International Journal of Food Microbiology 144 (2010) 118–125
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International Journal of Food Microbiology
j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro
Inactivation of Staphylococcus aureus and Escherichia coli by the synergistic action ofhigh hydrostatic pressure and dissolved CO2
Li Wang a,b, Jian Pan a,⁎, Huiming Xie a, Yi Yang a, Chunming Lin a
a Engineering Research Centre of Bio-Process, Ministry of Education of China, Hefei University of Technology, 193 Tun Xi Road, Hefei, Anhui 230009, PR Chinab School of Chemical Engineering, Hefei University of Technology, 193 Tun Xi Road, Hefei, Anhui 230009, PR China
Article history:Received 24 June 2010Received in revised form 5 September 2010Accepted 6 September 2010
Keywords:Synergistic inactivationDissolved CO2
High hydrostatic pressureStaphylococcus aureusEscherichia coli
This study focused on the synergistic inactivation effects of combined treatment of HHP and dissolved CO2 onmicroorganisms. The aimwas to reduce the treatment pressure of the traditional HHP technology andmake itmore economically feasible. The combined treatment showed a strong bactericidal effect on Staphylococcusaureus and Escherichia coli in liquid culture, which usually have high levels of barotolerance under pressurealone. To identify the influence of CO2, a new setup to dissolve, retain and measure the concentration of CO2
was constructed. The results demonstrated that an inactivation rate of more than 8 log units was obtained forE. coli both at 300 MPa with 1.2 NL/L CO2 and at 250 MPa with 3.2 NL/L CO2, while only 2.2 and 1.8 logreductions were observed at 300 MPa and 250 MPa, respectively, for the HHP treatments alone. For S. aureus,the inactivation rate of more than 7 log units was found at 350 MPa with 3.8 NL/L CO2, while only a 0.9 logreduction was achieved at this pressure in the absence of CO2. The SEM photographs showed seriouslydeformed cells after the synergistic treatments. In contrast, the cells treated with individual HHPmaintained arelatively smooth surface with invaginations. Propidium iodide staining and fluorescence observation wasperformed after pressure treatments. The results demonstrated that the combination of CO2 with HHP alsopromoted pressure induced cell membrane permeabilization greatly. It was deduced that the enrichment ofCO2 on the cell surface and its penetration into the cells at high pressure accounted for the membrane damageand cell death.
High hydrostatic pressure (HHP), as an alternative to heatpasteurisation, is one of the most promising non-thermal processingtechniques for the inactivation of microorganisms in liquid and solidfood systems while preserving nutritional and sensory characteristics.To improve HHP efficacy, it is necessary to reduce the operationpressure or dwell time and, in turn, the processing costs. Regarding thetreatment pressure, the barotolerance of bacteria is an importantparameter that varies largely depending on the species and treatmentconditions (Alpas et al., 1999; Patterson et al., 1995; Wuytack et al.,2002). In general, the high pressures of 600 MPa or more required forefficient microbial inactivation has limited the commercial break-through of HHP technology. The application of hurdle technology hasbeen proposed to increase the microbicidal effect of the process atlower pressures. Recently, to reduce the inactivation pressure, variouseffective synergistic treatments have attracted much more attention,
and several combined treatments have been investigated to optimisethe processes and elucidate the mechanism of HHP treatment. Thesecombined factors include antimicrobials, pH and moderate tempera-ture. For the antimicrobials from natural sources, such as nisin,pediocin, lysozyme and lactoperoxidase, have been used to combinewith HHP for microbial inactivation. It has been found that severalnatural biopreservatives, such as lysozyme and bacteriocins, areeffective against some gram-positive bacteria (Garcia-Graells et al.,2000). Due to the protection from the outer membrane against thepenetration of the above peptides and enzymes, gram-negativebacteria are normally insensitive to these antimicrobials, althoughhigh pressure can enhance their sensitivity to a certain extent(Masschalck et al., 2001). The combination of lactoperoxidase withHHP was found not to increase inactivation of several Escherichia colistrains even at pressure of 400 MPa (Garcia-Graells et al., 2000). Forthe combinations of pH and moderate temperature, it has beenreported that the high hydrostatic pressure applied in conjunctionwithmild heat and acidity in organic acid solutions can be an effectiveroute for inactivating strains of Staphylococcus aureus, Listeria mono-cytogenes and E. coli (Alpas et al., 2000; Koseki and Yamamoto, 2006;Somolinos et al., 2008). All these combined hurdle factors exhibit
Fig. 1. Schematic diagrams of the synergistic treatment of HHP and dissolved CO2 for microbial inactivation. After the sample was carbonated, the angle needle valve was closed andthe sample was pressurized in the high pressure vessel.
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different interactions with HHP to cause injury and death of thepressure-resistant and pressure-sensitive strains of food-bornepathogens.
Carbon dioxide is another inhibitory compound formicroorganisms.Compared to the mentioned bacteriocins and lysozyme, small andnonpolar CO2molecules can be expected to penetrate themembrane ofboth gram-positive and gram-negative bacteria under high pressure,which would result in the effective destruction of bacteria cellmembranes. The inactivation effects of CO2 combined with HHP onseveralmicroorganisms includingBacillus subtilis, S. aureus, Lactobacillusplantarum and E. coli have been demonstrated (Corwin and Shellham-mer, 2002; Park et al., 2003). In contrast to the dense-phase CO2 (DPCD)treatment, in which supercritical or near-critical CO2 was employed toinactivate microbes, this combined treatment employed high hydro-static pressure in thepresence of dissolvedCO2. In theDPCD technology,CO2 gas pressure is used in a less range of 10–25 MPa at a highertemperature range of 30–50 °C, and for a longer dwell time ofmore than1 h formicrobial inactivation usually (Bertoloni et al., 2006;Dillowet al.,1999; Hong and Pyun, 1999; Liao et al., 2007; Spilimbergo and Bertucco,2003). The HHP treatment could be considered to be safer and moreeconomical than CO2 gas pressure. However, the challenge remains tomaintain the dissolved CO2 at a given concentration in the flexiblepackage required by HHP treatment. Thus, the efficacy of synergisticaction has been limited for low CO2 concentrations in the combinedtreatment (Park et al., 2003).
In the present work, we developed a new method to dissolve andmaintain CO2 in the aqueous treatment medium. The strongsynergistic inactivation effect of HHP and dissolved CO2 at differentconcentrations on S. aureus and E. coli was demonstrated. The cellmorphological changes and membrane permeabilization induced byHHP treatmentswith or without CO2were also studied using scanningelectron microscopy (SEM) and propidium iodide (PI) staining, whichcompared and assessed the cell damage resulting from bothtreatments. These results may provide a new route to the effectivemicrobial inactivation at mild pressure for HHP technology and betterinsight into the mode of synergistic action of dissolved CO2 and HHP.
2. Materials and methods
2.1. Microorganism and growth conditions
Two microbial strains were used as the test organisms, S. aureus1.2465 and E. coli ATCC11775. They were inoculated from sub-culturedagar plates to 500 ml Luria Bertani (LB) broth at a pH of approximately
6.7 (not adjusted) in a narrow neck flask and incubated withoutagitation for 16 h at 37 °C until they reached stationary phase. The cellconcentration in the final culture was generally about 108–109 CFU/mL.
2.2. CO2 solubilization and HHP treatment
The two steps of CO2 solubilization and HHP treatment are shown inFig. 1. To dissolve CO2 to certain concentrations and maintain it in thebroth culture during consequent pressurization, we designed andconstructed a thin-wall cylinder (TWC) with an internal volume of100 ml, an outside diameter of 35 mm and a wall thickness of 0.35 mmmade of stainless steel. The bacterial culture was transferred asepticallyinto the TWC for carbonation. The TWCwas connected to a CO2 cylinderthrough a sterilised pipe to disperse CO2 into the culture in athermostatic bath at 1.5 MPa and 4 °C for 10 to 30 min. Then the angleneedle valvewas closed, and the TWCwas placed in HHP equipment forpressurization. The TWC transferred high pressure into the cellsuspension by deformation when subjected to external pressure inthe high pressure vessel. The permissible internal and external pressureof the TWCwaspreviously calculated to ensure that it couldwork underthe appointed internal pressures for CO2 dissolution and deform totransfer external pressure during theHHP treatments. The pressure unitis a custom built one which includes a 1 L high pressure vessel, withinternal size of 75 mm in diameter, 240 mm in height (Kefa, Bao Tou,China) and a high pressure intensifier pump (Dalong, Shanghai, China).The vessel was thermo regulated by a heating/cooling water jacket. Thepressure transfer fluid is dioctyl sebacate. At the beginning of theexperiment, the bacteria sample and the pressure medium in the vesselwere preheated to the experimental temperature using heating water.During pressurization, the vessel was thermo controlled with water inthe jacket at 30 °C and 20 °C for the two experimental temperatures,respectively. The adiabatic compression heat was transferred by thevessel wall andwater. The pressurization and depressurization time arewithin 10 s. The sample in the TWC may undergo a temporarytemperature increase of about 12 °C upon pressurization to 400 MPaas reported (Patazca et al., 2006; Shao et al., 2010) and was then cooledrapidly by the vessel and water in the jacket. The influence of adiabaticcompressionheat onpressure treatmentwasminimized in this study byheat transfer. Because the heat was dissipated, the final temperature ofthe sample after decompression was 4–6 °C below the initial temper-ature in each treatment (measured immediately after the sample wastaken out of the pressure vessel). The treatment temperature is referredto the initial treatment temperature in this paper. For comparison, thecontrol samples (without CO2) were aseptically filled into 30-ml
Table 1The effect of dissolved CO2 on the viability of the S. aureus and E. coli strains at 30 °Cwithout HHP treatment.
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sterilised polypropylene bottles and were treated under the sameconditions.
Fig. 3. Effect of CO2 concentrations on the inactivation of S. aureus and E. coli underpressures of 350 MPa and 250 MPa, respectively, at 30 °C for 10 min.
2.3. Measurement of the CO2 concentration
One of our main purposes was to test the synergistic effects ofdissolved CO2 and HHP on bacteria at different CO2 concentrations.Therefore, it was important to measure the exact quantity of thedissolved CO2 to identify the experimental conditions. The CO2 in theTWC was released and conducted into 150 mL 0.5 M NaOH solutionfor absorption and neutralization reaction. The absorption solution,which contained the mixed components of NaHCO3 and the excessNaOH, was then titrated with 0.5 M HCl. The two end points oftitration were at pH 8.32 and 3.89. The concentration of CO2 in thesample (v/v) was calculated and expressed as the normal litre per litreof sample (NL/L), the volume of CO2 under standard state of 20 °C,1 atm in the sample.
Fig. 2. Inactivation of S. aureus (A and C) and E. coli (B and D) as a function of the treatment p
2.4. Viability counts
The cell suspensions were serially diluted, and 1 ml of appropriatedilutions were inoculated onto nutrient agar plates. The plates wereincubated for 24 h at 37 °C, and the colony forming units (CFU) werecounted. The microbial survival ratio is expressed as the logarithmic
ressure with and without dissolved CO2 at 30 °C (A and B) or 20 °C (C and D) for 10 min.
Fig. 4. SEM micrographs of E. coli and S. aureus cells. (A), (B) and (C) are E. coli cells, (A) untreated, (B) treated with a combination of dissolved 3.2 NL/L CO2 and 250 MPa (lgN/N0=8.1) and (C) treated with 250 MPa (lgN/N0=1.8). (D), (E) and (F) are S. aureus cells, (D) untreated, (E) treated with a combination of dissolved 3.8 NL/L CO2 and 350 MPa (lgN/N0=7.8) and (F) treated with 350 MPa (lgN/N0=0.9). All of the HHP treatment conditions were at 30 °C for 10 min.
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(N/N0) with N and N0 representing the counts (CFU/mL) after andbefore the treatment.
2.5. Fixation and SEM observation
Both treated and untreated cell suspensions were centrifuged at3500×g for 10 min and resuspended in phosphate-buffered saline(PBS). The suspension was dropped to a fragment of cover slip platedwith Formvarmembrane, and the cover slipwas held still for 15 min todeposit cells on the membrane. The cells were immediately prefixedwith 0.1% glutaraldehyde in 100 mM phosphate buffer, pH 7.4, for 1 h
and then were fixed with 0.5% glutaraldehyde for 2 h. The glass pieceswere rinsed three times in 10 mM PBS. The samples were thendehydrated in increasing concentrations of ethanol as follows: 30%,50%, 70%, 80%, 90%, 95%, and 100% ethanol twice for 10 min each. Then,the cells were dried lyophilized, gold-coated and observed by SEM.
2.6. Cell staining and fluorescence examination
Cell membrane permeability was assessed using PI staining.Samples were centrifuged and resuspended in phosphate-bufferedsaline (PBS). A 1.5 mM stock solution of PI in PBS was added to a final
Table 2Morphological changes examined by SEM in E. coli and S. aureus cells subjected to thecombined treatments and the individual HHP treatments at 30 °C for 10 min.a
Bacteria Treatmentconditions
% of cells withdeformedconfigurations
% of cells withinvaginationsbut smooth surface
Inactivation ratein log units
S. aureus 350 MPa 69 0.95.5 NL/L CO2+350 MPa
99 7.9
E. coli 250 MPa 98 1.83.2 NL/L CO2+250 MPa
100 8.1
a The percentages of treated cells were calculated with respect to the total number ofcells examined for each SEM micrograph (approximately 200 cells).
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concentration of 75 mM. Then cells suspensions with PI wereincubated for 15 min at 4 °C in the dark and observed with a confocallaser scanning microscope (FV1000, Olympus, Japan). Black-and-white images were also captured to show the total cell population.The ratio of PI permeable cells to total cell counts represented thepercentage of membrane permeability. For each measurement, atleast 300 cells were counted.
3. Results
3.1. Effect of CO2 on S. aureus and E. coli strains viability
To determine the effect of dissolved CO2 alone on the viability ofthe two strains during and after the carbonation process, bacteriasuspensions were treated only with soluble CO2 at 30 °C without HHP.The viable cells were examined at several intervals after carbonation.The results are listed in Table 1. During the first 4 h of storage, the cellnumbers remained constant for S. aureus and increased a little forE. coli. There was a slight decrease in population after the 8-h storagefor both bacteria. The dissolved CO2 at these concentrations exerted abacteriostatic but no bactericidal effect on both strains for at least 24 hat the test temperature.
Fig. 5. Membrane permeabilization of E. coli at 250 MPa, with 3.2 NL/L CO2 (white),without CO2 (grey), and S. aureus at 350 MPa, with 3.8NL/L CO2 (white), without CO2
(grey), at 30 °C for 10 min. Permeabilization induced by heat treatment at 95 °C for10 min is shown for comparison.
3.2. Inactivation effect of the synergistic treatments on S. aureus and E.coli
The bacteria suspensions of S. aureuswith different concentrationsof CO2 or without CO2 were subjected to HHP ranging from 250 to440 MPa for 10 min. The results are shown in Fig. 2A. The controlsamples without CO2 showed high levels of barotolerance as inprevious reports (Alpas et al., 2000; Park et al., 2003; Wuytack et al.,2002). An inactivation rate of only 1.7 log units was achieved forS. aureus via HHP treatment with 440 MPa at 30 °C. Combinedtreatments were conducted at three CO2 concentrations, 2.6, 3.8 and5.5 NL/L, at 30 °C. With the dissolved CO2 present in the treatmentmedium, the synergistic inactivation effect on S. aureus was strong,achieving more than 7 log cycles under HHP of 350 MPa at 30 °C.Compared with the 0.9 log unit reduction under the 350-MPa HHPtreatment alone, the synergistic action of CO2 caused an additionalreduction of more than 6 log units.
The E. coli suspensions were also exposed to HHP from 100 to440 MPa, with or without combined CO2. As shown in Fig. 2B, theobvious synergy appeared at 150 MPa, and the inactivation rate ofmore than 8 log units was achieved both at 300 MPawith 1.2 NL/L CO2
and at 250 MPa with 3.2 or 4.5 NL/L CO2. The results showed that theE. coli cells were much sensitive to high hydrostatic pressure than theS. aureus but also highly sensitive to the combined treatment. Forpressures ranging from 250 to 300 MPa, the synergistic action alsocaused an additional reduction of approximately 6 log units whencompared to the individual HHP treatment. Considering a similarinactivation rate, these lethal pressures for the S. aureus and E. coliwere about 250 MPa lower than the reported pressures of about600 MPa and 500 MPa, respectively, required for elimination by HHPalone (Park et al., 2003; Wuytack et al., 2002).
The synergistic inactivation effects on S. aureus and E. coli declinedsignificantly at 20 °C, as shown in Fig. 2C and D, respectively. Weselected the two highest CO2 concentrations from the previous twogroups of experiments at 30 °C to investigate the influence oftemperature. For S. aureus, at the same CO2 concentration of 5.5 NL/Land pressure of 350 MPa, a 7.8 log unit reductionwas obtained at 30 °C,but only a 3.5 log unit reduction was obtained at 20 °C (Fig. 2C). For E.coli, at the same CO2 concentration of 4.5 NL/L and pressure of 250 MPa,there was an 8.2 log unit reduction at 30 °C but 3.9 log units at 20 °C(Fig. 2D). When the treatment temperature was reduced from 30 °C to20 °C, the elimination pressure of E. coli increased from 250 MPa to350 MPa.
3.3. The effect of the CO2 concentration on the synergistic inactivation
The S. aureus and E. coli strains were not effectively inactivated bythe individual HHP treatments of 350 and 250 MPa at 30 °C, whichcaused 0.9 log unit and 1.8 log unit reductions, respectively. Theinactivation rate increased rapidly with the increasing of the CO2
content and levelled off, reaching the maximum inactivation at 5.5NL/L for S. aureus and 3.2 NL/L for E. coli, as shown in Fig. 3. S. aureusseemed to need a slightly higher CO2 concentration for the effectivesynergy. In other words, the S. aureus exhibited more resistance bothin the combined and individual treatments than E. coli.
3.4. Cell morphological changes after the HHP alone and the synergistictreatments
The scanning electron microscope (SEM) images of the E. coli andS. aureus cells are shown in Fig. 4. Untreated S. aureus and E. coli cellspresented smooth and uniform configurations (Fig. 4A and D). Therewere obvious differences in the morphological changes between thecells treated by HHP with or without the synergy of dissolved CO2 atthe same pressure. The E. coli cells treated at 250 MPa in the presenceof CO2 showed a highly deformedmorphology of a rough surface with
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many concave defects and ruptured cell wall, whereas the treatmentwith the pressure of 250 MPa alone only induced cell invaginationswith the cell surface remaining smooth and continuous (Fig. 4C).Table 2 shows that these morphological changes on the combinedtreated cells occurred in nearly 100% of cells. SEM micrographs alsorevealed that the S. aureus cells treated at 350 MPa largely maintainedtheir aspects except for some invaginations (Fig. 4F). However, withCO2 present in the treatment medium under the same pressure,serious invaginations and shrinkages took place on cells, indicatingcell membrane or wall damage (Fig. 4E). No cell disruption wasobserved with SEM in our experiments, which was different from theformer reports (Park et al., 2003; Ritz et al., 2002).
3.5. Cell membrane integrity
Fig. 5 shows the cell membrane permeability via varioustreatments. Untreated samples were taken as controls which showedno uptake of PI. Only a small proportion in HHP alone treated cellswere permeabilized with PI, which indicated most of the cells kepttheir membrane integrity. The combination of dissolved CO2 withHHP increased themembrane permeability from 12% to 87% and 5% to69% for E. coli and S. aureus, with 3.2 NL/L and 3.8 NL/L CO2,respectively. However, the permeability did not reach an entireextent, as observed for heat treatment, although the inactivation wascomplete in both cases.
4. Discussion
S. aureus, a spherically shaped, Gram-positive facultative anaerobe,is one of themost pressure-resistant bacteria. It has been reported thatHHP of 400 MPa caused an approximately 1-log unit reduction in S.aureus in phosphate-buffered saline at room temperature, and it waseffectively inactivated at 600 MPa by HHP alone (Alpas et al., 2000;Park et al., 2003;Wuytack et al., 2002).WhenHHPwas combinedwithcarbonation at very low concentration of 5–6% in v/v, the inactivationpressure was first demonstrated to decrease to 500 MPa (Park et al.,2003). In this study, we observed an inactivation rate of greater than 7log units for S. aureus in the combined treatments at 350 MPa.However, with increasing the CO2 concentration, no further pressuredecrease was found obviously for the similar inactivation rate.
E. coliwas chosen as the target bacteria for its common existence infood, the considerable baroresistance among Gram-negative bacteriaand the insensitivity to some antimicrobials. In our experiments, E. colishowed a similar sensitivity response as S. aureus to this combinedtreatment at lower CO2 concentrations. These behaviours of the twostrains seemed to differ from the combined effects of high pressure andother antimicrobial compounds, as previously reported (Garcia-Graellset al., 2000;Masschalck et al., 2001). For E. coli, an additional inactivationof 6.4 log units at 250 MPa in the presence of CO2 demonstrated that thesynergy reached a higher level, which implies a differentmode of actionthan antimicrobials. We ascribed this effect to the ready penetration ofCO2 through the outer membrane of E. coli cells under high pressure, asdescribed further in the following discussion. In addition, the treatmenttemperature had a significant effect on the synergistic inactivation. Asimilar influence of temperature occurred in the dense-phase CO2
(DPCD) exposure (Dillow et al., 1999; Enomoto et al., 1997; Hong et al.,1997; Liao et al., 2007; Shimoda et al., 2002). Higher temperaturesenhanced thediffusivity of CO2 and could also increase thefluidity of cellmembrane tomake its penetration of the cell easier (Hong et al., 1997).
SEM photographs have been used to demonstrate morphologicalchanges in cells exposed to various treatments (Dillow et al., 1999;Espinasse et al., 2008; Park et al., 2003; Ritz et al., 2002; Tholozan et al.,2000). Inour experiments, theHHP treatments of 350 MPaand250 MPawere found to be only sub-lethal pressures for S. aureus and E. coli,respectively. A considerable population can survive these pressures,including the fraction resumed from sub-lethal damage in a suitable
culture (Kiliman et al., 2006; Shearer et al., 2009; Somolinos et al.,2008). Accordingly, the SEM micrographs (Fig. 4C and F) of cellstreated byHHP alone showed less change in the cellular features thanthose undergoing the synergistic treatments. After the synergisticaction of dissolved CO2, no cells survived, and the seriously deformedcells were observed (Fig. 4B and E). Though the SEM photographscould not correspond to the inactivation rate directly, theydemonstrated the significantly different changes from the differenttreatments, which can reveal the degree of cell damage. The resultsalso showed that cell rupture was more easily achieved in E. coli, theGram-negative bacteria, than in S. aureus, the Gram-positive bacteria,in the combined treatments due to the thinner cell wall in Gram-negative bacteria.
The cell membrane integrity was assessed by the exclusion of PI.The results in Fig. 5 showed a low uptake of PI in cells treated by HHPalone. The similar observations have been reported by other authors(Ananta et al., 2004; Moussa et al., 2007; Ritz et al., 2002). The lowuptake of PI in cellsmeansweak permeabilization of cellmembrane byHHP, which mechanism has not been clearly demonstrated. It wassuggested that the membranes physically reseal after decompressionto an extent where they exclude PI molecules. In addition, althoughthese cells did not take up PI, they may be permeabilized to the extentthat ions, water, and other small molecules can leak into or out of thecell cytoplasm, leading to cell death (Moussa et al., 2007). In our study,the increase in membrane permeabilization with the addition of CO2
was consistent with the increase in cell inactivation. The cellmembrane has been considered to be one of the major targets ofHHP treatment (Macdonald, 1984; Moussa et al., 2007; Ritz et al.,2002; Tholozan et al., 2000). When the membrane is extensivelypermeabilized, cell death occurs.
It has been found that CO2 inhibited microorganisms at very lowconcentration such as in fermentation and the mechanism stillremains to be unclear. The antimicrobial effects of compressed CO2
in supercritical or near-critical states have been extensively studied toelucidate the mechanism. The possible mechanisms of DPCD processinvolved the ability of supercritical fluid (SCF) CO2 to diffuse readilyinto the cell and alter the pH within the cell, the inactivation of keyenzymes of cells caused by the dissolved CO2, the extraction ofintracellular substances, or cell rupture due to the expansion of CO2
within the cells (Bertoloni et al., 2006; Dillow et al., 1999; Hong andPyun, 1999; Liao et al., 2007; Shimoda et al., 2002). In ourexperiments, much lower CO2 concentration under high pressurewas considered to result in the decrease of intracellular pH, thediffusion of CO2 into cells and cell rupture. However, the driving force,characterization and action mode are different, resulting in a differentinactivation effect compared with DPCD.
The CO2 concentrations in solutions vary largely depending on thetemperature, the pressure, the contacting time and the interface areaof the gas and liquid phases. It is well known that CO2 dissolves in theaqueous phase and forms carbonic acid, which further dissociates togive bicarbonate, carbonate and H+ ions, thus lowering the pH of thesolution. It has reported that the reduced pH may alter enzymeconformations and membrane permeability, and this will increase thesusceptibility of bacteria to high pressures (Alpas et al., 2000; Hongand Pyun, 1999; Koseki and Yamamoto, 2006; Somolinos et al., 2008).To discuss the effect of the pH in our experiments, the chemicalequations and standard equilibrium constants (named as KΘ, 25 °C) ofCO2 dissolving reactions can be listed as following:
CO2ðaqÞ + H2OðlÞ↔H2CO3ðaqÞ;ð1ÞKΘh
H2CO3ðaqÞ↔HþðaqÞ + HCO−3 ðaqÞ;ð2ÞKΘ
a1
HCO−3 ðaqÞ↔HþðaqÞ + CO2−
3 ðaqÞ;ð3ÞKΘa2
KΘh = 1:7 × 10−3
; KΘa1 = 2:5 × 10−4
; KΘa2 = 5:61 × 10−11
:
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Theoretically, the basic chemical equilibrium equation can be writtenas following:
LnKa = ΔGΘ= RT;
where Ka is the equilibrium constant of the reaction, ΔGΘ is thestandard free energy change. Making partial derivative with pressurefor above equation, we can get the equation as follows:
∂Ka
∂P
� �T= −ΔVΘ
RT;
where ΔVΘ is the sum changes of partial molar volume of all reactioncompounds. In high pressure liquid reaction system, it may not beignored for the change of partial molar volume of every composition.
Usually, in this case, ΔVΘb0, so, ∂Ka∂P
� �TN 0, which means the
equilibrium constants will increase with the increasing of pressureat a given reaction temperature, (Atkins, 1982).
In our experiments, after the initial CO2 was dissolved, the TWCwas sealed. The reaction system became a single aqueous solutionphase in unsaturation state. In this case, no additional CO2 gas present,thus, the concentration of dissolved CO2 no longer increased.However, due to ionization equilibrium constant would increasewith the high pressure, the acidity of the solution was increased.Because the compressibility of the liquid is small, the acidity increasewould be in a limited range. On the other hand, in our experiment, theprotein and amino acid in the treatment medium, Luria Bertani broth,acted as natural buffer system to prevent the decrease of pH. In thiscase, actually, the bactericidal effect of the low pH was reduced. Infact, the calculated pHs from above equilibrium constants range from3.9 to 3.5 for CO2 concentrations of 1–5 NL/L in pure water, yet, theinstant measured pH values of the combined treated samples afterdecompression (saturated with CO2 at atmosphere pressure) areabout 5.2 in our experiments. The results indicated not only theobvious buffer effect of the suspension medium but also lesssynergistic bactericidal effect of the low pH.
Compared with other organic acids used as acidulants, the specificinactivation effect of CO2 has been demonstrated by some authors(Becker, 1936; Erkmen, 2000; King and Mabitt, 1982; Lin et al., 1993;Sears and Eisenberg, 1961). It is extensively accepted that the actionof CO2 on cells correlates to its penetration of cells (Enomoto et al.,1997; Hong and Pyun, 1999; Liao et al., 2007; Shimoda et al., 2002).The specific cell-solution interfacial properties in carbonated suspen-sions, broth culture in our experiments, would contribute to themicrobial inactivation, which can be explained by the following twoaspects.
(i) The enrichment of CO2 on the surface of the bacterial cells.Generally, because the equilibrium constant of the reaction toform carbonic acid is very small (1.70×10−3), most of thedissolved CO2 molecules, existing in weak hydration state,should be taken into consideration for the inactivation.Furthermore, there exists a concentration redistribution ofCO2 in the cell suspension. The treatment medium, LuriaBertani broth in our experiments, is a solution of thehomogeneous phase, in which bacteria cells suspend andthus form a multiphase system. The cells then act asheterotypic particles in the solution, and the nucleation andgrowth of CO2 micro-bubbles occur with a decrease of Gibbsfree energy on their surface spontaneously. The CO2 moleculesmove towards the cell surface and grow there due to afavourable thermodynamic factor. Therefore, the CO2 concen-tration on the interface is actually much higher than that in thebulk solution, and an enriched layer of CO2 forms between the
cell and solution. The large specific surface of the bacterial cellalso favours this enrichment. The action of aggregated CO2 oncells would be explained as follows. The increase of CO2 woulddecrease the water miscibility of the cell membrane and theease of ionic penetration by altering the contact between thecells and their external aqueous environment (Sears andEisenberg, 1961). The interfacial tension between the cellsand the environment has been found to be very low, usuallyless than 1 dyne per cm (Harvey and Danielli, 1938). Thiswould suggest near miscibility between cells and the aqueousenvironment. Such close contact may facilitate metabolicexchanges. The surrounding CO2 molecules would shield thecells from these exchanges and cause cell narcosis, which isgenerally reversible (Sears and Eisenberg, 1961).
(ii) Cellmembranedisorganisationbypenetration anddiffusionof theCO2 under high pressure. However, it is not the same case underhigh pressure, which would propel the small and lipophilic CO2
molecules to penetrate into the bacteria cells. The CO2 moleculesaccumulate and form carbonic acid to reduce the intracellular pH,with the majority existing freely. The pressure also facilitates thediffusion of the CO2 molecules in the phospholipid and conse-quently destroys the arrangement and interaction of thephospholipid bilayer. When the high hydrostatic pressure isreleased, the excess intracellular CO2 can impinge or shear the cellenvelopewith a larger gas expanding velocity than the cytoplasm,causing cell membrane or wall damage, which results in celldeformation, as presented in Fig. 4B and E. It has been suggestedpreviously that the cell membrane could be a target for highpressure by disorganisation among membrane phospholipids(Macdonald, 1984). The synergistic action of dissolved CO2
reasonably enhanced this disorganisation, and thus the bacteri-cidal effect was enhanced.
5. Conclusions
In summary, our results have shown that the synergistic treatmentof HHP and dissolved CO2 can inactivate S. aureus 1.2465 and E. coliATCC11775 effectively. An originally designed method made itpossible to demonstrate this inactivation effect by dissolving andmaintaining CO2 in the treatment medium during pressurization atthe given concentrations. The serious cell deformation of the twostrains was observed from the SEM images after the synergistictreatment. Membrane permeabilization indicated by PI staining alsoincreased significantly. Our results suggest that the cell death andmembrane damage correlate with the CO2 enrichment and penetra-tion of the membrane under pressure. In the presence of CO2, which ischeap and safe for food and easy to remove, much lower pressureswere needed to achieve the desired inactivation. Further work will beundertaken to study the combined effects on other microbial models,particularly more resistant ones such as spores. The aim will be tovalidate the new process and make the high hydrostatic pressuretechnology more economically feasible.
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