence of 2H2O or 2H2O-sucrose (Fig4) If sucrose was added to 2H2O the peak was shifted tolower frequencies Therefore a difference in the frequencyresponse was observed between 2H2O and 2H2O-sucrose Theosmolyte induced a difference of 1 cmminus1 and 2 5 cmminus1 atlow and high temperatures respectively as was previouslyreported [1718] The temperature-induced shift of the peakmaxima to higher frequencies corresponds to ldquomeltingrdquo of theacyl chains of the phospholipids and the high conformationaldisorder of the phospho-lipids at high temperatures [44] ForL lactis suspended in 2H2O the phase transition was detectedbetween 7 degC (gel phase) and 35 degC (liquid crystalline phase)If sucrose was added to 2H2O the phase transition occurredbetween 8 degC and 30 degC (Fig 4)

34 Detection of the temperature-dependent conformationalchanges in the protein structure by the use of FT-IRspectroscopy

FT-IR spectra were recorded in the temperature range from30 degC to 65 degC and from 30 degC to 75 degC in 2H2O and 2H2O-sucrose respectively The temperature range was chosen toinclude the optimal temperature of growth of L lactis and thetemperatures that allowed a complete inactivation of thepopulation in less than 2 min (Figs 1 and 2)

35 Analysis of the amide IIprime- and the amide II band

The amide II band and its corresponding amide IIprime bandare sensitive indicators of hydrogen-deuterium (1H2H)exchange in proteins To highlight the temperature induced1H2H exchange in L lactis proteins difference spectra weregenerated by subtracting data obtained at 30 degC from thoseobtained at elevated temperatures (Fig 5) The centralfrequency corresponding to the amide II and amide IIprime

Table 2Protein secondary structure elements and the corresponding peak frequencies inabsorption FT-IR spectra

Structure element Wave number (cmminus1) Width (cmminus1)

α-helix sim1652 1649ndash1653β-sheet sim1681 1680ndash1683β-sheet sim1636 ndashβ-sheet sim1618 ndashTurn sim1687 ndashRandom coil sim1658 ndashUnordered sim1642 ndash

bands changed significantly with increase in temperature Theprotonated fraction (1550 cmminus1) decreased with increasingtemperature The amide IIprime band at 1450 cmminus1 representingthe deuterated fraction of proteins increased with increasingtemperature These spectral changes were observed in both

Fig 5 Difference FT-IR spectra of amide II and amide IIprime region (1360 cmminus1 to1600 cmminus1) generated by subtraction of the spectrum at 30 degC from the spectra atelevated temperatures A data generated in 2H2O B data generated in 2H2O-sucrose

1193KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

solvents 2H2O and 2H2O-sucrose however in the presenceof sucrose higher temperatures were needed to induce thehydrogen exchange To visualise the effect of sucrose on the1H2H exchange in L lactis proteins the area ratio of the amideII band (1510 cmminus1 to 1590 cmminus1) relative to the correspondingband areas at 30 degC is shown in Fig 6 The amide II peak area isdecreasing due to the increasing 1H2H exchange and acorresponding increase of the amide IIprime peak areas (1410 cmminus1

to 1490 cmminus1) was apparent (data not shown) The difference inthe relative peak areas suggests an enhanced 1H2H exchange in2H2O compared to 2H2O-sucrose For example in 2H2O adecrease of the protonated fraction of about 33 is observed at65degC whereas only a reduction of about 20 is seen with cellssuspended in 2H2O-sucrose

Fig 7 Difference FT-IR spectra of the amide Iprime region (1600 cmminus1 to 1700 cmminus1)generated by subtraction of the spectrum at 30 degC from the spectra at elevatedtemperatures A measurement data generated in 2H2O B measurement datagenerated in 2H2O-sucrose

36 Analysis of the amide Iprime band

The amide Iprime region detected in L lactis shows a broadand asymmetric band with the maximum around located near1645 cmminus1 (Figs 3D and 8) To emphasise temperaturedependent changes difference FT-IR spectra were calculatedby subtracting the spectrum measured at 30 degC from thespectra obtained at elevated temperatures (Fig 7) For Llactis suspended in 2H2O spectral changes were detected attemperatures above 45 degC whereas temperatures above 50 degCwere required to induce spectral changes with L lactis in2H2O-sucrose (Fig 7) This result suggests sucrose enhancesthe thermal stability of intra-cellular proteins

The peak at sim1645 cmminus1 derives from to α-helices andpolypeptide chains with random structure (Table 2) [3235414248] The temperature-induced changes of the spectrasuggest a decrease of the structural elements detected withinthis range of frequency with increase in temperature Theemerging new bands around 1620 cmminus1 and 1683 cmminus1 athigh temperatures reflect the formation of a hydrogen-bonded

Fig 6 Temperature effect on the amide II peak areas at elevated temperaturerelative to the amide II peak area at 30 degC Amide II peak areas were calculatedfor wave numbers ranging from 1510 cmminus1 to 1590 cmminus1 for data generated in2H2O () and 2H2O-sucrose ()

β-sheet structure leading to an irreversible re-association ofunfolded peptide segments (Table 2) [45ndash47]

A second-derivative-analysis of the amide Iprime regionprovided a more detailed decomposition of various second-ary structure elements in L lactis proteins (Table 2) Acomparison of the spectra of the amide Iprime region and thecorresponding second derivative spectra recorded at 32 degCand 74 degC is shown in Fig 8 Conformational changes thatwere induced by a temperature up-shift indicate theformation of β-sheet-structures at sim1683 cmminus1 (Fig 8)The new bands at 1658 cmminus1 and 1642 cmminus1 emerging athigh temperatures suggest a growing fraction of disorderedsegments Moreover the second derivative analysis reveals aloss in α-helical structures (1649 cmminus1) and a gain in (inter-molecular) β-sheet-structures (1618 cmminus1)

4 Discussion

The thermal stability of proteins can be modifiedintrinsically by altering the sequence amino acids orextrinsically by suitable stabilising effectors eg osmolytesThe general mechanism of protein stabilisation by solutesand carbohydrates in particular is the exclusion of solutefrom the protein domain [23] The ability of carbohydratesto stabilise proteins has been attributed to this preferentialhydration of proteins [212249] Bacteria are required to

Fig 8 Amide Iprime band of the infrared spectra obtained from L lactis at 32 degC and75 degC corresponding to viable and inactivated cells respectively The FT-IRspectra (A) and their second derivatives (B) were recorded in 2H2O-sucrose

Fig 9 Temperature effect on the different parameters describing thermalinactivation data of bacterial cell suspensions and temperature inducedconformational changes of the protein structure A intensity of the amide IIprimeband (1440 cmminus1) at elevated temperatures corrected for the peak intensity at30 degC B intensity of the amide Iprime band (1620 cmminus1) at elevated temperaturescorrected for the peak intensity at 30 degC C rate constants k describing lethal ()and sublethal () injury of the bacteria The filled symbols represent datagenerated in 2H2O-sucrose the empty symbols represent data generated in2H2O

1194 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

adapt to osmotic up-shift by accumulating compatible solutes[1213] This accumulation of solutes equilibrates theintracellular osmotic pressure to the environmental osmoticpressure and additionally provides protection to other stressconditions such as heat freezing drying and highhydrostatic pressure [56151718] The protective effect ofcompatible solutes on bacterial survival was previouslycorrelated with the protective effect of solutes on thebacterial membrane [151729] This study confirmed aprotective effect of sucrose on the bacterial survival andcytoplasmic membranes but the membrane phase transitionoccurred in the temperature range of 5ndash30 degC and thusappeared to be unrelated to the heat inactivation of L lactis

Conformational changes in the proteins of L lactis weredetermined with FT-IR spectroscopy The data obtained inthis work with L lactis are in excellent agreement with thespectra of purified proteins [32] and correspond to previousFT-IR measurements on bacterial cells [17]

The analysis of heat induced conformational changes ofthe proteins of L lactis enables a comparison of thetemperature effects on protein structure with bacterial survivaland sublethal injury (Fig 9) The difference in the intensitiesof the amide II and IIprime peaks (1550 cmminus1 and 1440 cmminus1

respectively) at elevated temperature compared to the peakintensity at 30 degC were used as an index for heat-induced1H2H exchange The buried residues of the protein structurebecame accessible to deuterium upon temperature up-shift andwere exchanged by the surrounding 2H2O (Figs 5 9A)[36373940] Modifications of the secondary structure of theproteins were detected by analysis of the amide Iprime band (Figs7 and 9B) To obtain a rate constant as an indicator for thetemperature effects on bacterial viability the experimentaldata on bacterial inactivation was fitted to a first orderequation (Fig 9C) Although first order models may beinadequate to describe inactivation curves with pronouncednon-linearities they have been widely used to describe

1195KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

thermal death time data and provided a reasonable agreementto our experimental data (correlation coefficient r2 gt087) Incontrast to more elaborate models that take into account non-linear survivor curves a simple first order model allows therepresentation of the inactivation kinetics by a singleparameter the rate constant k and thus greatly facilitatesthe comparison with the FT-IR data on protein structureHowever it should be stressed that the extensive washingsteps that were required to replace 1H2O by 2H2O for FT-IRspectroscopy of the cultures likely affected various aspects ofbacterial physiology

In the presence of sucrose the heat-induced structuralchanges in L lactis proteins detected by the amide Iprime and theamide IIprime bands were shifted to higher temperatures by about10 degC Inhibition of the 1H2H exchange by sucrose was moreprominent at lethal temperatures above 50 degC compared toambient temperatures of 30ndash45 degC (Figs 6 and 9) Thepresence of high (intracellular) sucrose concentration thusincreased the stability of L lactis proteins at denaturingtemperature Furthermore the bacterial viability was protectedby sucrose A temperature increase of about 10 degC wasrequired to achieve the same lethal effect in milk buffer sucrosecompared to milk buffer (Fig 9C)

The heat induced conformational changes of the L lactisproteins are related to the survival of this organism at lethaltemperatures No conformational changes in the L lactisproteins were detected by analysis of the amide Iprime band atsublethal temperatures The onset of conformational changescould be located between 50 and 60 degC in 2H2O and 2H2O-sucrose respectively These temperatures correspond to thelowest temperatures at which substantial inactivation of Llactis were detected in milk buffer and milk buffer withsucrose respectively (compare Fig 9B and C) Temperatureup-shift induced an increase of structural disorder andintermolecular β-sheet-structures in L lactis proteins a clearsign of irreversible protein aggregation and a decrease ofordered structural components mainly α-helices A moredetailed interpretation of the conformational changes ofprotein secondary structures is not feasible because thecombined spectra of the more than 2000 proteins expressedby L lactis [50] were determined and the structural changesof individual proteins overlap These results indicate heat-induced protein denaturation to an extent which is beyondthe cellular capacity of protein repair or de-novo synthesisand hence contributes to cell death The presence ofintracellular unordered or denatured proteins is attributableto heat denaturation of existing proteins or mis-translationoccurring at the ribosome Ribosome denaturation inEscherichia coli occurs in the range of 50ndash90 degC and waspreviously identified as a direct cause for heat-induced celldeath [51]

The 1H2H exchange was more susceptible to temperaturethan the spectral bands relating to protein secondarystructure Remarkably an increased 1H2H exchange wasobserved already in the range of 32ndash42 degC which is abovethe optimal temperature of growth but below the maximumgrowth temperature of L lactis This indicates that

conformational instabilities of proteins that may interferewith their respective functions occur already at temperaturespermitting growth of the organisms In keeping with thisinterpretation the primary response of L lactis to temper-ature upshift is the overexpression of heat shock proteinsincluding the DnaK-GrpE-DnaJ and the GroELS chaperonecomplexes [5253] In Lactobacillus sakei the induction ofheat shock proteins was apparent already at a temperature afew degrees above the optimum temperature of growth [54]The molecular chaperones are highly conserved families ofproteins that maintain or restore native protein conformationeven at high temperatures or assist in the degradation ofunfolded and aggregated proteins [55] As the ribosome hasbeen described as (heat) stress sensor and ribosome functionis coupled to protein quality control via trans-translation theexpression of chaperons is also a reaction to impairedtranslation by stalled ribosomes at sublethal stress tempera-tures [356]

In conclusion in this work demonstrated that molarconcentrations of sucrose increase the heat resistance of Llactis This finding extends previous findings on the role ofcompatible solutes for the bacterial resistance to freezingdrying and heat Additionally it demonstrates that highintracellular sucrose concentrations prevent the denaturationor misfolding of L lactis proteins Remarkably the use ofFT-IR spectroscopy enabled the detection of proteinconformational instabilities occurring already at elevatedtemperatures within the growth range of L lactis It isanticipated that these results will facilitate the process designfor thermal treatments to eliminate or stabilise bacterial cellsin food and biomedical applications

Acknowledgements

We gratefully acknowledge the support of DeutscheForschungsgemeinschaft Grant No FOR 3582 and SFB 533B11 and we wish to thank Gesa Groumlhnke for her excellentlaboratory work

References

[1] CE Dodd TG Aldsworth The importance of RpoS in the survival ofbacteria through food processing Int J Food Microbiol 74 (2002)189ndash194

[2] JD Taylor-Robinson M Child R Pickup P Strike C Edwards Cellndashcell interactions influence resistance and survival of Salmonella serotypeTyphimurium to environmental stress J Appl Microbiol 94 (2003)95ndash102

[3] M De Angelis R Di Cagno C Huet C Crecchio PF Fox M GobbettiHeat shock response in Lactobacillus plantarum Appl EnvironMicrobiol 70 (2004) 1336ndash1346

[4] CW Blackburn LM Curtis L Humpheson C Billon PJ McClureDevelopment of thermal inactivation models for Salmonella enteritidis andEscherichia coli O157H7 with temperature pH and NaCl as controllingfactors Int J Food Microbiol 38 (1997) 31ndash44

[5] KLMattick F Jorgensen JD Legan HM Lappin-Scott TJ HumphreyHabituation of Salmonella spp at reduced water activity and its effect onheat tolerance Appl Environ Microbiol 66 (2000) 4921ndash4925

1196 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

[6] L Coroller I Leguerinel P Mafart Effect of water activities of heatingand recovery media on apparent heat resistance of Bacillus cereus sporesAppl Environ Microbiol 67 (2001) 317ndash322

[7] C Laroche P Gervais Unexpected thermal destruction of dried glassbead-immobilized microorganisms as a function of water activity ApplEnviron Microbiol 69 (2003) 3015ndash3019

[8] EY Wuytack LD Phuong A Aertsen KM Reyns D Marquenie BDe Ketelaere B Masschalck I Van Opstal AM Diels CW MichielsComparison of sublethal injury induced in Salmonella enterica serovarTyphimurium by heat and by different nonthermal treatments J Food Prot66 (2003) 31ndash37

[9] RC Williams SS Sumner DA Golden Survival of Escherichiacoli O157H7 and salmonella in apple cider and orange juice asaffected by ozone and treatment temperature J Food Prot 67 (2004)2381ndash2386

[10] TB Hansen S Knochel Factors influencing resuscitation and growth ofheat injured Listeria monocytogenes 13-249 in sous vide cooked beef IntJ Food Microbiol 63 (2001) 135ndash147

[11] A Ikari M Nakano M Ishibashi K Kawano Y Suketa H Harada KTakagi Recovery from heat shock injury by activation of Na+ndashglucosecotransporter in renal epithelial cells Biochim Biophys Acta 1643 (2003)47ndash53

[12] E Glaasker FS Tjan PF Ter Steeg WN Konings B PoolmanPhysiological response of Lactobacillus plantarum to salt and nonelec-trolyte stress J Bacteriol 180 (1998) 4718ndash4723

[13] J Palmfeldt P Radstrom B Hahn-Hagerdal Optimisation of initial cellconcentration enhances freeze-drying tolerance of Pseudomonas chloro-raphis Cryobiology 47 (2003) 21ndash29

[14] MF Roberts Organic compatible solutes of halotolerant and halophilicmicroorganisms Saline Sys 1 (2005) 5

[15] AMolina-HoumlppnerWDosterRFVogelMGGaumlnzleProtectiveeffectofsucroseandsodiumchlorideforLactococcuslactisduringsublethalandlethalhigh-pressuretreatmentsApplEnvironMicrobiol70(2004)2013ndash2020

[16] G Zhao G Zhang Effect of protective agents freezing temperaturerehydration media on viability of malolactic bacteria subjected to freeze-drying J Appl Microbiol 99 (2005) 333ndash338

[17] LJM Linders WF Wolkers FA Hoekstra K Vant Riet Effect ofadded carbohydrates on membrane phase behavior and survival of driedLactobacillus plantarum Cryobiology 35 (1997) 31ndash40

[18] Y Mille L Beney P Gervais Viability of Escherichia coli after combinedosmotic and thermal treatment a plasma membrane implication BiochimBiophys Acta 1567 (2002) 41ndash48

[19] TJ Ahern AM Klibanov The mechanisms of reversible enzymeinactivation at 100 degC Science 228 (1985) 1280ndash1284

[20] GN Somero Proteins and temperature Annu Rev Physiol 57 (1995)43ndash68

[21] M Sola-Penna JR Meyer-Fernades Protective role of trehalose inthermal denaturation of yeast pyrophosphatase Z Naturforsch 49c (1994)327ndash330

[22] J Saad-Nehme AL Bezerra LA Fornells JL Silva JR Meyer-Fernandes A contribution of the mitochondrial adenosinetriphosphataseinhibitor protein to the thermal stability of the F0F1ndashATPase complexZ Naturforsch 52c (1997) 459ndash465

[23] SN Timasheff The control of protein stability and association by weak-interactions with watermdashHow do solvents affect these processes AnnuRev Biophys Biomol Struct 22 (1993) 67ndash97

[24] H-U Gremlich B Yan Infrared and raman spectroscopy of biologicalmaterials Marcel Dekker New York 2000

[25] H Fabian W Mantele Infrared spectroscopy of proteins Handbook ofVibrational Spectroscopy vol 5 John Wiley and Sons Chichester 2002pp 3424ndash3999

[26] K Murayama M Tomida Heat-induced secondary structure andconformation change of bovine serum albumin investigated by Fouriertransform infrared spectroscopy Biochemistry 43 (2004) 11526ndash11532

[27] SM Choi CY Ma Conformational study of globulin from commonbuckwheat (Fagopyrum esculentum Moench) by Fourier transforminfrared spectroscopy differential scanning calorimetry J Agric FoodChem 53 (2005) 8046ndash8053

[28] C Dirix T Duvetter A Van Loey M Hendrickx K Heremans The insitu observation of the temperature and pressure stability of recombinantAspergillus aculeatus pectin methylesterase with Fourier transforminfrared spectroscopy reveals an unusual pressure stability of beta-helicesBiochem J 392 (2005) 565ndash571

[29] SB Leslie E Israeli B Lighthart JH Crowe LM Crowe Trehaloseand sucrose protect both membranes and proteins in intact bacteria duringdrying Appl Environ Microbiol 61 (1995) 3592ndash3597

[30] A Molina-Houmlppner Physiological response of Lactococcus lactis to highpressure Doctoral thesis Technische Universitaumlt Muumlnchen Centre for Lifeand Food Sciences Weihenstephan 2002

[31] JM Jay MJ Loessner DA Golden (Eds) Modern food microbiologySpringer Berlin 2005

[32] A Barth C Zscherp What vibrations tell us about proteins Q RevBiophys 35 (2002) 369ndash430

[33] GR Snyder Vibrational spectra of crystalline n-paraffins J MolSpectrosc 7 (1961) 116ndash144

[34] O Reis R Winter TW Zerda The effect of high external pressure onDPPC-cholesterol multilamellar vesicles a pressure-tuning Fouriertransform infrared spectroscopy study Biochim Biophys Acta 1279(1996) 5ndash16

[35] Anonymous Determination of secondary structure in proteins by Fouriertransform infrared spectroscopy (FTIR) imb Jena without publishingDate download 27022005 URL httpwwwimb-jenadeImgLibDocftirIMAGE_FTIRhtml

[36] TM Raschke S Marqusee Hydrogen exchange studies of proteinstructure Curr Opin Biotechnol 9 (1998) 80ndash86

[37] E Goormaghtigh V Raussens JM Ruysschaert Attenuated totalreflection infrared spectroscopy of proteins and lipids in biologicalmembranes Biochim Biophys Acta 1422 (1999) 105ndash185

[38] H Susi The strength of hydrogen bonding infrared spectroscopyMethods A Compan Methods Enzymol 26 (Pt C) (1972) 381ndash391

[39] L Smeller P Rubens J Frank J Fidy K Heremans Two-dimensional Fourier-transform infrared correlation spectroscopy studyof the high-pressure tuning of proteins Vibr Spectrosc 22 (2000)119ndash125

[40] L Smeller F Meersman J Fidy K Heremans High-pressure FTIR studyof the stability of horseradish peroxidase Effect of heme substitutionligand binding Ca++ removal and reduction of the disulfide bondsBiochemistry 42 (2002) 553ndash561

[41] N Takeda M Kato Y Taniguchi Pressure-induced secondary structurechanges of ribonuclease A and ribonuclease S studied by FTIRspectroscopy Biospectroscopy 1 (1995) 207ndash216

[42] N Takeda M Kato Y Taniguchi Pressure and thermally-inducedreversible changes in the secondary structure of ribonuclease A studied byFTIR spectroscopy Biochemistry 34 (1995) 5980ndash5987

[43] M Jackson HH Mantsch Beware of proteins in DMSO BiochimBiophys Acta 1078 (1991) 231ndash235

[44] HM Ulmer H Herberhold S Fahsel MG Gaumlnzle R Winter RF VogelEffects of pressure-induced membrane phase transitions on inactivation ofHorA an ATP dependent multidrug resistance transporter in Lactobacillusplantarum Appl Environ Microbiol 68 (2002) 1088ndash1095

[45] M Jackson PI Haris D Chapman Fourier transform infraredspectroscopic studies of Ca(2+)-binding proteins Biochemistry 30(1991) 9681ndash9686

[46] S Luo CF Huang JF McClelland JD Graves A study of proteinsecondary structure by Fourier transform infraredphotoacoustic spectros-copy and its application for recombinant proteins Anal Biochem 216(1994) 67ndash76

[47] K Goosens L Smeller J Frank K Heremans Pressure-tuning theconformation of bovine pancreatic trypsin inhibitor studied by Fourier-transform infrared spectroscopy Eur J Biochem 236 (1996) 236ndash262

[48] R Gilmanshin M Gulotta RB Dyer RH Callender Structures ofapomyoglobins various acid-destabilized forms Biochemistry 40 (2001)5127ndash5136

[49] JH Crowe LM Crowe D Chapman Preservation of membranes inanhydrobiotic organismsmdashThe role of trehalose Science 223 (1984)701ndash703

1193KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

solvents 2H2O and 2H2O-sucrose however in the presenceof sucrose higher temperatures were needed to induce thehydrogen exchange To visualise the effect of sucrose on the1H2H exchange in L lactis proteins the area ratio of the amideII band (1510 cmminus1 to 1590 cmminus1) relative to the correspondingband areas at 30 degC is shown in Fig 6 The amide II peak area isdecreasing due to the increasing 1H2H exchange and acorresponding increase of the amide IIprime peak areas (1410 cmminus1

to 1490 cmminus1) was apparent (data not shown) The difference inthe relative peak areas suggests an enhanced 1H2H exchange in2H2O compared to 2H2O-sucrose For example in 2H2O adecrease of the protonated fraction of about 33 is observed at65degC whereas only a reduction of about 20 is seen with cellssuspended in 2H2O-sucrose

Fig 7 Difference FT-IR spectra of the amide Iprime region (1600 cmminus1 to 1700 cmminus1)generated by subtraction of the spectrum at 30 degC from the spectra at elevatedtemperatures A measurement data generated in 2H2O B measurement datagenerated in 2H2O-sucrose

36 Analysis of the amide Iprime band

The amide Iprime region detected in L lactis shows a broadand asymmetric band with the maximum around located near1645 cmminus1 (Figs 3D and 8) To emphasise temperaturedependent changes difference FT-IR spectra were calculatedby subtracting the spectrum measured at 30 degC from thespectra obtained at elevated temperatures (Fig 7) For Llactis suspended in 2H2O spectral changes were detected attemperatures above 45 degC whereas temperatures above 50 degCwere required to induce spectral changes with L lactis in2H2O-sucrose (Fig 7) This result suggests sucrose enhancesthe thermal stability of intra-cellular proteins

The peak at sim1645 cmminus1 derives from to α-helices andpolypeptide chains with random structure (Table 2) [3235414248] The temperature-induced changes of the spectrasuggest a decrease of the structural elements detected withinthis range of frequency with increase in temperature Theemerging new bands around 1620 cmminus1 and 1683 cmminus1 athigh temperatures reflect the formation of a hydrogen-bonded

Fig 6 Temperature effect on the amide II peak areas at elevated temperaturerelative to the amide II peak area at 30 degC Amide II peak areas were calculatedfor wave numbers ranging from 1510 cmminus1 to 1590 cmminus1 for data generated in2H2O () and 2H2O-sucrose ()

β-sheet structure leading to an irreversible re-association ofunfolded peptide segments (Table 2) [45ndash47]

A second-derivative-analysis of the amide Iprime regionprovided a more detailed decomposition of various second-ary structure elements in L lactis proteins (Table 2) Acomparison of the spectra of the amide Iprime region and thecorresponding second derivative spectra recorded at 32 degCand 74 degC is shown in Fig 8 Conformational changes thatwere induced by a temperature up-shift indicate theformation of β-sheet-structures at sim1683 cmminus1 (Fig 8)The new bands at 1658 cmminus1 and 1642 cmminus1 emerging athigh temperatures suggest a growing fraction of disorderedsegments Moreover the second derivative analysis reveals aloss in α-helical structures (1649 cmminus1) and a gain in (inter-molecular) β-sheet-structures (1618 cmminus1)

4 Discussion

The thermal stability of proteins can be modifiedintrinsically by altering the sequence amino acids orextrinsically by suitable stabilising effectors eg osmolytesThe general mechanism of protein stabilisation by solutesand carbohydrates in particular is the exclusion of solutefrom the protein domain [23] The ability of carbohydratesto stabilise proteins has been attributed to this preferentialhydration of proteins [212249] Bacteria are required to

Fig 8 Amide Iprime band of the infrared spectra obtained from L lactis at 32 degC and75 degC corresponding to viable and inactivated cells respectively The FT-IRspectra (A) and their second derivatives (B) were recorded in 2H2O-sucrose

Fig 9 Temperature effect on the different parameters describing thermalinactivation data of bacterial cell suspensions and temperature inducedconformational changes of the protein structure A intensity of the amide IIprimeband (1440 cmminus1) at elevated temperatures corrected for the peak intensity at30 degC B intensity of the amide Iprime band (1620 cmminus1) at elevated temperaturescorrected for the peak intensity at 30 degC C rate constants k describing lethal ()and sublethal () injury of the bacteria The filled symbols represent datagenerated in 2H2O-sucrose the empty symbols represent data generated in2H2O

1194 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

adapt to osmotic up-shift by accumulating compatible solutes[1213] This accumulation of solutes equilibrates theintracellular osmotic pressure to the environmental osmoticpressure and additionally provides protection to other stressconditions such as heat freezing drying and highhydrostatic pressure [56151718] The protective effect ofcompatible solutes on bacterial survival was previouslycorrelated with the protective effect of solutes on thebacterial membrane [151729] This study confirmed aprotective effect of sucrose on the bacterial survival andcytoplasmic membranes but the membrane phase transitionoccurred in the temperature range of 5ndash30 degC and thusappeared to be unrelated to the heat inactivation of L lactis

Conformational changes in the proteins of L lactis weredetermined with FT-IR spectroscopy The data obtained inthis work with L lactis are in excellent agreement with thespectra of purified proteins [32] and correspond to previousFT-IR measurements on bacterial cells [17]

The analysis of heat induced conformational changes ofthe proteins of L lactis enables a comparison of thetemperature effects on protein structure with bacterial survivaland sublethal injury (Fig 9) The difference in the intensitiesof the amide II and IIprime peaks (1550 cmminus1 and 1440 cmminus1

respectively) at elevated temperature compared to the peakintensity at 30 degC were used as an index for heat-induced1H2H exchange The buried residues of the protein structurebecame accessible to deuterium upon temperature up-shift andwere exchanged by the surrounding 2H2O (Figs 5 9A)[36373940] Modifications of the secondary structure of theproteins were detected by analysis of the amide Iprime band (Figs7 and 9B) To obtain a rate constant as an indicator for thetemperature effects on bacterial viability the experimentaldata on bacterial inactivation was fitted to a first orderequation (Fig 9C) Although first order models may beinadequate to describe inactivation curves with pronouncednon-linearities they have been widely used to describe

1195KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

thermal death time data and provided a reasonable agreementto our experimental data (correlation coefficient r2 gt087) Incontrast to more elaborate models that take into account non-linear survivor curves a simple first order model allows therepresentation of the inactivation kinetics by a singleparameter the rate constant k and thus greatly facilitatesthe comparison with the FT-IR data on protein structureHowever it should be stressed that the extensive washingsteps that were required to replace 1H2O by 2H2O for FT-IRspectroscopy of the cultures likely affected various aspects ofbacterial physiology

In the presence of sucrose the heat-induced structuralchanges in L lactis proteins detected by the amide Iprime and theamide IIprime bands were shifted to higher temperatures by about10 degC Inhibition of the 1H2H exchange by sucrose was moreprominent at lethal temperatures above 50 degC compared toambient temperatures of 30ndash45 degC (Figs 6 and 9) Thepresence of high (intracellular) sucrose concentration thusincreased the stability of L lactis proteins at denaturingtemperature Furthermore the bacterial viability was protectedby sucrose A temperature increase of about 10 degC wasrequired to achieve the same lethal effect in milk buffer sucrosecompared to milk buffer (Fig 9C)

The heat induced conformational changes of the L lactisproteins are related to the survival of this organism at lethaltemperatures No conformational changes in the L lactisproteins were detected by analysis of the amide Iprime band atsublethal temperatures The onset of conformational changescould be located between 50 and 60 degC in 2H2O and 2H2O-sucrose respectively These temperatures correspond to thelowest temperatures at which substantial inactivation of Llactis were detected in milk buffer and milk buffer withsucrose respectively (compare Fig 9B and C) Temperatureup-shift induced an increase of structural disorder andintermolecular β-sheet-structures in L lactis proteins a clearsign of irreversible protein aggregation and a decrease ofordered structural components mainly α-helices A moredetailed interpretation of the conformational changes ofprotein secondary structures is not feasible because thecombined spectra of the more than 2000 proteins expressedby L lactis [50] were determined and the structural changesof individual proteins overlap These results indicate heat-induced protein denaturation to an extent which is beyondthe cellular capacity of protein repair or de-novo synthesisand hence contributes to cell death The presence ofintracellular unordered or denatured proteins is attributableto heat denaturation of existing proteins or mis-translationoccurring at the ribosome Ribosome denaturation inEscherichia coli occurs in the range of 50ndash90 degC and waspreviously identified as a direct cause for heat-induced celldeath [51]

The 1H2H exchange was more susceptible to temperaturethan the spectral bands relating to protein secondarystructure Remarkably an increased 1H2H exchange wasobserved already in the range of 32ndash42 degC which is abovethe optimal temperature of growth but below the maximumgrowth temperature of L lactis This indicates that

conformational instabilities of proteins that may interferewith their respective functions occur already at temperaturespermitting growth of the organisms In keeping with thisinterpretation the primary response of L lactis to temper-ature upshift is the overexpression of heat shock proteinsincluding the DnaK-GrpE-DnaJ and the GroELS chaperonecomplexes [5253] In Lactobacillus sakei the induction ofheat shock proteins was apparent already at a temperature afew degrees above the optimum temperature of growth [54]The molecular chaperones are highly conserved families ofproteins that maintain or restore native protein conformationeven at high temperatures or assist in the degradation ofunfolded and aggregated proteins [55] As the ribosome hasbeen described as (heat) stress sensor and ribosome functionis coupled to protein quality control via trans-translation theexpression of chaperons is also a reaction to impairedtranslation by stalled ribosomes at sublethal stress tempera-tures [356]

In conclusion in this work demonstrated that molarconcentrations of sucrose increase the heat resistance of Llactis This finding extends previous findings on the role ofcompatible solutes for the bacterial resistance to freezingdrying and heat Additionally it demonstrates that highintracellular sucrose concentrations prevent the denaturationor misfolding of L lactis proteins Remarkably the use ofFT-IR spectroscopy enabled the detection of proteinconformational instabilities occurring already at elevatedtemperatures within the growth range of L lactis It isanticipated that these results will facilitate the process designfor thermal treatments to eliminate or stabilise bacterial cellsin food and biomedical applications

Acknowledgements

We gratefully acknowledge the support of DeutscheForschungsgemeinschaft Grant No FOR 3582 and SFB 533B11 and we wish to thank Gesa Groumlhnke for her excellentlaboratory work

References

[1] CE Dodd TG Aldsworth The importance of RpoS in the survival ofbacteria through food processing Int J Food Microbiol 74 (2002)189ndash194

[2] JD Taylor-Robinson M Child R Pickup P Strike C Edwards Cellndashcell interactions influence resistance and survival of Salmonella serotypeTyphimurium to environmental stress J Appl Microbiol 94 (2003)95ndash102

[3] M De Angelis R Di Cagno C Huet C Crecchio PF Fox M GobbettiHeat shock response in Lactobacillus plantarum Appl EnvironMicrobiol 70 (2004) 1336ndash1346

[4] CW Blackburn LM Curtis L Humpheson C Billon PJ McClureDevelopment of thermal inactivation models for Salmonella enteritidis andEscherichia coli O157H7 with temperature pH and NaCl as controllingfactors Int J Food Microbiol 38 (1997) 31ndash44

[5] KLMattick F Jorgensen JD Legan HM Lappin-Scott TJ HumphreyHabituation of Salmonella spp at reduced water activity and its effect onheat tolerance Appl Environ Microbiol 66 (2000) 4921ndash4925

1196 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

[6] L Coroller I Leguerinel P Mafart Effect of water activities of heatingand recovery media on apparent heat resistance of Bacillus cereus sporesAppl Environ Microbiol 67 (2001) 317ndash322

[7] C Laroche P Gervais Unexpected thermal destruction of dried glassbead-immobilized microorganisms as a function of water activity ApplEnviron Microbiol 69 (2003) 3015ndash3019

[8] EY Wuytack LD Phuong A Aertsen KM Reyns D Marquenie BDe Ketelaere B Masschalck I Van Opstal AM Diels CW MichielsComparison of sublethal injury induced in Salmonella enterica serovarTyphimurium by heat and by different nonthermal treatments J Food Prot66 (2003) 31ndash37

[9] RC Williams SS Sumner DA Golden Survival of Escherichiacoli O157H7 and salmonella in apple cider and orange juice asaffected by ozone and treatment temperature J Food Prot 67 (2004)2381ndash2386

[10] TB Hansen S Knochel Factors influencing resuscitation and growth ofheat injured Listeria monocytogenes 13-249 in sous vide cooked beef IntJ Food Microbiol 63 (2001) 135ndash147

[11] A Ikari M Nakano M Ishibashi K Kawano Y Suketa H Harada KTakagi Recovery from heat shock injury by activation of Na+ndashglucosecotransporter in renal epithelial cells Biochim Biophys Acta 1643 (2003)47ndash53

[12] E Glaasker FS Tjan PF Ter Steeg WN Konings B PoolmanPhysiological response of Lactobacillus plantarum to salt and nonelec-trolyte stress J Bacteriol 180 (1998) 4718ndash4723

[13] J Palmfeldt P Radstrom B Hahn-Hagerdal Optimisation of initial cellconcentration enhances freeze-drying tolerance of Pseudomonas chloro-raphis Cryobiology 47 (2003) 21ndash29

[14] MF Roberts Organic compatible solutes of halotolerant and halophilicmicroorganisms Saline Sys 1 (2005) 5

[15] AMolina-HoumlppnerWDosterRFVogelMGGaumlnzleProtectiveeffectofsucroseandsodiumchlorideforLactococcuslactisduringsublethalandlethalhigh-pressuretreatmentsApplEnvironMicrobiol70(2004)2013ndash2020

[16] G Zhao G Zhang Effect of protective agents freezing temperaturerehydration media on viability of malolactic bacteria subjected to freeze-drying J Appl Microbiol 99 (2005) 333ndash338

[17] LJM Linders WF Wolkers FA Hoekstra K Vant Riet Effect ofadded carbohydrates on membrane phase behavior and survival of driedLactobacillus plantarum Cryobiology 35 (1997) 31ndash40

[18] Y Mille L Beney P Gervais Viability of Escherichia coli after combinedosmotic and thermal treatment a plasma membrane implication BiochimBiophys Acta 1567 (2002) 41ndash48

[19] TJ Ahern AM Klibanov The mechanisms of reversible enzymeinactivation at 100 degC Science 228 (1985) 1280ndash1284

[20] GN Somero Proteins and temperature Annu Rev Physiol 57 (1995)43ndash68

[21] M Sola-Penna JR Meyer-Fernades Protective role of trehalose inthermal denaturation of yeast pyrophosphatase Z Naturforsch 49c (1994)327ndash330

[22] J Saad-Nehme AL Bezerra LA Fornells JL Silva JR Meyer-Fernandes A contribution of the mitochondrial adenosinetriphosphataseinhibitor protein to the thermal stability of the F0F1ndashATPase complexZ Naturforsch 52c (1997) 459ndash465

[23] SN Timasheff The control of protein stability and association by weak-interactions with watermdashHow do solvents affect these processes AnnuRev Biophys Biomol Struct 22 (1993) 67ndash97

[24] H-U Gremlich B Yan Infrared and raman spectroscopy of biologicalmaterials Marcel Dekker New York 2000

[25] H Fabian W Mantele Infrared spectroscopy of proteins Handbook ofVibrational Spectroscopy vol 5 John Wiley and Sons Chichester 2002pp 3424ndash3999

[26] K Murayama M Tomida Heat-induced secondary structure andconformation change of bovine serum albumin investigated by Fouriertransform infrared spectroscopy Biochemistry 43 (2004) 11526ndash11532

[27] SM Choi CY Ma Conformational study of globulin from commonbuckwheat (Fagopyrum esculentum Moench) by Fourier transforminfrared spectroscopy differential scanning calorimetry J Agric FoodChem 53 (2005) 8046ndash8053

[28] C Dirix T Duvetter A Van Loey M Hendrickx K Heremans The insitu observation of the temperature and pressure stability of recombinantAspergillus aculeatus pectin methylesterase with Fourier transforminfrared spectroscopy reveals an unusual pressure stability of beta-helicesBiochem J 392 (2005) 565ndash571

[29] SB Leslie E Israeli B Lighthart JH Crowe LM Crowe Trehaloseand sucrose protect both membranes and proteins in intact bacteria duringdrying Appl Environ Microbiol 61 (1995) 3592ndash3597

[30] A Molina-Houmlppner Physiological response of Lactococcus lactis to highpressure Doctoral thesis Technische Universitaumlt Muumlnchen Centre for Lifeand Food Sciences Weihenstephan 2002

[31] JM Jay MJ Loessner DA Golden (Eds) Modern food microbiologySpringer Berlin 2005

[32] A Barth C Zscherp What vibrations tell us about proteins Q RevBiophys 35 (2002) 369ndash430

[33] GR Snyder Vibrational spectra of crystalline n-paraffins J MolSpectrosc 7 (1961) 116ndash144

[34] O Reis R Winter TW Zerda The effect of high external pressure onDPPC-cholesterol multilamellar vesicles a pressure-tuning Fouriertransform infrared spectroscopy study Biochim Biophys Acta 1279(1996) 5ndash16

[35] Anonymous Determination of secondary structure in proteins by Fouriertransform infrared spectroscopy (FTIR) imb Jena without publishingDate download 27022005 URL httpwwwimb-jenadeImgLibDocftirIMAGE_FTIRhtml

[36] TM Raschke S Marqusee Hydrogen exchange studies of proteinstructure Curr Opin Biotechnol 9 (1998) 80ndash86

[37] E Goormaghtigh V Raussens JM Ruysschaert Attenuated totalreflection infrared spectroscopy of proteins and lipids in biologicalmembranes Biochim Biophys Acta 1422 (1999) 105ndash185

[38] H Susi The strength of hydrogen bonding infrared spectroscopyMethods A Compan Methods Enzymol 26 (Pt C) (1972) 381ndash391

[39] L Smeller P Rubens J Frank J Fidy K Heremans Two-dimensional Fourier-transform infrared correlation spectroscopy studyof the high-pressure tuning of proteins Vibr Spectrosc 22 (2000)119ndash125

[40] L Smeller F Meersman J Fidy K Heremans High-pressure FTIR studyof the stability of horseradish peroxidase Effect of heme substitutionligand binding Ca++ removal and reduction of the disulfide bondsBiochemistry 42 (2002) 553ndash561

[41] N Takeda M Kato Y Taniguchi Pressure-induced secondary structurechanges of ribonuclease A and ribonuclease S studied by FTIRspectroscopy Biospectroscopy 1 (1995) 207ndash216

[42] N Takeda M Kato Y Taniguchi Pressure and thermally-inducedreversible changes in the secondary structure of ribonuclease A studied byFTIR spectroscopy Biochemistry 34 (1995) 5980ndash5987

[43] M Jackson HH Mantsch Beware of proteins in DMSO BiochimBiophys Acta 1078 (1991) 231ndash235

[44] HM Ulmer H Herberhold S Fahsel MG Gaumlnzle R Winter RF VogelEffects of pressure-induced membrane phase transitions on inactivation ofHorA an ATP dependent multidrug resistance transporter in Lactobacillusplantarum Appl Environ Microbiol 68 (2002) 1088ndash1095

[45] M Jackson PI Haris D Chapman Fourier transform infraredspectroscopic studies of Ca(2+)-binding proteins Biochemistry 30(1991) 9681ndash9686

[46] S Luo CF Huang JF McClelland JD Graves A study of proteinsecondary structure by Fourier transform infraredphotoacoustic spectros-copy and its application for recombinant proteins Anal Biochem 216(1994) 67ndash76

[47] K Goosens L Smeller J Frank K Heremans Pressure-tuning theconformation of bovine pancreatic trypsin inhibitor studied by Fourier-transform infrared spectroscopy Eur J Biochem 236 (1996) 236ndash262

[48] R Gilmanshin M Gulotta RB Dyer RH Callender Structures ofapomyoglobins various acid-destabilized forms Biochemistry 40 (2001)5127ndash5136

[49] JH Crowe LM Crowe D Chapman Preservation of membranes inanhydrobiotic organismsmdashThe role of trehalose Science 223 (1984)701ndash703

Fig 8 Amide Iprime band of the infrared spectra obtained from L lactis at 32 degC and75 degC corresponding to viable and inactivated cells respectively The FT-IRspectra (A) and their second derivatives (B) were recorded in 2H2O-sucrose

Fig 9 Temperature effect on the different parameters describing thermalinactivation data of bacterial cell suspensions and temperature inducedconformational changes of the protein structure A intensity of the amide IIprimeband (1440 cmminus1) at elevated temperatures corrected for the peak intensity at30 degC B intensity of the amide Iprime band (1620 cmminus1) at elevated temperaturescorrected for the peak intensity at 30 degC C rate constants k describing lethal ()and sublethal () injury of the bacteria The filled symbols represent datagenerated in 2H2O-sucrose the empty symbols represent data generated in2H2O

1194 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

adapt to osmotic up-shift by accumulating compatible solutes[1213] This accumulation of solutes equilibrates theintracellular osmotic pressure to the environmental osmoticpressure and additionally provides protection to other stressconditions such as heat freezing drying and highhydrostatic pressure [56151718] The protective effect ofcompatible solutes on bacterial survival was previouslycorrelated with the protective effect of solutes on thebacterial membrane [151729] This study confirmed aprotective effect of sucrose on the bacterial survival andcytoplasmic membranes but the membrane phase transitionoccurred in the temperature range of 5ndash30 degC and thusappeared to be unrelated to the heat inactivation of L lactis

Conformational changes in the proteins of L lactis weredetermined with FT-IR spectroscopy The data obtained inthis work with L lactis are in excellent agreement with thespectra of purified proteins [32] and correspond to previousFT-IR measurements on bacterial cells [17]

The analysis of heat induced conformational changes ofthe proteins of L lactis enables a comparison of thetemperature effects on protein structure with bacterial survivaland sublethal injury (Fig 9) The difference in the intensitiesof the amide II and IIprime peaks (1550 cmminus1 and 1440 cmminus1

respectively) at elevated temperature compared to the peakintensity at 30 degC were used as an index for heat-induced1H2H exchange The buried residues of the protein structurebecame accessible to deuterium upon temperature up-shift andwere exchanged by the surrounding 2H2O (Figs 5 9A)[36373940] Modifications of the secondary structure of theproteins were detected by analysis of the amide Iprime band (Figs7 and 9B) To obtain a rate constant as an indicator for thetemperature effects on bacterial viability the experimentaldata on bacterial inactivation was fitted to a first orderequation (Fig 9C) Although first order models may beinadequate to describe inactivation curves with pronouncednon-linearities they have been widely used to describe

1195KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

thermal death time data and provided a reasonable agreementto our experimental data (correlation coefficient r2 gt087) Incontrast to more elaborate models that take into account non-linear survivor curves a simple first order model allows therepresentation of the inactivation kinetics by a singleparameter the rate constant k and thus greatly facilitatesthe comparison with the FT-IR data on protein structureHowever it should be stressed that the extensive washingsteps that were required to replace 1H2O by 2H2O for FT-IRspectroscopy of the cultures likely affected various aspects ofbacterial physiology

In the presence of sucrose the heat-induced structuralchanges in L lactis proteins detected by the amide Iprime and theamide IIprime bands were shifted to higher temperatures by about10 degC Inhibition of the 1H2H exchange by sucrose was moreprominent at lethal temperatures above 50 degC compared toambient temperatures of 30ndash45 degC (Figs 6 and 9) Thepresence of high (intracellular) sucrose concentration thusincreased the stability of L lactis proteins at denaturingtemperature Furthermore the bacterial viability was protectedby sucrose A temperature increase of about 10 degC wasrequired to achieve the same lethal effect in milk buffer sucrosecompared to milk buffer (Fig 9C)

The heat induced conformational changes of the L lactisproteins are related to the survival of this organism at lethaltemperatures No conformational changes in the L lactisproteins were detected by analysis of the amide Iprime band atsublethal temperatures The onset of conformational changescould be located between 50 and 60 degC in 2H2O and 2H2O-sucrose respectively These temperatures correspond to thelowest temperatures at which substantial inactivation of Llactis were detected in milk buffer and milk buffer withsucrose respectively (compare Fig 9B and C) Temperatureup-shift induced an increase of structural disorder andintermolecular β-sheet-structures in L lactis proteins a clearsign of irreversible protein aggregation and a decrease ofordered structural components mainly α-helices A moredetailed interpretation of the conformational changes ofprotein secondary structures is not feasible because thecombined spectra of the more than 2000 proteins expressedby L lactis [50] were determined and the structural changesof individual proteins overlap These results indicate heat-induced protein denaturation to an extent which is beyondthe cellular capacity of protein repair or de-novo synthesisand hence contributes to cell death The presence ofintracellular unordered or denatured proteins is attributableto heat denaturation of existing proteins or mis-translationoccurring at the ribosome Ribosome denaturation inEscherichia coli occurs in the range of 50ndash90 degC and waspreviously identified as a direct cause for heat-induced celldeath [51]

The 1H2H exchange was more susceptible to temperaturethan the spectral bands relating to protein secondarystructure Remarkably an increased 1H2H exchange wasobserved already in the range of 32ndash42 degC which is abovethe optimal temperature of growth but below the maximumgrowth temperature of L lactis This indicates that

conformational instabilities of proteins that may interferewith their respective functions occur already at temperaturespermitting growth of the organisms In keeping with thisinterpretation the primary response of L lactis to temper-ature upshift is the overexpression of heat shock proteinsincluding the DnaK-GrpE-DnaJ and the GroELS chaperonecomplexes [5253] In Lactobacillus sakei the induction ofheat shock proteins was apparent already at a temperature afew degrees above the optimum temperature of growth [54]The molecular chaperones are highly conserved families ofproteins that maintain or restore native protein conformationeven at high temperatures or assist in the degradation ofunfolded and aggregated proteins [55] As the ribosome hasbeen described as (heat) stress sensor and ribosome functionis coupled to protein quality control via trans-translation theexpression of chaperons is also a reaction to impairedtranslation by stalled ribosomes at sublethal stress tempera-tures [356]

In conclusion in this work demonstrated that molarconcentrations of sucrose increase the heat resistance of Llactis This finding extends previous findings on the role ofcompatible solutes for the bacterial resistance to freezingdrying and heat Additionally it demonstrates that highintracellular sucrose concentrations prevent the denaturationor misfolding of L lactis proteins Remarkably the use ofFT-IR spectroscopy enabled the detection of proteinconformational instabilities occurring already at elevatedtemperatures within the growth range of L lactis It isanticipated that these results will facilitate the process designfor thermal treatments to eliminate or stabilise bacterial cellsin food and biomedical applications

Acknowledgements

We gratefully acknowledge the support of DeutscheForschungsgemeinschaft Grant No FOR 3582 and SFB 533B11 and we wish to thank Gesa Groumlhnke for her excellentlaboratory work

References

[1] CE Dodd TG Aldsworth The importance of RpoS in the survival ofbacteria through food processing Int J Food Microbiol 74 (2002)189ndash194

[2] JD Taylor-Robinson M Child R Pickup P Strike C Edwards Cellndashcell interactions influence resistance and survival of Salmonella serotypeTyphimurium to environmental stress J Appl Microbiol 94 (2003)95ndash102

[3] M De Angelis R Di Cagno C Huet C Crecchio PF Fox M GobbettiHeat shock response in Lactobacillus plantarum Appl EnvironMicrobiol 70 (2004) 1336ndash1346

[4] CW Blackburn LM Curtis L Humpheson C Billon PJ McClureDevelopment of thermal inactivation models for Salmonella enteritidis andEscherichia coli O157H7 with temperature pH and NaCl as controllingfactors Int J Food Microbiol 38 (1997) 31ndash44

[5] KLMattick F Jorgensen JD Legan HM Lappin-Scott TJ HumphreyHabituation of Salmonella spp at reduced water activity and its effect onheat tolerance Appl Environ Microbiol 66 (2000) 4921ndash4925

1196 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

[6] L Coroller I Leguerinel P Mafart Effect of water activities of heatingand recovery media on apparent heat resistance of Bacillus cereus sporesAppl Environ Microbiol 67 (2001) 317ndash322

[7] C Laroche P Gervais Unexpected thermal destruction of dried glassbead-immobilized microorganisms as a function of water activity ApplEnviron Microbiol 69 (2003) 3015ndash3019

[8] EY Wuytack LD Phuong A Aertsen KM Reyns D Marquenie BDe Ketelaere B Masschalck I Van Opstal AM Diels CW MichielsComparison of sublethal injury induced in Salmonella enterica serovarTyphimurium by heat and by different nonthermal treatments J Food Prot66 (2003) 31ndash37

[9] RC Williams SS Sumner DA Golden Survival of Escherichiacoli O157H7 and salmonella in apple cider and orange juice asaffected by ozone and treatment temperature J Food Prot 67 (2004)2381ndash2386

[10] TB Hansen S Knochel Factors influencing resuscitation and growth ofheat injured Listeria monocytogenes 13-249 in sous vide cooked beef IntJ Food Microbiol 63 (2001) 135ndash147

[11] A Ikari M Nakano M Ishibashi K Kawano Y Suketa H Harada KTakagi Recovery from heat shock injury by activation of Na+ndashglucosecotransporter in renal epithelial cells Biochim Biophys Acta 1643 (2003)47ndash53

[12] E Glaasker FS Tjan PF Ter Steeg WN Konings B PoolmanPhysiological response of Lactobacillus plantarum to salt and nonelec-trolyte stress J Bacteriol 180 (1998) 4718ndash4723

[13] J Palmfeldt P Radstrom B Hahn-Hagerdal Optimisation of initial cellconcentration enhances freeze-drying tolerance of Pseudomonas chloro-raphis Cryobiology 47 (2003) 21ndash29

[14] MF Roberts Organic compatible solutes of halotolerant and halophilicmicroorganisms Saline Sys 1 (2005) 5

[15] AMolina-HoumlppnerWDosterRFVogelMGGaumlnzleProtectiveeffectofsucroseandsodiumchlorideforLactococcuslactisduringsublethalandlethalhigh-pressuretreatmentsApplEnvironMicrobiol70(2004)2013ndash2020

[16] G Zhao G Zhang Effect of protective agents freezing temperaturerehydration media on viability of malolactic bacteria subjected to freeze-drying J Appl Microbiol 99 (2005) 333ndash338

[17] LJM Linders WF Wolkers FA Hoekstra K Vant Riet Effect ofadded carbohydrates on membrane phase behavior and survival of driedLactobacillus plantarum Cryobiology 35 (1997) 31ndash40

[18] Y Mille L Beney P Gervais Viability of Escherichia coli after combinedosmotic and thermal treatment a plasma membrane implication BiochimBiophys Acta 1567 (2002) 41ndash48

[19] TJ Ahern AM Klibanov The mechanisms of reversible enzymeinactivation at 100 degC Science 228 (1985) 1280ndash1284

[20] GN Somero Proteins and temperature Annu Rev Physiol 57 (1995)43ndash68

[21] M Sola-Penna JR Meyer-Fernades Protective role of trehalose inthermal denaturation of yeast pyrophosphatase Z Naturforsch 49c (1994)327ndash330

[22] J Saad-Nehme AL Bezerra LA Fornells JL Silva JR Meyer-Fernandes A contribution of the mitochondrial adenosinetriphosphataseinhibitor protein to the thermal stability of the F0F1ndashATPase complexZ Naturforsch 52c (1997) 459ndash465

[23] SN Timasheff The control of protein stability and association by weak-interactions with watermdashHow do solvents affect these processes AnnuRev Biophys Biomol Struct 22 (1993) 67ndash97

[24] H-U Gremlich B Yan Infrared and raman spectroscopy of biologicalmaterials Marcel Dekker New York 2000

[25] H Fabian W Mantele Infrared spectroscopy of proteins Handbook ofVibrational Spectroscopy vol 5 John Wiley and Sons Chichester 2002pp 3424ndash3999

[26] K Murayama M Tomida Heat-induced secondary structure andconformation change of bovine serum albumin investigated by Fouriertransform infrared spectroscopy Biochemistry 43 (2004) 11526ndash11532

[27] SM Choi CY Ma Conformational study of globulin from commonbuckwheat (Fagopyrum esculentum Moench) by Fourier transforminfrared spectroscopy differential scanning calorimetry J Agric FoodChem 53 (2005) 8046ndash8053

[28] C Dirix T Duvetter A Van Loey M Hendrickx K Heremans The insitu observation of the temperature and pressure stability of recombinantAspergillus aculeatus pectin methylesterase with Fourier transforminfrared spectroscopy reveals an unusual pressure stability of beta-helicesBiochem J 392 (2005) 565ndash571

[29] SB Leslie E Israeli B Lighthart JH Crowe LM Crowe Trehaloseand sucrose protect both membranes and proteins in intact bacteria duringdrying Appl Environ Microbiol 61 (1995) 3592ndash3597

[30] A Molina-Houmlppner Physiological response of Lactococcus lactis to highpressure Doctoral thesis Technische Universitaumlt Muumlnchen Centre for Lifeand Food Sciences Weihenstephan 2002

[31] JM Jay MJ Loessner DA Golden (Eds) Modern food microbiologySpringer Berlin 2005

[32] A Barth C Zscherp What vibrations tell us about proteins Q RevBiophys 35 (2002) 369ndash430

[33] GR Snyder Vibrational spectra of crystalline n-paraffins J MolSpectrosc 7 (1961) 116ndash144

[34] O Reis R Winter TW Zerda The effect of high external pressure onDPPC-cholesterol multilamellar vesicles a pressure-tuning Fouriertransform infrared spectroscopy study Biochim Biophys Acta 1279(1996) 5ndash16

[35] Anonymous Determination of secondary structure in proteins by Fouriertransform infrared spectroscopy (FTIR) imb Jena without publishingDate download 27022005 URL httpwwwimb-jenadeImgLibDocftirIMAGE_FTIRhtml

[36] TM Raschke S Marqusee Hydrogen exchange studies of proteinstructure Curr Opin Biotechnol 9 (1998) 80ndash86

[37] E Goormaghtigh V Raussens JM Ruysschaert Attenuated totalreflection infrared spectroscopy of proteins and lipids in biologicalmembranes Biochim Biophys Acta 1422 (1999) 105ndash185

[38] H Susi The strength of hydrogen bonding infrared spectroscopyMethods A Compan Methods Enzymol 26 (Pt C) (1972) 381ndash391

[39] L Smeller P Rubens J Frank J Fidy K Heremans Two-dimensional Fourier-transform infrared correlation spectroscopy studyof the high-pressure tuning of proteins Vibr Spectrosc 22 (2000)119ndash125

[40] L Smeller F Meersman J Fidy K Heremans High-pressure FTIR studyof the stability of horseradish peroxidase Effect of heme substitutionligand binding Ca++ removal and reduction of the disulfide bondsBiochemistry 42 (2002) 553ndash561

[41] N Takeda M Kato Y Taniguchi Pressure-induced secondary structurechanges of ribonuclease A and ribonuclease S studied by FTIRspectroscopy Biospectroscopy 1 (1995) 207ndash216

[42] N Takeda M Kato Y Taniguchi Pressure and thermally-inducedreversible changes in the secondary structure of ribonuclease A studied byFTIR spectroscopy Biochemistry 34 (1995) 5980ndash5987

[43] M Jackson HH Mantsch Beware of proteins in DMSO BiochimBiophys Acta 1078 (1991) 231ndash235

[44] HM Ulmer H Herberhold S Fahsel MG Gaumlnzle R Winter RF VogelEffects of pressure-induced membrane phase transitions on inactivation ofHorA an ATP dependent multidrug resistance transporter in Lactobacillusplantarum Appl Environ Microbiol 68 (2002) 1088ndash1095

[45] M Jackson PI Haris D Chapman Fourier transform infraredspectroscopic studies of Ca(2+)-binding proteins Biochemistry 30(1991) 9681ndash9686

[46] S Luo CF Huang JF McClelland JD Graves A study of proteinsecondary structure by Fourier transform infraredphotoacoustic spectros-copy and its application for recombinant proteins Anal Biochem 216(1994) 67ndash76

[47] K Goosens L Smeller J Frank K Heremans Pressure-tuning theconformation of bovine pancreatic trypsin inhibitor studied by Fourier-transform infrared spectroscopy Eur J Biochem 236 (1996) 236ndash262

[48] R Gilmanshin M Gulotta RB Dyer RH Callender Structures ofapomyoglobins various acid-destabilized forms Biochemistry 40 (2001)5127ndash5136

[49] JH Crowe LM Crowe D Chapman Preservation of membranes inanhydrobiotic organismsmdashThe role of trehalose Science 223 (1984)701ndash703

1195KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

thermal death time data and provided a reasonable agreementto our experimental data (correlation coefficient r2 gt087) Incontrast to more elaborate models that take into account non-linear survivor curves a simple first order model allows therepresentation of the inactivation kinetics by a singleparameter the rate constant k and thus greatly facilitatesthe comparison with the FT-IR data on protein structureHowever it should be stressed that the extensive washingsteps that were required to replace 1H2O by 2H2O for FT-IRspectroscopy of the cultures likely affected various aspects ofbacterial physiology

In the presence of sucrose the heat-induced structuralchanges in L lactis proteins detected by the amide Iprime and theamide IIprime bands were shifted to higher temperatures by about10 degC Inhibition of the 1H2H exchange by sucrose was moreprominent at lethal temperatures above 50 degC compared toambient temperatures of 30ndash45 degC (Figs 6 and 9) Thepresence of high (intracellular) sucrose concentration thusincreased the stability of L lactis proteins at denaturingtemperature Furthermore the bacterial viability was protectedby sucrose A temperature increase of about 10 degC wasrequired to achieve the same lethal effect in milk buffer sucrosecompared to milk buffer (Fig 9C)

The heat induced conformational changes of the L lactisproteins are related to the survival of this organism at lethaltemperatures No conformational changes in the L lactisproteins were detected by analysis of the amide Iprime band atsublethal temperatures The onset of conformational changescould be located between 50 and 60 degC in 2H2O and 2H2O-sucrose respectively These temperatures correspond to thelowest temperatures at which substantial inactivation of Llactis were detected in milk buffer and milk buffer withsucrose respectively (compare Fig 9B and C) Temperatureup-shift induced an increase of structural disorder andintermolecular β-sheet-structures in L lactis proteins a clearsign of irreversible protein aggregation and a decrease ofordered structural components mainly α-helices A moredetailed interpretation of the conformational changes ofprotein secondary structures is not feasible because thecombined spectra of the more than 2000 proteins expressedby L lactis [50] were determined and the structural changesof individual proteins overlap These results indicate heat-induced protein denaturation to an extent which is beyondthe cellular capacity of protein repair or de-novo synthesisand hence contributes to cell death The presence ofintracellular unordered or denatured proteins is attributableto heat denaturation of existing proteins or mis-translationoccurring at the ribosome Ribosome denaturation inEscherichia coli occurs in the range of 50ndash90 degC and waspreviously identified as a direct cause for heat-induced celldeath [51]

The 1H2H exchange was more susceptible to temperaturethan the spectral bands relating to protein secondarystructure Remarkably an increased 1H2H exchange wasobserved already in the range of 32ndash42 degC which is abovethe optimal temperature of growth but below the maximumgrowth temperature of L lactis This indicates that

conformational instabilities of proteins that may interferewith their respective functions occur already at temperaturespermitting growth of the organisms In keeping with thisinterpretation the primary response of L lactis to temper-ature upshift is the overexpression of heat shock proteinsincluding the DnaK-GrpE-DnaJ and the GroELS chaperonecomplexes [5253] In Lactobacillus sakei the induction ofheat shock proteins was apparent already at a temperature afew degrees above the optimum temperature of growth [54]The molecular chaperones are highly conserved families ofproteins that maintain or restore native protein conformationeven at high temperatures or assist in the degradation ofunfolded and aggregated proteins [55] As the ribosome hasbeen described as (heat) stress sensor and ribosome functionis coupled to protein quality control via trans-translation theexpression of chaperons is also a reaction to impairedtranslation by stalled ribosomes at sublethal stress tempera-tures [356]

In conclusion in this work demonstrated that molarconcentrations of sucrose increase the heat resistance of Llactis This finding extends previous findings on the role ofcompatible solutes for the bacterial resistance to freezingdrying and heat Additionally it demonstrates that highintracellular sucrose concentrations prevent the denaturationor misfolding of L lactis proteins Remarkably the use ofFT-IR spectroscopy enabled the detection of proteinconformational instabilities occurring already at elevatedtemperatures within the growth range of L lactis It isanticipated that these results will facilitate the process designfor thermal treatments to eliminate or stabilise bacterial cellsin food and biomedical applications

Acknowledgements

We gratefully acknowledge the support of DeutscheForschungsgemeinschaft Grant No FOR 3582 and SFB 533B11 and we wish to thank Gesa Groumlhnke for her excellentlaboratory work

References

[1] CE Dodd TG Aldsworth The importance of RpoS in the survival ofbacteria through food processing Int J Food Microbiol 74 (2002)189ndash194

[2] JD Taylor-Robinson M Child R Pickup P Strike C Edwards Cellndashcell interactions influence resistance and survival of Salmonella serotypeTyphimurium to environmental stress J Appl Microbiol 94 (2003)95ndash102

[3] M De Angelis R Di Cagno C Huet C Crecchio PF Fox M GobbettiHeat shock response in Lactobacillus plantarum Appl EnvironMicrobiol 70 (2004) 1336ndash1346

[4] CW Blackburn LM Curtis L Humpheson C Billon PJ McClureDevelopment of thermal inactivation models for Salmonella enteritidis andEscherichia coli O157H7 with temperature pH and NaCl as controllingfactors Int J Food Microbiol 38 (1997) 31ndash44

[5] KLMattick F Jorgensen JD Legan HM Lappin-Scott TJ HumphreyHabituation of Salmonella spp at reduced water activity and its effect onheat tolerance Appl Environ Microbiol 66 (2000) 4921ndash4925

1196 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

[6] L Coroller I Leguerinel P Mafart Effect of water activities of heatingand recovery media on apparent heat resistance of Bacillus cereus sporesAppl Environ Microbiol 67 (2001) 317ndash322

[7] C Laroche P Gervais Unexpected thermal destruction of dried glassbead-immobilized microorganisms as a function of water activity ApplEnviron Microbiol 69 (2003) 3015ndash3019

[8] EY Wuytack LD Phuong A Aertsen KM Reyns D Marquenie BDe Ketelaere B Masschalck I Van Opstal AM Diels CW MichielsComparison of sublethal injury induced in Salmonella enterica serovarTyphimurium by heat and by different nonthermal treatments J Food Prot66 (2003) 31ndash37

[9] RC Williams SS Sumner DA Golden Survival of Escherichiacoli O157H7 and salmonella in apple cider and orange juice asaffected by ozone and treatment temperature J Food Prot 67 (2004)2381ndash2386

[10] TB Hansen S Knochel Factors influencing resuscitation and growth ofheat injured Listeria monocytogenes 13-249 in sous vide cooked beef IntJ Food Microbiol 63 (2001) 135ndash147

[11] A Ikari M Nakano M Ishibashi K Kawano Y Suketa H Harada KTakagi Recovery from heat shock injury by activation of Na+ndashglucosecotransporter in renal epithelial cells Biochim Biophys Acta 1643 (2003)47ndash53

[12] E Glaasker FS Tjan PF Ter Steeg WN Konings B PoolmanPhysiological response of Lactobacillus plantarum to salt and nonelec-trolyte stress J Bacteriol 180 (1998) 4718ndash4723

[13] J Palmfeldt P Radstrom B Hahn-Hagerdal Optimisation of initial cellconcentration enhances freeze-drying tolerance of Pseudomonas chloro-raphis Cryobiology 47 (2003) 21ndash29

[14] MF Roberts Organic compatible solutes of halotolerant and halophilicmicroorganisms Saline Sys 1 (2005) 5

[15] AMolina-HoumlppnerWDosterRFVogelMGGaumlnzleProtectiveeffectofsucroseandsodiumchlorideforLactococcuslactisduringsublethalandlethalhigh-pressuretreatmentsApplEnvironMicrobiol70(2004)2013ndash2020

[16] G Zhao G Zhang Effect of protective agents freezing temperaturerehydration media on viability of malolactic bacteria subjected to freeze-drying J Appl Microbiol 99 (2005) 333ndash338

[17] LJM Linders WF Wolkers FA Hoekstra K Vant Riet Effect ofadded carbohydrates on membrane phase behavior and survival of driedLactobacillus plantarum Cryobiology 35 (1997) 31ndash40

[18] Y Mille L Beney P Gervais Viability of Escherichia coli after combinedosmotic and thermal treatment a plasma membrane implication BiochimBiophys Acta 1567 (2002) 41ndash48

[19] TJ Ahern AM Klibanov The mechanisms of reversible enzymeinactivation at 100 degC Science 228 (1985) 1280ndash1284

[20] GN Somero Proteins and temperature Annu Rev Physiol 57 (1995)43ndash68

[21] M Sola-Penna JR Meyer-Fernades Protective role of trehalose inthermal denaturation of yeast pyrophosphatase Z Naturforsch 49c (1994)327ndash330

[22] J Saad-Nehme AL Bezerra LA Fornells JL Silva JR Meyer-Fernandes A contribution of the mitochondrial adenosinetriphosphataseinhibitor protein to the thermal stability of the F0F1ndashATPase complexZ Naturforsch 52c (1997) 459ndash465

[23] SN Timasheff The control of protein stability and association by weak-interactions with watermdashHow do solvents affect these processes AnnuRev Biophys Biomol Struct 22 (1993) 67ndash97

[24] H-U Gremlich B Yan Infrared and raman spectroscopy of biologicalmaterials Marcel Dekker New York 2000

[25] H Fabian W Mantele Infrared spectroscopy of proteins Handbook ofVibrational Spectroscopy vol 5 John Wiley and Sons Chichester 2002pp 3424ndash3999

[26] K Murayama M Tomida Heat-induced secondary structure andconformation change of bovine serum albumin investigated by Fouriertransform infrared spectroscopy Biochemistry 43 (2004) 11526ndash11532

[27] SM Choi CY Ma Conformational study of globulin from commonbuckwheat (Fagopyrum esculentum Moench) by Fourier transforminfrared spectroscopy differential scanning calorimetry J Agric FoodChem 53 (2005) 8046ndash8053

[28] C Dirix T Duvetter A Van Loey M Hendrickx K Heremans The insitu observation of the temperature and pressure stability of recombinantAspergillus aculeatus pectin methylesterase with Fourier transforminfrared spectroscopy reveals an unusual pressure stability of beta-helicesBiochem J 392 (2005) 565ndash571

[29] SB Leslie E Israeli B Lighthart JH Crowe LM Crowe Trehaloseand sucrose protect both membranes and proteins in intact bacteria duringdrying Appl Environ Microbiol 61 (1995) 3592ndash3597

[30] A Molina-Houmlppner Physiological response of Lactococcus lactis to highpressure Doctoral thesis Technische Universitaumlt Muumlnchen Centre for Lifeand Food Sciences Weihenstephan 2002

[31] JM Jay MJ Loessner DA Golden (Eds) Modern food microbiologySpringer Berlin 2005

[32] A Barth C Zscherp What vibrations tell us about proteins Q RevBiophys 35 (2002) 369ndash430

[33] GR Snyder Vibrational spectra of crystalline n-paraffins J MolSpectrosc 7 (1961) 116ndash144

[34] O Reis R Winter TW Zerda The effect of high external pressure onDPPC-cholesterol multilamellar vesicles a pressure-tuning Fouriertransform infrared spectroscopy study Biochim Biophys Acta 1279(1996) 5ndash16

[35] Anonymous Determination of secondary structure in proteins by Fouriertransform infrared spectroscopy (FTIR) imb Jena without publishingDate download 27022005 URL httpwwwimb-jenadeImgLibDocftirIMAGE_FTIRhtml

[36] TM Raschke S Marqusee Hydrogen exchange studies of proteinstructure Curr Opin Biotechnol 9 (1998) 80ndash86

[37] E Goormaghtigh V Raussens JM Ruysschaert Attenuated totalreflection infrared spectroscopy of proteins and lipids in biologicalmembranes Biochim Biophys Acta 1422 (1999) 105ndash185

[38] H Susi The strength of hydrogen bonding infrared spectroscopyMethods A Compan Methods Enzymol 26 (Pt C) (1972) 381ndash391

[39] L Smeller P Rubens J Frank J Fidy K Heremans Two-dimensional Fourier-transform infrared correlation spectroscopy studyof the high-pressure tuning of proteins Vibr Spectrosc 22 (2000)119ndash125

[40] L Smeller F Meersman J Fidy K Heremans High-pressure FTIR studyof the stability of horseradish peroxidase Effect of heme substitutionligand binding Ca++ removal and reduction of the disulfide bondsBiochemistry 42 (2002) 553ndash561

[41] N Takeda M Kato Y Taniguchi Pressure-induced secondary structurechanges of ribonuclease A and ribonuclease S studied by FTIRspectroscopy Biospectroscopy 1 (1995) 207ndash216

[42] N Takeda M Kato Y Taniguchi Pressure and thermally-inducedreversible changes in the secondary structure of ribonuclease A studied byFTIR spectroscopy Biochemistry 34 (1995) 5980ndash5987

[43] M Jackson HH Mantsch Beware of proteins in DMSO BiochimBiophys Acta 1078 (1991) 231ndash235

[44] HM Ulmer H Herberhold S Fahsel MG Gaumlnzle R Winter RF VogelEffects of pressure-induced membrane phase transitions on inactivation ofHorA an ATP dependent multidrug resistance transporter in Lactobacillusplantarum Appl Environ Microbiol 68 (2002) 1088ndash1095

[45] M Jackson PI Haris D Chapman Fourier transform infraredspectroscopic studies of Ca(2+)-binding proteins Biochemistry 30(1991) 9681ndash9686

[46] S Luo CF Huang JF McClelland JD Graves A study of proteinsecondary structure by Fourier transform infraredphotoacoustic spectros-copy and its application for recombinant proteins Anal Biochem 216(1994) 67ndash76

[47] K Goosens L Smeller J Frank K Heremans Pressure-tuning theconformation of bovine pancreatic trypsin inhibitor studied by Fourier-transform infrared spectroscopy Eur J Biochem 236 (1996) 236ndash262

[48] R Gilmanshin M Gulotta RB Dyer RH Callender Structures ofapomyoglobins various acid-destabilized forms Biochemistry 40 (2001)5127ndash5136

[49] JH Crowe LM Crowe D Chapman Preservation of membranes inanhydrobiotic organismsmdashThe role of trehalose Science 223 (1984)701ndash703

1196 KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

[6] L Coroller I Leguerinel P Mafart Effect of water activities of heatingand recovery media on apparent heat resistance of Bacillus cereus sporesAppl Environ Microbiol 67 (2001) 317ndash322

[7] C Laroche P Gervais Unexpected thermal destruction of dried glassbead-immobilized microorganisms as a function of water activity ApplEnviron Microbiol 69 (2003) 3015ndash3019

[8] EY Wuytack LD Phuong A Aertsen KM Reyns D Marquenie BDe Ketelaere B Masschalck I Van Opstal AM Diels CW MichielsComparison of sublethal injury induced in Salmonella enterica serovarTyphimurium by heat and by different nonthermal treatments J Food Prot66 (2003) 31ndash37

[9] RC Williams SS Sumner DA Golden Survival of Escherichiacoli O157H7 and salmonella in apple cider and orange juice asaffected by ozone and treatment temperature J Food Prot 67 (2004)2381ndash2386

[10] TB Hansen S Knochel Factors influencing resuscitation and growth ofheat injured Listeria monocytogenes 13-249 in sous vide cooked beef IntJ Food Microbiol 63 (2001) 135ndash147

[11] A Ikari M Nakano M Ishibashi K Kawano Y Suketa H Harada KTakagi Recovery from heat shock injury by activation of Na+ndashglucosecotransporter in renal epithelial cells Biochim Biophys Acta 1643 (2003)47ndash53

[12] E Glaasker FS Tjan PF Ter Steeg WN Konings B PoolmanPhysiological response of Lactobacillus plantarum to salt and nonelec-trolyte stress J Bacteriol 180 (1998) 4718ndash4723

[13] J Palmfeldt P Radstrom B Hahn-Hagerdal Optimisation of initial cellconcentration enhances freeze-drying tolerance of Pseudomonas chloro-raphis Cryobiology 47 (2003) 21ndash29

[14] MF Roberts Organic compatible solutes of halotolerant and halophilicmicroorganisms Saline Sys 1 (2005) 5

[15] AMolina-HoumlppnerWDosterRFVogelMGGaumlnzleProtectiveeffectofsucroseandsodiumchlorideforLactococcuslactisduringsublethalandlethalhigh-pressuretreatmentsApplEnvironMicrobiol70(2004)2013ndash2020

[16] G Zhao G Zhang Effect of protective agents freezing temperaturerehydration media on viability of malolactic bacteria subjected to freeze-drying J Appl Microbiol 99 (2005) 333ndash338

[17] LJM Linders WF Wolkers FA Hoekstra K Vant Riet Effect ofadded carbohydrates on membrane phase behavior and survival of driedLactobacillus plantarum Cryobiology 35 (1997) 31ndash40

[18] Y Mille L Beney P Gervais Viability of Escherichia coli after combinedosmotic and thermal treatment a plasma membrane implication BiochimBiophys Acta 1567 (2002) 41ndash48

[19] TJ Ahern AM Klibanov The mechanisms of reversible enzymeinactivation at 100 degC Science 228 (1985) 1280ndash1284

[20] GN Somero Proteins and temperature Annu Rev Physiol 57 (1995)43ndash68

[21] M Sola-Penna JR Meyer-Fernades Protective role of trehalose inthermal denaturation of yeast pyrophosphatase Z Naturforsch 49c (1994)327ndash330

[22] J Saad-Nehme AL Bezerra LA Fornells JL Silva JR Meyer-Fernandes A contribution of the mitochondrial adenosinetriphosphataseinhibitor protein to the thermal stability of the F0F1ndashATPase complexZ Naturforsch 52c (1997) 459ndash465

[23] SN Timasheff The control of protein stability and association by weak-interactions with watermdashHow do solvents affect these processes AnnuRev Biophys Biomol Struct 22 (1993) 67ndash97

[24] H-U Gremlich B Yan Infrared and raman spectroscopy of biologicalmaterials Marcel Dekker New York 2000

[25] H Fabian W Mantele Infrared spectroscopy of proteins Handbook ofVibrational Spectroscopy vol 5 John Wiley and Sons Chichester 2002pp 3424ndash3999

[26] K Murayama M Tomida Heat-induced secondary structure andconformation change of bovine serum albumin investigated by Fouriertransform infrared spectroscopy Biochemistry 43 (2004) 11526ndash11532

[27] SM Choi CY Ma Conformational study of globulin from commonbuckwheat (Fagopyrum esculentum Moench) by Fourier transforminfrared spectroscopy differential scanning calorimetry J Agric FoodChem 53 (2005) 8046ndash8053

[28] C Dirix T Duvetter A Van Loey M Hendrickx K Heremans The insitu observation of the temperature and pressure stability of recombinantAspergillus aculeatus pectin methylesterase with Fourier transforminfrared spectroscopy reveals an unusual pressure stability of beta-helicesBiochem J 392 (2005) 565ndash571

[29] SB Leslie E Israeli B Lighthart JH Crowe LM Crowe Trehaloseand sucrose protect both membranes and proteins in intact bacteria duringdrying Appl Environ Microbiol 61 (1995) 3592ndash3597

[30] A Molina-Houmlppner Physiological response of Lactococcus lactis to highpressure Doctoral thesis Technische Universitaumlt Muumlnchen Centre for Lifeand Food Sciences Weihenstephan 2002

[31] JM Jay MJ Loessner DA Golden (Eds) Modern food microbiologySpringer Berlin 2005

[32] A Barth C Zscherp What vibrations tell us about proteins Q RevBiophys 35 (2002) 369ndash430

[33] GR Snyder Vibrational spectra of crystalline n-paraffins J MolSpectrosc 7 (1961) 116ndash144

[34] O Reis R Winter TW Zerda The effect of high external pressure onDPPC-cholesterol multilamellar vesicles a pressure-tuning Fouriertransform infrared spectroscopy study Biochim Biophys Acta 1279(1996) 5ndash16

[35] Anonymous Determination of secondary structure in proteins by Fouriertransform infrared spectroscopy (FTIR) imb Jena without publishingDate download 27022005 URL httpwwwimb-jenadeImgLibDocftirIMAGE_FTIRhtml

[36] TM Raschke S Marqusee Hydrogen exchange studies of proteinstructure Curr Opin Biotechnol 9 (1998) 80ndash86

[37] E Goormaghtigh V Raussens JM Ruysschaert Attenuated totalreflection infrared spectroscopy of proteins and lipids in biologicalmembranes Biochim Biophys Acta 1422 (1999) 105ndash185

[38] H Susi The strength of hydrogen bonding infrared spectroscopyMethods A Compan Methods Enzymol 26 (Pt C) (1972) 381ndash391

[39] L Smeller P Rubens J Frank J Fidy K Heremans Two-dimensional Fourier-transform infrared correlation spectroscopy studyof the high-pressure tuning of proteins Vibr Spectrosc 22 (2000)119ndash125

[40] L Smeller F Meersman J Fidy K Heremans High-pressure FTIR studyof the stability of horseradish peroxidase Effect of heme substitutionligand binding Ca++ removal and reduction of the disulfide bondsBiochemistry 42 (2002) 553ndash561

[41] N Takeda M Kato Y Taniguchi Pressure-induced secondary structurechanges of ribonuclease A and ribonuclease S studied by FTIRspectroscopy Biospectroscopy 1 (1995) 207ndash216

[42] N Takeda M Kato Y Taniguchi Pressure and thermally-inducedreversible changes in the secondary structure of ribonuclease A studied byFTIR spectroscopy Biochemistry 34 (1995) 5980ndash5987

[43] M Jackson HH Mantsch Beware of proteins in DMSO BiochimBiophys Acta 1078 (1991) 231ndash235

[44] HM Ulmer H Herberhold S Fahsel MG Gaumlnzle R Winter RF VogelEffects of pressure-induced membrane phase transitions on inactivation ofHorA an ATP dependent multidrug resistance transporter in Lactobacillusplantarum Appl Environ Microbiol 68 (2002) 1088ndash1095

[45] M Jackson PI Haris D Chapman Fourier transform infraredspectroscopic studies of Ca(2+)-binding proteins Biochemistry 30(1991) 9681ndash9686

[46] S Luo CF Huang JF McClelland JD Graves A study of proteinsecondary structure by Fourier transform infraredphotoacoustic spectros-copy and its application for recombinant proteins Anal Biochem 216(1994) 67ndash76

[47] K Goosens L Smeller J Frank K Heremans Pressure-tuning theconformation of bovine pancreatic trypsin inhibitor studied by Fourier-transform infrared spectroscopy Eur J Biochem 236 (1996) 236ndash262

[48] R Gilmanshin M Gulotta RB Dyer RH Callender Structures ofapomyoglobins various acid-destabilized forms Biochemistry 40 (2001)5127ndash5136

[49] JH Crowe LM Crowe D Chapman Preservation of membranes inanhydrobiotic organismsmdashThe role of trehalose Science 223 (1984)701ndash703

1197KV Kilimann et al Biochimica et Biophysica Acta 1764 (2006) 1188ndash1197

[50] O Drews G Reil H Parlar A Goumlrg Setting up standards and a referencemap for the alkaline proteome of the Gram-positive bacterium Lactococcuslactis Proteomics 4 (2004) 1293ndash1304

[51] TT Nielen S Molin Role of ribosome degradation in the death of heat-stressed Salmonella typhimurium FEMS Microbiol Lett 142 (1996)155ndash160

[52] B Koch M Kilstrup FK Vogensen K Hammer Induces levels of heatshock proteins in a dnaK mutant of Lactococcus lactis J Bacteriol 180(1998) 3873ndash3881

[53] RD Whitaker CA Batt Characterization of the heat shock response inLactococcus lactis subsp lactis Appl EnvironMicrobiol 57 (1991) 1408ndash1412

[54] G Schmidt C Hertel WP Hammes Molecular characterisation of thednaK operon of Lactobacillus sakei LTH681 Syst Appl Microbiol 22(1999) 321ndash328

[55] X Zhang F Beuron PS Freemont Machinery of protein folding andunfolding Curr Opin Struct Biol 12 (2002) 231ndash238

[56] JH Whithey DI Friedman The biological roles of trans-translationCurr Opin Microbiol 5 (2002) 154ndash159