International Journal of Food Microbiology 197 (2015) 4552

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r .com/ locate / i j foodmicroCarvacrol suppresses high pressure high temperature inactivation ofBacillus cereus sporesHue Luu-Thi a, Jorinde Corthouts a, Ioannis Passaris a, Tara Grauwet b, Abram Aertsen a,Marc Hendrickx b, Chris W. Michiels a,a Laboratory of Food Microbiology, Leuven Food Science and Nutrition Research Center (LFoRCe), Department of Microbial and Molecular Systems (M2S), KU Leuven, Kasteelpark Arenberg 22,B-3001 Heverlee, Belgiumb Laboratory of Food Technology, Leuven Food Science and Nutrition Research Center (LFoRCe), Department of Microbial and Molecular Systems (M2S), KU Leuven, Kasteelpark Arenberg 22,B-3001 Heverlee, Belgium Corresponding author. Tel.: +32 16 321578; fax: +3E-mail address: Chris.michiels@biw.kuleuven.be (C.W

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.12.0160168-1605/ 2014 Elsevier B.V. All rights reserved.a b s t r a c ta r t i c l e i n f oArticle history:Received 7 July 2014Received in revised form 20 October 2014Accepted 14 December 2014Available online 17 December 2014

Keywords:Bacillus cereus sporesHigh pressure high temperature treatmentDipicolinic acidSpore refractilityGermination and inactivationCarvacrolThe inactivation of bacterial spores generally proceeds faster and at lower temperatures when heat treatmentsare conducted under high pressure, and high pressure high temperature (HPHT) processing is, therefore, receiv-ing an increased interest from food processors. However, the mechanisms of spore inactivation by HPHT treat-ment are poorly understood, particularly at moderately elevated temperature. In the current work, we studiedinactivation of the spores of Bacillus cereus F4430/73 byHPHT treatment for 5min at 600MPa in the temperaturerange of 50100 C, using temperature increments of 5 C. Additionally, we investigated the effect of the naturalantimicrobial carvacrol on spore germination and inactivation under these conditions. Spore inactivation byHPHT was less than about 1 log unit at 50 to 70 C, but gradually increased at higher temperatures up to about5 log units at 100 C. DPA release and loss of spore refractility in the spore population were higher at moderate(65 C) than at high (70 C) treatment temperatures, and we propose that moderate conditions inducedthe normal physiological pathway of spore germination resulting in fully hydrated spores, while at higher tem-peratures this pathwaywas suppressed and replaced by anothermechanism of pressure-induced dipicolinic acid(DPA) release that results only in partial spore rehydration, probably because spore cortex hydrolysis is inhibited.Carvacrol strongly suppressed DPA release and spore rehydration during HPHT treatment at 65 C and alsopartly inhibited DPA release at 65 C. Concomitantly, HPHT spore inactivation was reduced by carvacrol at6590 C but unaffected at 95100 C.

2014 Elsevier B.V. All rights reserved.1. Introduction

High pressure treatment at elevated temperature (HPHT) has gainedan increasing interest as an alternative for thermal appertization (HT) oflow acid foods. In the HPHT process, products are first preheated to amoderate initial temperature (typically 7090 C), and subsequentlypressurized for a fewminutes at 500800MPa. Because pressure build-up is rapid, the products heat up by compression heating and reach pro-cess temperatures of 100120 C. Similarly, product temperaturerapidly drops to approximately the initial temperature during pressurerelease. Because adiabatic heating and cooling are volumetric, solidproducts can be heated more rapidly by HPHT than by conventionalheating, and,when considering the thermal component only, HPHTpro-cesses will have a lower total thermal impact on the product than HTprocesseswith the same process value (F0). As a result, HPHTprocessingis generally advantageous for the retention of flavor, color, texture, and2 16 321960.. Michiels).nutritional value. Besides a thermal component, HPHT processes alsohave a pressure component that potentially contributes to bacterialinactivation or has an influence on the rate of thermal inactivation.While most studies report increased bacterial spore inactivation byHPHT compared to HT treatment with the same thermal impact(Olivier et al., 2011; Patazca et al., 2006; Zhu et al., 2008), some studiesreport an opposite effect (Margosch et al., 2006), and it is currentlyunclear which factors are at the origin of a synergistic or antagonisticinteraction of HP and HT, respectively.

An issue of interest is themechanismof spore inactivation. It has beenwell documented that high pressure (HP) treatment at ambient or slight-ly elevated temperature induces spore germination in Bacillus subtilisand some other Bacillus species (Paidhungat et al., 2002; Setlow, 2003;Wuytack et al., 2000). At moderate pressures between 50 MPa and300400MPa, this germination requires the spore's germinant receptorsalthough it occurs even in the absence of nutrients (Black et al., 2005).Germination induced by moderate pressure proceeds through thesame steps as the physiological germinant-induced germinationprocess. Activation of the germinant receptors is followed by releaseof dipicolinic acid (DPA), which activates cortex lytic enzymes (CLEs).

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46 H. Luu-Thi et al. / International Journal of Food Microbiology 197 (2015) 4552Cortex hydrolysis leads to core rehydration,which in turn allows the deg-radation of small acid soluble proteins (SASPs), ATP generation and, final-ly, resumption of DNA, RNA and protein synthesis when the spore core isfully hydrated. During germination, spores gradually lose their extremeresistance and they can subsequently be inactivated by UV radiation, hy-drogen peroxide, heat or pressure (Paidhungat et al., 2002; Reineke et al.,2013c; Wuytack et al., 1998).

HP treatment at500 MPa and moderate temperature also triggersspore germination but, at least in B. subtilis, this germination processdiffers in several features from that induced at moderate pressures.One important difference is that spores lacking nutrient receptors alsogerminate at very high pressures. The earliest detectable event underthese conditions is the release of DPA, and the current view is thatpressures N500 MPa induce the direct opening of DPA channels inthe spore's inner membrane by a physicochemical mechanism as op-posed to the physiological process that takes place at low pressures(Paidhungat et al., 2002; Reineke et al., 2011, 2012; Setlow, 2013).Cortex hydrolysis and spore core rehydration also take place, butspore germination is not complete or is delayed, since no degradationof SASPs and ATP production are observed immediately after treatment,possibly because of inactivation of Gpr, the germination proteasethat normally degrades the SASPs (Black et al., 2005; Reineke et al.,2013c; Wuytack et al., 1998). Moderately increased temperatures(up to 60 C) generally stimulate HP-induced spore germination, in par-ticular the physiological germination induced by moderate pressures(Wuytack et al., 1998). On the other hand, higher temperatures, likethose prevailing in HPHT processes (typically 90 C) probably sup-press physiological spore germination because they may irreversiblydenature proteins of the germination system like Gpr and the cortex-lytic enzymes. Nevertheless, even at very high temperature and pres-sure, HPHT treatment triggers Ca-DPA release and a partial rehydrationof the spores, and this is believed to be the reason for the higher sporeinactivation efficiency of HPHT treatment compared to HT treatment(Margosch et al., 2004; Reineke et al., 2013a, 2013b, 2013c).

The combination of HP treatment with natural antimicrobial com-pounds has been explored as a strategy to enhance microbial inactiva-tion, and several studies have reported synergistic effects. While mostof these studies have been conducted with vegetative bacterial cells,synergistic effects have also been observed with spores. Nisin enhancedHP inactivation of spores from Alicyclobacillus acidoterrestris, B. subtilis,Bacillus cereus, Bacillus amyloliquefaciens and Clostridium sporogenes,while for some bacteria, no enhancement of spore inactivation wasseen (Black et al., 2008; Hofstetter et al., 2013a; Sokolowska et al.,2012). The effect of reutericyclin was also species dependent, with anenhanced inactivation in Clostridium beijerinckii, but reduced inactiva-tion in C. sporogenes (Hofstetter et al., 2013a). Olive powder also slightlyreduced spore inactivation by HP treatment in B. cereus (Marco et al.,2011). A group of natural antimicrobials that have received growingattention is composed of essential oils from herbs and spices, becauseof their ability to control spoilage bacteria, inhibit foodborne pathogensand extend shelf-life (Gayan et al., 2012). Essential oils have beenreported to increase the inactivation of bacterial spores by heat. Forinstance, the time needed for a 6 log reduction of Bacillus coagulansspores at 100 C was reduced from 3.25min to 1.67min in the presenceof 400 g/g oregano essential oil in nutrient broth (Haberbeck et al.,2012). However, to our knowledge, no information is available on thecombination of essential oils with HPHT treatment to enhance bacterialspore inactivation.

In the present study, we investigate the effect of carvacrol on theHPHT inactivation of B. cereus spores. Besides quantifying spore inacti-vation and germination by plating methods, we also report the releaseof DPA and the loss of spore refractility. Together, these data providenovel insights in themechanisms of B. cereus spore inactivation and ger-mination at different treatment conditions (600 MPa, 50100 C withholding time of 5 min), and in the way by which carvacrol interfereswith these mechanisms.2. Material and methods

2.1. Bacterial strain and preparation of spores

B. cereus F4430/73, a strain that was isolated from a diarrheal out-break and belongs to phylogenetic group IV of B. cereus sensu lato,was kindly provided by M. H. Guinebretire (INRA, Avignon, France).To induce sporulation, a loopful of cells from a 80 C glycerol stockwas streaked on a BHI plate and incubated at 30 C for 24 h. A single col-ony was then grown for 48 h at 30 C with shaking (200 rpm) in BrainHeart Infusion broth (BHI, Oxoid, Basingstoke, United Kingdom). A hun-dred microliters of this culture was diluted into 1 ml of sterile distilledwater. From that, 50 l was surface-plated on nutrient agar CM0003(Oxoid) using sterile glass beads and incubated at 30 C. Samples wereexamined microscopically daily until more than 95% of the cellsconsisted of phase bright spores, which was after about 45 days of in-cubation. The plates were then flooded with 3 ml sterile cold deionizedwater and the spores were dislodged and suspended with a sterile bentglass rod. Pooled suspensions from several plates were washed and re-suspended in one fifth of the original volume in cold sterile deionizedwater. To separate spores from cell debris and vegetative cells, 10 mlof this suspension was gently deposited on top of 50 ml 40% sucroseand centrifuged at 4000 g for 10 min at 4 C (procedure adaptedfrom Sorg and Sonenshein (2010)). The pellet was resuspended in ster-ile cold deionized water, washed four additional times, and stored at20 C. These spore suspensions contained nomore than 1% vegetativecells and were used within a week after harvesting for thermal andHPHT treatments. No changes in microscopical spore appearance andin the fraction of heat-sensitive spores occurred during storage.

2.2. Thermal treatment

Before use, spores were collected by centrifugation at 4000 g for10 min and resuspended in 0.1 M 2-(N-morpholino) ethanesulfonicacid (MES) buffer (pH 6.1) at approximately 108 cfu ml1. MES bufferwas chosen for both HT and HPHT experiments because its pH variesonly slightly with temperature and pressure (pKa / C = 0.011;V = 3.9 cm3 mol1) (Bruins et al., 2007). Therefore, the pH of thespore suspensions is predicted to be in the range of 5.35.9 under theconditions of our HPHT and HT experiments. Moreover, MES is nontox-ic, does not permeate membranes and does not form complexes withmetal ions like Ca2+ from the spores.

Where applicable, 5 mM carvacrol (Sigma-Aldrich, Diegem,Belgium) was added to the cell suspensions using a 1 M carvacrolstock solution in 97% ethanol. This concentration is about three timeslower than the minimal inhibitory concentration for B. cereusATCC11778 (Rosato et al., 2007). Eightymicroliters of the spore suspen-sionswas transferred into a glass capillary, whichwas then heat-sealed,immersed in an oil bath at 50100 C (with interval of 5 C) for 5 minand put in ice-water slurry immediately after treatment. The treatedspore suspensions were serially diluted in sterile potassium phosphatebuffer (pH 7.0), plated on Tryptone Soya Agar (TSA, Oxoid) and incubat-ed at 30 C for 24 h to enumerate survivors. Prolonged incubation timedid not yield higher spore viability. All thermal experiments wereconducted in triplicate using the same spore suspension.

2.3. HPHT equipment and procedure

HPHT treatment was conducted using a custom-made apparatusconsisting of six parallel vertical vessels equipped with an externalheating coil (HPIU-10.000, maximum temperature 120 C, maximumpressure 800MPa, Resato, The Netherlands), and using propylene glycolas pressure-transmitting medium. One of the six vessels was used for adummy sample with a thermocouple, prepared in the same way as thereal samples, to allow inline temperature registration inside the sample

47H. Luu-Thi et al. / International Journal of Food Microbiology 197 (2015) 4552holder during processing. Pressure was also continuously recorded forall individual vessels during HPHT treatment.

Three hundred microliter aliquots of the spore suspensions (withor without carvacrol 5 mM) were sealed in a sterile polyethylenepouch. All high pressure treatments were done in triplicate fromthe same spore suspension. The samples were placed in cylindricalpolytetrafluoroethylene (Teflon) sample holders with an inner diam-eter of 12 mm, length of 85 mm and wall thickness of 4 mm (Vink,Belgium). The sample holders were further filled with water whileavoiding inclusion of air bubbles, closed with moveable caps and vacu-um sealed in double plastic pouches to prevent contamination of theequipment in case of leakage.

The following procedurewas used to ensure that isothermal and iso-baric conditions were rapidly reached and stably maintained (Grauwetet al., 2011; Vervoort et al., 2011). After being pre-equilibrated at 4 C,the filled sample holders were loaded into the HP vessels that werepreheated at the target process temperature. When the samples hadwarmed up to a predetermined temperature Ti (period t1, Fig. 1), pres-sure was built up immediately to 150MPa, and then further to 600MPaat a rate of 10 MPa/s. Based on preliminary experiments, the tempera-ture Ti for each treatment was selected in the way that the heat gener-ated by compression raised the temperature inside the sample holdersto the desired value (period t2, Fig. 1). After reaching 600MPa, an isola-tion time of 90 s was used to allow further temperature equilibration inorder to reachmore accurately the target temperature (period t3, Fig. 1).From that point onwards, individual vesselswere isolated from the highpressure circuit and held for 5 min of holding time (period t4, Fig. 1.),after which pressure was released instantaneously, resulting in animmediate temperature drop. As can be seen for the treatment at600 MPa, 80 C in Fig. 1, this procedure ensured stable isothermal andisobaric conditions.

After decompression, the samples were immediately removed fromthe vessels and cooled in ice-water slurry for further analysis. A portionof this spore suspension was taken immediately for examination byFig. 1. Recorded pressure and temperature history of sample subjected to (A) 600MPa, 50 C,5minand (B)600MPa, 95 C, 5min: t1: preheating timeat atmospheric pressure from4C toTi(only part of t1 shown); t2: time of pressure build-up; t3: isolation time (90 s); t4: isobaric andisothermal treatment time. Inactivation is studied from the start of period t4, indicated ast = 0 min.phase contrast microscopy. A second and a third portion were used toenumerate the survivors and theungerminated spores among the survi-vors by performing a plate count, directly or after an additional heattreatment at 70 C for 10 min, respectively. The enumeration of sporeswas done as described above for thermal treatments. Finally, the lastportion was centrifuged (10,600 g for 4 min) to remove the spore pel-let and the supernatant was diluted 1:10 in TrisHCl buffer (pH 7.5) andstored at20 C for DPA analysis.

2.4. Analysis of loss of spore refractility by phase contrast microscopy

Immediately after HPHT treatment, spore samples were immobilizedon thin pads of 2% agarose (Eurogentec, Belgium) on a microscopy slideand mounted with a cover glass. Phase contrast microscopy (Eclipse Tiinverted microscope, Nikon, Champigny-sur-Marne, France) was usedto acquire images of five different fields from each sample whichcontained a sufficient number of well isolated spores. Images were visu-alized using NIS-Elements software (Nikon). To quantify the loss of sporerefractility, pixel intensity was measured in a square of 3 3 pixels(or 0.22 0.22 m) in the center of 80 individual randomly picked sporesfor each sample, using the open source software ImageJ. The center of thespore was chosen because the light path through the spore core is longerat the center than at the edges, and consequently the pixel intensity at thecenter is most representative for the state of the spore core. From thesemeasurements, a distribution of spore refractility was constructed foreach treated sample (and for an untreated control sample) using tenequally sized intervals of pixel intensities ranging between the lowestand the highest values observed.

2.5. DPA measurement

DPAmeasurementwas based on the formation of a fluorescent com-plex with Tb3+ (Kort et al., 2005). The spore supernatants from20 Cwere diluted 1:25 in TrisHCl buffer (pH 7.5), and 100 l of this suspen-sion was transferred into a 96-well black microtiter plate (GreinerBio-one, Belgium) and mixed with an equal volume of 20 mM TbCl3 inTrisHCl buffer (pH 7.5). Fluorescence measurements were done in aspectrofluorometer (Synergy Mx-biotek, USA) with excitation andemission wavelengths of 270 and 545 nm, respectively. DPA releasefrom the spores by HPHT or HT treatment was expressed relative tothe value obtained for the supernatant of an autoclaved spore suspen-sion, which is assumed to have released its entire DPA depot. The pres-ence of 5 mM carvacrol did not interfere with the quantification of DPAby this method (data not shown).

2.6. Statistical analysis

Spore inactivation and DPA release data were analyzed statisticallyusing one-way analysis of variance (ANOVA), followed by TukeyKramer's post-hoc test formultiple comparisonwith a 5% level of signif-icance (p b 0.05).

3. Result and discussion

3.1. Loss of viability and heat resistance after HPHT and HT treatment

In a first experiment, we determined spore inactivation and germi-nation after HPHT treatments at a single pressure (600 MPa) but overa wide range of different temperatures (50100 C). In these experi-ments, germination is operationally defined as the loss of resistance toa 10 min/70 C heat treatment, and it should be emphasized that thisdoes not necessarily imply involvement of all the germination proteinsof the physiological germination process induced by germinants. Whenwe first consider the results in the absence of carvacrol, a number ofobservations can be made (Fig. 2). The treatments at 5060 C cause asmall reduction of 0.40.7 log units that could be due to the residual

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Fig. 2. Surviving heat resistant and heat sensitive spores (log N) after HPHT treatment at 600MPa, 50100 C for 5min. (a) HPHT treatment; (b) HPHT treatmentwith additional heating at70 C for 10min; (c)HPHT treatment in the presenceof 5mMcarvacrol; (d)HPHT treatment in the presenceof 5mMcarvacrolwith additional heating at 70 C for 10min. Initial refers tothe spore count of the untreated spore suspension. Error bars represent standard deviation (N= 3).

48 H. Luu-Thi et al. / International Journal of Food Microbiology 197 (2015) 4552presence of vegetative cells or spores that have not developed full resis-tance. Additional heat treatment before plating further reduced thenumber of survivors by 0.81.1 log after HPHT at 55 and 60 C, indicat-ing that these treatments induced spore germination. The HPHT treat-ments at 65 and 70 C also caused germination, but at the same timethey also caused more inactivation, probably because the germinatedsporeswere partially killed during the treatment. At still higher temper-atures (75100 C), there was progressively more inactivation, up toabout 5 log at 95 and 100 C. As expected, heat sensitive survivors areno longer detected after these treatments, and whether or not inactiva-tion is preceded by germination under these conditions cannot bededuced from the data. Treatment at 100 C did not enhance spore inac-tivation compared to at 95 C, probably due to the presence of asuperdormant spore fraction.

Carvacrol had a pronounced effect on spore germination and inacti-vation, except at the highest process temperatures (95 and 100 C).First, it completely inhibited spore germination by HPHT at 5570 C.As a result, spore inactivation under these process conditions remainedvery low, and can probably be entirely ascribed to the residual vegeta-tive cells. Interestingly, also at 7590 C, carvacrol suppressed sporeinactivation by 0.81.4 log units, and this suggests that inactivationat these temperatures is also dependent on germination. At 95 and100 C, finally, carvacrol had no effect on HPHT inactivation. Thiscould be because themechanismof germination under these conditionsis different, or because the mechanism by which carvacrol suppressesgermination is not working.

For comparison, we also conducted thermal treatment at thesame temperatures (Fig. 3). In line with the high spore heat resistanceof B. cereus F4430/73, a considerable level of spore inactivationwas only observed at 100 C (4.2 log). It can also be seen that HPHT0

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Fig. 3. Surviving spores (log N) after HT treatment at 50100 C for 5min. (a) Treatment in the aspore count of the untreated spore suspension. Error bars represent standard deviation (N = 3treatment is more effective than HT treatment at the same temperature(p b 0.05), as is the case for most bacterial spores, at least at tempera-tures below about 100 C (Olivier et al., 2011; Patazca et al., 2006; Zhuet al., 2008). However, the effect of carvacrol was opposite to whatwas observed for HPHT treatment, since carvacrol increased inactiva-tion by HT, particularly at high temperatures. For example, at 95 and100 C, inactivation was increased by 3.4 and 1.8 log units, respectively.This is in linewith findings in other studies where essential oils or plantextracts have been combined with heat treatment to inactivate spores(Bevilacqua et al., 2009; Haberbeck et al., 2012). As a result, the differ-ence in spore inactivation between HT and HPHT treatments at thesame temperature became much smaller in the presence of carvacroland, in fact, inactivation by HT is even significantly higher than byHPHT at 100 C in the presence of carvacrol (p b 0.05).

3.2. DPA release from HPHT and HT treated spores

With few exceptions, spores are more efficiently inactivated byHPHT than by HT treatment at the same temperature. This has been at-tributed to the release of the spore's large depot of Ca-DPA under pres-sure, which leads to a partial rehydration and consequently a partial lossof heat resistance of the spores (Black et al., 2007; Paidhungat et al.,2002; Reineke et al., 2011; Setlow, 2013). Therefore, to further investi-gate why carvacrol inhibited spore germination and inactivation byHPHT treatment, we analyzed the amount of DPA in supernatants pre-pared fromspore suspensions immediately after theHPHTandHT treat-ments (Figs. 4 and 5). The result for HPHT treatment is a relativelycomplex trend of DPA release exhibiting two distinct maxima as a func-tion of temperature. DPA release first increased from 75 to almost 100%(5065 C), then abruptly decreased to 56% (75 C), and steadilya b a b a b a b a b a b

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Fig. 4. Percentage of DPA release from spores after HPHT treatment at 600 MPa, 50100 C for 5 min, compared to control sample of autoclaved spores (121 C for 20 min): (a) in theabsence of carvacrol; (b) in the presence of 5 mM carvacrol. Error bars represent standard deviation (N = 3).

49H. Luu-Thi et al. / International Journal of Food Microbiology 197 (2015) 4552increased again up to 90% at 100 C. DPA releasewas significantly higherat 65 C than at 75 and 80 C and, vice-versa, DPA release at 75 C wassignificantly lower than at 60 and 65 C (p b 0.05). This trend is differentfrom that of B. subtilis spores, for which the rate of DPA release was re-ported to increase with increasing HPHT (600 MPa) process tempera-ture from 40 to 80 C (Reineke et al., 2013b). The reason for thisdifference is not clear, but the results suggest that two different mecha-nisms of DPA release are active in B. cereus as opposed to only onemechanism in B. subtilis. The first mechanism is dominant in the mildtemperature range (5070 C) and includes a heat sensitive step orcomponent because it is strongly suppressed at 75 C. The secondmechanism becomes gradually more important as the process temper-ature increases, and dominates at 75 C. These results may indicatethat the physiological germination process can be induced in B. cereusF4430/73 bymild temperatureHPHT conditions, and itwould beworth-while to investigatewhether this induction proceeds through activationof the germinant receptors by making use of receptorless mutants. InB. subtilis, in contrast, only pressures 200 MPa induce germinationby activation of germinant receptors, while the pressure and tempera-ture limits to activate these receptors in spores of other sporeformershave not been investigated.

In the presence of carvacrol, DPA release was strongly suppressed inthe moderate temperature range (5070 C) (p b 0.05). This is compat-ible with the above stated hypothesis that physiological germinationtakes place under these conditions, and with reports in the literature0%

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Fig. 5. Percentage of DPA release from spores after HT treatment at 50100 C for 5 min, comcarvacrol; (b) in the presence of 5 mM carvacrol. Error bars represent standard deviation (N =that carvacrol inhibits physiological germination induced by germinants(Juneja et al., 2006; Juneja and Friedman, 2007; Periago et al., 2006).DPA release in the presence of carvacrol increased with increasing pro-cess temperature up to about 68% at 75 C, and then remained at aboutthe same level at higher temperatures, except for one deviating value(46%) at 90 C. DPA release was significantly reduced by carvacrol at90100 C (p b 0.05), suggesting that carvacrol also at least partiallysuppresses the germinant receptor independent mechanism of DPA re-lease which was proposed to proceed by direct opening of membraneDPA channels or creation of pores in the membrane (Black et al.,2007). Comparison of Figs. 4 and 2 also shows that spore inactivationcannot be predicted by DPA release alone. For example, although DPArelease at 75 and 80 C was about equal in the absence and in the pres-ence of carvacrol, inactivation was about tenfold higher in the absenceof carvacrol.

Compared to HPHT treatment, HT treatment caused much less DPArelease, and this DPA release was not affected by carvacrol (Fig. 5).Therefore, the mechanism of DPA release by HT treatment is probablydifferent from that by HPHT treatment in the investigated temperaturerange. This could be related to the different effects of HPHT and HTtreatment on the spore membrane (Hofstetter et al., 2013a, 2013b).Comparison of Figs. 5 and 3 also shows that DPA release is not correlatedto HT inactivation and thus DPA release is not contributing to the lethaleffect of HT treatment on spores. This conclusion confirms earlier find-ings of Coleman et al. (2007, 2010), who showed that heat-treatedb a b a b a b a b a b

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pared to control sample of autoclaved spores (121 C for 20 min): (a) in the absence of3).

50 H. Luu-Thi et al. / International Journal of Food Microbiology 197 (2015) 4552spore suspensions of B. subtilis, B. cereus and Bacillus megateriumcontained a large fraction of killed spores that had retained their DPA.These authors concluded that wet heat inactivated spores through pro-tein denaturation and that DPA release took place after the spores werekilled.

3.3. Spore refractility distribution after HPHT treatment

Spore suspensions subjected to HPHT treatment were also analyzedby phase contrast microscopy to assess the loss of refractility, which re-sults from the release of solutes and the uptake of water in the sporecore. As such, loss of refractility is also a marker of spore germination(Paidhungat and Setlow, 2002). Microscopical analysis has the advan-tage that it allows to determine not only the average, but also the distri-bution of the loss of refractility in the spore population.

In the absence of carvacrol, treatment at 50 C already caused an im-portant loss of refractility, with the average pixel intensity decreasingfrom 11,126 to 6879 pixel units (Fig. 6). With increasing process tem-perature, the loss of refractility first rapidly increased (5060 C), thendecreased (6085 C), and finally remained at approximately thesame level (85100 C). In the presence of carvacrol, the loss ofrefractility after HPHT treatment was always lower than in its absence,0.0

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Fig. 6.Microscopically determined pixel intensity distributions of spores in the absence (A) andnonpressurized control sample. Eighty spores were analyzed for each treatment and for the cothe darkest and the brightest spore, respectively.but the effect of carvacrol was larger at lower than at higher processtemperatures. At 50 and 55 C, the loss of spore refractility wascompletely inhibited. At higher temperatures, the trend was similar tothat observed in the absence of carvacrol, with an increase (6065 C)followed by a decrease (6585 C) and a plateau (85100 C).

A comparison of the patterns of DPA release and spore refractilityloss (Figs. 4 and 6) shows that, although the general trends showsome resemblance, both indices are not strictly correlated. Most nota-bly, spores treated at 5560 C incurred a much larger refractility lossthan spores treated at 95100 C, despite a similar degree of about80% DPA release. Loss of spore refractility during spore germination isdue to replacement of solutes bywater. The first event during spore ger-mination that results in loss of refractility is the release of Ca-DPA and itsreplacement by water, but this results only in a partial rehydration ofthe spore core and thus a partial loss of refractility. Ca-DPA subsequentlyactivates the cortex hydrolases and the hydrolysis of the spore cortexleads to further spore rehydration (Kong et al., 2010; Reineke et al.,2013b, 2013c). Therefore, our data seem to indicate that all the HPHTtreatments trigger DPA release, but the released DPA triggers cortexhydrolysis only at moderate temperatures. A possible explanation isthat the cortex lytic enzymes are inactivated during HPHT treatmentat N65 C. The spore refractility data therefore further support the600 MPa

13195-14566

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10454-11825

9084-10454

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3601-4972

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600 MPa

13195-14566

11825-13195

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presence (B) of 5mM carvacrol after HPHT treatment at 600MPa, 50100 C for 5min andntrol sample. Minimum and maximum pixel intensities recorded were 860 and 14,566 for

51H. Luu-Thi et al. / International Journal of Food Microbiology 197 (2015) 4552hypothesis formulated above that HPHT treatment at mild tempera-tures may induce a physiological germination process by activatingthe germinant receptors.

The spore refractility data also confirm that spore germination byHPHT at mild temperatures is strongly inhibited by carvacrol. The effectof various organic compounds on spore germination by nutrient andnon-nutrient germinants has been investigated previously (Cortezzoet al., 2004; van Melis et al., 2011a, 2012), and carvacrol and other es-sential oil components can inhibit nutrient-induced germination ofspores from some Bacillus and Clostridium spp. (Bevilacqua et al.,2011; Juneja et al., 2006; Periago et al., 2006). Interference with HP in-duced germination has been studied for sorbic acid (van Melis et al.,2011b). In this study, undissociated sorbic acid (3 mM, pH 5.5) blockedthe germination of B. cereus by various nutrient germinants as well asby mild HP treatment (150 MPa), but not by severe HP treatment(500 MPa) and Ca-DPA. Since only the former triggers germination viathe germinant receptors, this led the authors to propose that sorbicacid may interfere with the signaling pathway initiated by activationof a germinant receptor and leading to the opening of the Ca-DPA chan-nels. Unfortunately, DPA release was not reported in this study. At firstsight, our results with carvacrol are different, since we observed stronginhibition of germination at 600MPa (5065 C). However, as discussedabove, the patterns of DPA release and loss of refractility suggest thatgermination under these conditions may proceed through activationof the germinant receptors. Why this was not the case in the study ofvan Melis et al. (2011b) is not clear, but it could be because of strain-specific behavior or differences in experimental conditions such astreatment temperature ormedium. In any case, if we accept the hypoth-esis of receptor-dependent germination by HPHT at 65 C, carvacrolmay have the samemode of action as sorbic acid, i.e., it may accumulatein the spore inner membrane and interfere with the signaling betweennutrient receptors and the Ca-DPA channel (van Melis et al., 2011a,2011b). Also the effects of nisin and reutericyclin on DPA release andspore inactivation by HPHT treatment may be related to their effectson the spore membrane (Hofstetter et al., 2013a, 2013b).

At higher temperatures, in the absence of carvacrol, there is less DPArelease and themechanismprobably does not involve germinant recep-tors and cortex hydrolases, as discussed above. Our observations are inline with previous studies with B. subtilis spores (Reineke et al., 2013a,2013c) reporting that HP treatment caused full spore hydration whenit was conducted at lower temperature (e.g., 37 C) but only partial hy-dration when it was conducted at higher temperature (e.g., N60 C).This was explained by assuming a delayed onset of stage II germinationas well as the inactivation of cortex lytic enzymes at high temperatures.Despite the differentmechanisms, carvacrol also partially inhibited DPArelease and spore rehydration under the high temperature conditions,probably because DPA release is affected by the impact of carvacrol onthe inner membrane. This may directly increase membrane permeabil-ity for Ca-DPA or indirectly promote opening Ca-DPA channels.

4. Conclusion

Plate counts of total survivors and heat resistant survivors, measure-ment of DPA release and microscopical analysis of spore refractility losshave provided insight in the effects of HPHT treatment at 600 MPa anddifferent temperatures on spores of B. cereus F4430/73. The results sug-gest that treatment at moderate temperatures (65 C) induces thenormal physiological pathway of spore germination by activating thegerminant receptors, and resulting in fully hydrated spores. At highertemperatures, physiological spore germination is suppressed and an-other mechanism of pressure-induced DPA release that results only inpartial spore rehydration takes over, probably because spore cortex hy-drolysis is inhibited. Carvacrol strongly suppressed the physiologicalgermination mechanism during HPHT treatment at 65 C, and alsopartly inhibited DPA release at 65 C. From an application point ofview, the addition of carvacrol does not increase the efficacy of sporeinactivation by HPHT treatment and, at 6590 C, even decreases theefficacy. This is in contrast to the stimulation of spore inactivation bycarvacrol during thermal inactivation.Acknowledgments

Thisworkwasfinancially supported by fellowships from theKULeu-ven Research Fund (DBOF/10/043) to Luu-Thi H., from the ResearchFoundation Flanders (FWO) to Tara Grauwet, and from the FlemishAgency for Innovation by Science and Technology (IWT-Vlaanderen)to Ioannis Passaris. Research grants were obtained from the KU LeuvenResearch Fund (METH/07/03, METH/14/03, STRT1/10/36 and IDO/10/012), and FWO (G.0C77.14). We thank M. H. Guinebretire (INRA,Avignon, France) for kindly providing the B. cereus strain F4430/73.References

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Carvacrol suppresses high pressure high temperature inactivation of Bacillus cereus spores1. Introduction2. Material and methods2.1. Bacterial strain and preparation of spores2.2. Thermal treatment2.3. HPHT equipment and procedure2.4. Analysis of loss of spore refractility by phase contrast microscopy2.5. DPA measurement2.6. Statistical analysis

3. Result and discussion3.1. Loss of viability and heat resistance after HPHT and HT treatment3.2. DPA release from HPHT and HT treated spores3.3. Spore refractility distribution after HPHT treatment

4. ConclusionAcknowledgmentsReferences