Real time intracellular pH dynamics in Listeria innocua under CO2 and N2O pressure

J. of Supercritical Fluids 58 (2011) 385–390

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Real time intracellular pH dynamics in Listeria innocua under CO2 and N2 O pressure Stefano Giulitti, Claudio Cinquemani, Alberto Quaranta, Sara Spilimbergo ∗ Dept. of Materials Engineering and Industrial Technologies, Faculty of Engineering, University of Trento, via Mesiano 77, 38123 Trento, Italy

a r t i c l e

i n f o

Article history: Received 30 March 2011 Received in revised form 12 July 2011 Accepted 13 July 2011 Keywords: Pressure CO2 N2 O Real-time, Intracellular pH Listeria innocua

a b s t r a c t This article investigates the inactivation mechanism of high-pressure food treatment, considered as alternative to conventional biocidal processes. We aimed to determine intracellular pH decrease under CO2 and N2 O pressure, so far postulated as one of the main causes of inactivation. Working with a lab-scale bioreactor in mild conditions – 25 ◦ C and pressures up to 8 MPa – we monitored – for the first time during pressurization – cytoplasmic pH variations of Listeria innocua labeled with pH-sensitive fluorophores based on fluorescein. We show that carbonic acid, due to solubilization of CO2 into the aqueous phase, causes a rapid pH drop in the cytosol, reaching pH 4.8 at 1 MPa and falls below the detection limit of the indicator fluorophore of pH 4.0. This correlates with a reduced viability (below 90%) in all the pressure ranges investigated. Contrarily, treatment under N2 O pressure reduces cell viability without significant pH-drop neither of intra- nor extra-cellular liquid at any pressure investigated. The pH value remains between 7 and 6 while an inactivation of more than 80% is achieved at 8 MPa. Our data clearly demonstrate that, as a critical pressure is achieved, microbial inactivation is mainly due to pressure-induced membrane permeation – stimulated by non-acidifying fluids as well, rather then cytoplasmic acidification, as widely argued so far. A definitive understanding of the microbial inactivation mechanism due to CO2 /N2 O under pressure has been advanced significantly. © 2011 Elsevier B.V. All rights reserved.

1. Introduction In almost all industrial bioprocesses, safe measures against proliferation of microorganisms are compulsory requirements to assure human health. According to the product to be sterilized, various measures are available to inactivate microorganisms [1]. State-of-the-art industrial processes for inactivation of microorganisms act drastically by ␥-rays DNA scission [2] or heating-denaturation of proteins [3]. In food applications, inactivation techniques such as the use of heat-treatment, biocidal gases or irradiation are commonly used, even though they frequently do not preserve nutritional, functional or physical–chemical properties of the product. Alternative processes include microwave treatment [4], ultrasound or pulsed electric fields [5], hydrostatic pressure treatment [6] and dense-gas (CO2 and N2 O) processes. Dense-gas processes represent an attractive alternative as they guarantee both inactivation of microorganisms and preservation of the product properties. Nevertheless the precise inactivation mechanism is still under debate

∗ Corresponding author. Tel.: +39 0461 882485; fax: +39 0461 281945. E-mail addresses: [email protected], [email protected] (S. Spilimbergo). 0896-8446/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2011.07.012

and the recent literature still lacks experimental data to confirm theoretical hypotheses. The use of compressed CO2 , which is regarded in the United States Food and Drug Administration (FDA) as a GRAS (Generally Recognized As Safe) substance, has been investigated intensively as agent in low-temperature and wide-target alternative processes to conventional ones [7–11]. While as concerns N2 O only limited studies have been conducted on its potentiality as bactericidal agent, nevertheless, as a non-acidifying gas, it can be considered a promising alternative to CO2 [12,13]. The inactivation mechanism lying under this innovative process remains unknown and it is largely dependent on the type of microorganism and treated medium [14]. Loss of membrane integrity and severe alteration of intracellular pH (pHi ) seem to play a fundamental role in microbial inactivation: it is not a case that these phenomena have major importance for various cellular processes including enzyme activity, gene transcription and protein synthesis. In normal conditions, the cell counteracts intracellular pH change by proton transport pathways [15] or by water in- or out-flux and buffering mechanisms. Membrane permeabilization kinetics has been recently measured, during CO2 treatment, indicating a progressive irreversible permeabilization of the cell envelope due to the strong modifications of its properties after the first minutes of treatment [16]; while, as concerns pHi , only preliminary studies have been

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published recently on pH decrease evaluation just after the treatment [17].What is known so far is that carbonic acid formation in water solutions increases the inactivation potential of pure CO2 [18], but it has not been clarified yet to which extent the formation of carbonic acid and it subsequent dissociation takes place in intra- and extracellular environment. Early techniques to measure pHi based on radio-labeled weak acids and 31 P nuclear magnetic resonance technique have been replaced by fluorescent techniques due to better time resolution and ease of use [19] and have since been applied in numerous investigations using carboxyfluorescein for pH ranges from 5.0 to 8.0 [20–23]. Recently, applying both CO2 and N2 O pressure, intracellular pH change has been measured after treatments using pH-sensitive and non-toxic dyes (cDCFDA-SE and cFDA-SE) in Listeria innocua [24]. Besides the importance of final results, off-line measurements are not sufficient to explain process kinetics as during system depressurization and sample preparation pHi changes cannot be avoided. Therefore, the aim of our study was to set up a stirred pilot-scale high-pressure reactor to allow on-line fluorescence measurement of reliable pHi changes in L. innocua. Treatments were performed at mild conditions (25 ◦ C and up to 8 MPa) to be able to follow pH variation kinetics, during the entire process, step by step, in order to clarify definitively if acidification can be considered or not a key factor of microbial inactivation, thus getting further insight into the mechanism underlying inactivation under CO2 and N2 O pressure. Exhibiting various similar physical properties but different reactivities in water, either acidifying CO2 or non-acidifying N2 O were used to clarify the inactivation process. Additionally, we focused our interest on which condition leads to an inactivation of the bacteria. Therefore, dynamics of intracellular pH were correlated to standard cell viability to assess separately pressure and acidification roles. 2. Materials and methods 2.1. Strains and growth conditions The non-pathogenic bacteria L. innocua (DSMZ, DSM 20649) was used as test-microorganism. Cells were grown in Brain Heart Infusion broth (Fluka, USA) at 37 ◦ C for 16 h (OD600 = 0.9) in Erlenmeyer flasks with a working volume of 200 mL without stirring. Cultures were centrifuged for 10 min at 8000 rcf and finally washed in phosphate buffer (PBS). 2.2. Cell staining with fluorescent probes Intracellular pH was measured by staining cells with pHsensitive and non-toxic fluorescent probes: cDCFDA-SE (5-(6)Carboxy-2 ,7 -dichlorofluorescein diacetate, N-succinimidyl ester), (Invitrogen, USA) or cFDA-SE (5-(6)-Carboxyfluorescein diacetate N-succinimidyl ester) (Invitrogen, USA) with a pH-range sensitivity of 3.5–5.0 and 5.0–8.0 respectively. Both molecules are cleaved by esterase and react with free amines, thus binding practically irreversibly, inside the cell. Compared to previous works [20,23], an improved method was adapted for cell staining. 150 mL of L. innocua culture in PBS was incubated at 37 ◦ C with 10 mM glucose and 3 mM cDCFDA-SE or cFDA-SE (dissolved in 50 ␮L of DMSO; Sigma–Aldrich, USA) for 45 and 30 min respectively. In order to remove unbound fluorescent probe cells were then centrifuged at 8000 rcf for 10 min and incubated again at 37 ◦ C for 30 min in PBS and 10 mM glucose. Stained cells were washed twice in PBS and re-suspended in Ringer solution to yield an OD600 of 0.38 which corresponds to a starting cell concentration of N0 = 109 CFU/mL. The obtained cell suspensions were immediately used to generate calibration curves and to perform

pressure treatments. In order to assess of the staining procedure caused damage to the bacteria we evaluated growth of stained and unstained cells but found no significant difference. 2.3. High-pressure apparatus The cylindrical stainless steel reactor (height: 110 mm, inner diameter: 60 mm) was set to constant temperature at 25 ◦ C by a water bath (MPM Instruments, Italy). The reactor was filled with 200 mL of cell suspension and temperature stability was awaited. During the experiment, 300 rpm magnetic stirring (Velp, USA) and baffles increased mixing of the solution, thus, assuring fast equilibration of pH throughout the reactor. Carbon dioxide, CO2 , 4.0 (Messer, Italy) and Nitrous oxide, N2 O, 4.0 (Messer, Italy) were used to treat either buffered and Ringer solution (Oxoid, UK). The gas was liquefied at 3 ◦ C using a chiller (MPM Instruments, Italy) and pumped into the reactor with a volumetric pump M910s (Lewa, Germany). Working with pressurized N2 O, the reactor was previously fluxed with N2 O to avoid medium acidification due to forced-dissolved atmospheric CO2 , purging gases through an exit valve. Pressure was measured using a manometric pressure indicator (Gefran, Italy), recorded with a data acquisition system (National Instruments, Field Point, FP-1000, USA) and processed with LabVIEW 5.0 software (National Instruments, USA). 2.4. cDCFDA fluorescence calibration and measurements Silica optical fibers (Ocean Optics) were used to send the excitation light from a halogen lamp light into the reactor and to collect the intensity of fluorescence light emitted by the cells at a 90◦ angle. Emission light was acquired by a spectrometer S2000 (Ocean Optics). Blank signal was obtained subtracting scattered light of an unstained sample with OD600 = 0.38. Spectra were processed through SpectraSuite software (Ocean Optics) with an integration time of 4 s. The mean value at 518 nm (isoemissive wavelength) was used as a fixed intensity reference. Vertical-shifted spectra were eventually corrected by subtracting the deviation from the mean intensity at 518 nm to the emission peak intensity at 548 nm. To obtain a reliable correlation between fluorescence intensity and pHi , both calibrations at atmospheric pressure and under elevated pressure were required. 2.5. cFDA fluorescence calibration and measurements The same procedures described for cDCFDA were followed. The mean value at 509 nm (isoemissive wavelength) was used as a reference. Vertical-shifted spectra were eventually corrected by subtracting the deviation from the mean intensity at 509 nm to the emission peak intensity at 528 nm. 2.6. Calibration curve at atmospheric pressure (CCAP) L. innocua cells were centrifuged and resuspended to a final OD600 of 0.38 in Ringer solution adjusted to pH 5.0. Triton X-100 (0.2% (v/v) final concentration) was added 30 min before fluorescence measurement to permeabilize cells in suspension. Since the intracellular pH and the extracellular pH is inherently the same after permeabilization, we in this way obtained a direct correlation between pHi , and fluorescence intensity. Regarding cDCFDA, fluorescence-emission spectra were firstly collected at pH 6.0 and then at different pH values down to 4.0 by adding the right amount of HCl 1 M. All the data reported have been normalized by the maximum intensity value collected (pH 6.0). Regarding cFDA, fluorescence-emission spectra were collected at pH 5.0 and then at different pH values up to 8.0 by adding KOH

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1 M. Data have been normalized by the maximum intensity value collected (pH 8.0). 2.7. Calibration curve under pressure (CCUP) We observed that pressurizing the medium shifts the calibration curves to lower I/I0 ratios. Therefore, we had to acquire a separate calibration curve for all pressures investigated. For this purpose we used either N2 O with cFDA or CO2 with cDCFDA. Ringer solution – despite providing a friendly environment for cells – has no buffer capacity. To prevent acidification, induced by either residual atmospheric or pumped CO2 , cells were resuspended at OD600 = 0.38 in stable citric acid/(di)sodium hydrogen phosphate buffer solutions at pH values ranging from 4.0 to 7.0. NaCl, KCl and 10 mM glucose were added in the buffer with total amounts of 135 mM sodium and 5 mM potassium. After permeabilization at atmospheric pressure with 0.2% (v/v) Triton X-100 for 30 min, pressurized gas was applied. For each sample fluorescence-emission spectra were acquired at atmospheric pressure (after permeabilization) and at 0.5, 1, 5 and 8 MPa after pressure stabilization at each stage.

Fig. 1. Calibration curves based on normalized intensities (I/I0 ) of cDCFDA in permeabilized cells versus pHi . Signal was acquired at 0.1 MPa (CCAP) and at 0.5, 1, 5, 8 MPa (CCUP) with CO2 in the pH range 4.0–6.0. CCAP follows a sigmoidal trend (correlation index 0.98); Carbonic acid, due to CO2 solubilization, significantly decreases pHex and CCUPs indicate a progressive and substantial fluorescence decay with increasing pressure. Error bars refer to standard deviations (n = 5).

2.8. pHi evaluation To evaluate pHi dynamics under pressure, experiments were performed resuspending cells in Ringer solution (OD600 0.38) with 10 mM glucose, adjusted to pH 6.2. After pressurization up to the set-up value, fluorescence-emission spectra were collected every 15 min. 2.9. Viability count To evaluate viability after pressure treatments, cells were plated in different dilutions of Ringer solution on 50 mm diameter Petri dishes with standard plate count agar (PCA). Colony forming units (CFU) were counted after 48 h of cell growth at 37 ◦ C. Viability (%) is expressed as ratio between initial concentration (N0 ) of CFU and final concentration of CFU after treatment (either at N2 O and CO2 pressurization). 3. Results 3.1. Fluorescence CCAP at pH 4–6 using cDCFDA as stain L. innocua cells were stained and subjected to membrane permeabilization with 0.2% (v/v) Triton X-100. CCAP was performed in Ringer solution (Fig. 1). Experimental data were found to be best fit by a sigmoidal curve – with a correlation index of 0.98 – and normalized to the maximum value measured at pHex 6.0. The sigmoidal curve at atmospheric pressure was used to interpolate discrete data point under pressure. 3.2. Fluorescence CCUP at pH 4–6 using cDCFDA as stain CCUP were performed at various pressures (0.5–8 MPa) under CO2 using resuspended cells in buffered solutions at pHex ranging from 4.0 to 6.0 (see Fig. 1, full shapes). Cells were permeabilized as described above with Triton X-100. Fluorescence signal was acquired before and after permeabilization at atmospheric pressure, i.e. an absolute pressure of 0.1 MPa, to confirm total equilibration between pHi and pHex . As previously demonstrated, L. innocua was able to maintain a neutral pHi [24]. CCAP signals at atmospheric pressure strongly correlate to the CCAP fit. Even thought pHex is stable, fluorescence intensity decreases as a higher pressure is applied, especially at 5 and 8 MPa. At pHex lower than

4.0 optical signal resulted unstable, therefore data were discarded as not reliable. 3.3. pHi determination under CO2 pressure Fig. 3 shows pHi kinetics versus time of samples treated with carbon dioxide (full shapes). Despite CO2 saturated solutions lie below pHex 3.3 after 15 min [25] at all applied pressures, pHi decreases following a kinetic rate which increases with the pressure applied: at both 0.5 and 1 MPa, pHi progressively decreased to a final value of 4.6 at 60 min, while at 8 MPa, the highest pHi drop within 1 min at pHi 5.0 was observed. pHi 4.0, the minimum detectable value, was reached at 35 and 25 min at 5 and 8 MPa respectively, however, data suggest a continuous cytosol acidification, probably reaching pHex at longer treatment times (Fig. 3, dotted lines). Due to the increased permeability during treatment, fluorescent probe attached to small cellular components can be released into free bulk solution, thus altering mean fluorescence intensity from the cytosol of cells. However, after 25 min of treatment at 8 MPa, we verified that cDCFDA release in the supernatant reached a maximum value of 4.7%. 3.4. Fluorescence CCAP at pH 5–7 using cFDA as stain Fig. 2 shows the calibration curve regarding cFDA. Five data replicates were fitted to a sigmoidal curve by Origin® resulting in a correlation index R2 of 0.99 – and normalized to the maximum value measured at pHex 8.0 (not shown). Origin’s nonlinear regression method is based on the Levenberg–Marquardt algorithm, which is the most widely used algorithm in nonlinear least squares fitting. 3.5. Fluorescence CCUP at pH 5–7 using cFDA as stain The same procedures followed for cDCFDA were used for online measurement with cFDA-stained cells. CCUP were performed at various pressures (0.5–8 MPa) under N2 O pressure, resuspending cells in buffered solutions at pH 5.0, 6.0, and 7.0 (Fig. 2, empty shapes). Fluorescence signal clearly decreased as the applied pressure was increased.

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Fig. 2. Calibration curves based on normalized intensities (I/I0 ) of cFDA in permeabilized cells versus pHi . Signal was acquired at 0.1 MPa (CCAP) and at 0.5, 1, 5, 8 MPa (CCUP) with N2 O in the pH range 5.0–7.0. CCAP follows a sigmoidal trend (correlation index 0.98); N2 O does not significantly affect pHex and CCUPs indicate a progressive fluorescence decay with increasing pressure. Error bars refer to standard deviations (n = 5).

Fig. 3. pHi kinetics at different pressure values of either N2 O or CO2 . Empty and full shapes represent CO2 and N2 O pressure values, respectively. Vertical error bars refer to pHi standard deviations (n = 5). Dashed horizontal lines refer to pHex value at the equilibrium in the system H2 O–N2 O/H2 O–CO2 . Dotted curves indicate supposed trends of pHi below the lower sensitivity limit of 4.0: continuous and slow decrease of I/I0 signal occurred during the experiments meaning a probable equilibration with extracellular pH as with N2 O-treated samples.

3.6. pHi determination under N2 O pressure In Fig. 3, pHi dynamics under N2 O pressure are compared with the CO2 ’s ones. Although pHex at equilibrium (upper dashed line) was stable at 6.2, progressive pHi decrease, and thus cellular permeabilization, was observed during the pressure treatment; furthermore the pHi decreases following a first order kinetic rate, with a slope proportional to the applied pressure: at 8 MPa the kinetic rate is the highest in the pressure range considered and the lowest pHi indicated was 6.2 at 60 min. After 25 min of treatment at 8 MPa, cFDA release (probably due to the bound to small peptides) in the supernatant was limited to 2.9%. 3.7. Cellular viability Cellular viability was tested, plating each sample after 60 min treatment and counting colonies after 48 h incubation (Fig. 4). On one hand, CO2 had a strong impact even at lower pressures, probably due to carbonic acid formation. Each pressure applied induced a viability drop down to approximately 1% at 8 MPa. On the other hand, N2 O showed limited effect at pressures up to 5 MPa with over

than 70% of cells alive. At 8 MPa, inactivation increases dramatically with only 18% of initial cells forming colonies. 4. Discussion High-pressure treatment is an emerging field that can be applied to several areas where microbial inactivation at low temperature is essential. However, the mechanisms underlying this process have not been studied in sufficient depth so far. Off-line investigations cannot explain without ambiguity physical or chemical variations in the cellular cytosol [16,26]. Several hypotheses about CO2 inactivation mechanism have been formulated and proposed but they are yet to be confirmed by suitable experimental evidence, while, as N2 O is concerning, the literature is almost inexistent. What has been postulated so far, is that, once CO2 has dissolved into the liquid phase, it probably acts through 6 steps to exert its ability to be lethal for the cell: (1) cell membrane modification, (2) intracellular pH (pHi ) decrease [23], (3) key enzyme inactivation/cellular metabolism inhibition due to pHi lowering [24], (4)

Fig. 4. Cellular viability after treatments with N2 O and CO2 for 60 min. Percentage of viability, compared to an untreated sample, is shown at 0.5, 1, 5, 8 MPa. Cells were plated on standard PCA agar and colonies were counted after 48 h of incubation at 37 ◦ C. N2 O becomes effective only close to critical pressure, whereas CO2 has a significant inactivation even at low pressure. Error bars represent standard deviations (n = 4).

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direct inhibitory effect of molecular CO2 and HCO3 − on metabolism [27], (5) disordering of the intracellular electrolyte balance [27], and (6) removal of vital constituents from cells and cell membranes [28,29]. After dissolution of CO2 inside the cell membrane, most of the other steps may not occur consecutively, but rather take place simultaneously in a very complex and interrelated manner [30]. The amount of CO2 accumulated in the lipid phase structurally and functionally disrupts the cell membrane due to a loss of order in the lipid layer, which may increase the fluidity [31]. The high affinity between CO2 and the plasma membrane has been theoretically and experimentally confirmed by Spilimbergo et al. [16,32], who calculated the amount of CO2 that can be dissolved into the phospholipids of a model cell membrane and evaluated CO2 diffusion and cell permeabilization caused by SC-CO2 , by real-time measurement of propidium iodide uptake by spectrometer analysis [16]. The theory, nevertheless, was still waiting to be completely verified by suitable on-line experiments focused on the evaluation of pH decrease of the intracellular environmental in real-time during the process. Thanks to an improved staining method and a novel spectroscopic technique, we analysed – in real-time – intracellular pH during pressurization towards supercritical conditions gaining deeper knowledge about intracellular mechanisms. At the process conditions selected (25 ◦ C, 0.5–8 MPa, 1–60 min), low protein release, slow pH decrease and an increasing inactivation allowed to follow progressive pHi dynamics in real-time both applying pressurized CO2 and N2 O. Using plate counting we were able to compare the pHi dynamic with microbial inactivation in the same treatment time condition. During treatment, it was clear that carbon dioxide caused a progressive decrease of pHi with a fast drop as rapid as the pressure applied was higher. Due to the acid-driven cellular modifications, which lead to an unstable optical output signal, and due to low sensitivity of the fluorescent dye at this low pH it was not possible to follow pHi below 4.0 during higher-pressure treatments. Low pressures (0.5–1 MPa) showed slower pHi dynamic acidification, while, at higher pressures (5–8 MPa), towards the critical pressure, pHi decrease followed significantly faster kinetics. As concerns cell viability, we observed a significant inactivation at 0.5 MPa, after 60 min of treatments, while at the same operative conditions but at higher pressure, the cell inactivation was almost total. This observation leads to the conclusion that far from critical conditions, although limited membrane chemical modification and pHi drop occur, low pHex plays a substantial role in microbial inactivation. Membrane integrity may be compromised only towards the critical pressure, thus leading to almost complete inactivation [16]. Many cell types are not able to counteract the ionic pressure through their proteic pumps. Channels and porins do not completely stop the influx of protons. A contribution of higher pressures on inactivation is masked by this early phenomenon (external pH dramatically decrease even at 0.5 MPa) leading to slower viability decrease seen at 1–8 MPa compared to 0.5 MPa. To confirm our hypothesis of the unique effect of high pressurized gases towards critical point on increased membrane permeability and subsequent inactivation, pressurized N2 O was applied: treatment at 0.5 MPa demonstrated that L. innocua is capable to counteract pHi variation in a non-starving condition stabilizing intracellular pH at 6.8 for 60 min. A more limited phenomenon happens treating cells at 5 MPa with almost pHi /pHex equilibration. Even if environmental conditions applied are subcritical (25 ◦ C), not reaching the critical point (N2 O: Tc = 36.4 ◦ C, pc = 7.25 MPa), complete membrane permeability was reached at 8 MPa, confirmed by complete equilibration between pHex and pHi . Membrane integrity failure is also supported by cell viability showing a substantial inactivation only at the highest pressure applied in this study (18% colony forming bacteria). Despite the acidifica-

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tion due to carbonic acid, this data confirm that the two gases have analog behaviour near the critical pressure and that towards supercritical state microbial inactivation is mainly affected by increased membrane’s permeability. In other words, as the supercritical pressure is achieved, dissolved gases play a major role in membrane’s increased permeability thus lowering pHi and providing possible positive feedbacks on inactivation. In conclusions, our study investigated deep further the inactivation mechanisms with both CO2 and N2 O. Owing to an improved staining method and a spectroscopic technique, we were able to precisely measure intracellular pH variation kinetics during CO2 /N2 O process. From the analysis of the experimental data obtained, we could demonstrate for the first time that pH decrease must be considered a key factor in microbial inactivation only at certain pressure condition: in near- and super-critical conditions, membrane permeability increase must be considered the determining factor of inactivation, with (CO2 ) or without (N2 O) active pH changes, while, far from critical pressure condition, low extracellular pH (due to CO2 dissolution and consecutively to some extent dissociation in the liquid phase), plays an primary role in microbial inactivation, as treatment with N2 O do not cause any significant microbial count decrease. These insights have a great importance in a growing industrial field such as supercritical fluids: knowing intracellular pH dynamic can be useful to get new strategies to manipulate microbial cytosol with the goal of optimizing CO2 technologies for microbial process such as pasteurization, intracellular product extraction via cell rupture or providing new non acidifying gases such as N2 O to be exploited in the same process where pH preservation has a critical role.

Acknowledgments We gratefully acknowledge the kind support of this project by Montana Alimentari S.p.A, Italy.

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