Inactivation of Microorganisms in Carbon Dioxide at Elevated

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Ind. Eng. Chem. Res. 2007, 46, 6345-6352

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Inactivation of Microorganisms in Carbon Dioxide at Elevated Pressure and Ambient Temperature Yin Wu, Shan-Jing Yao,* and Yi-Xin Guan Department of Chemical and Biochemical Engineering, Zhejiang UniVersity, Hangzhou 310027, China

Microorganisms are inactivated remarkably by carbon dioxide at the elevated pressure. The process can be divided into two phases based on the different rate of inactivation: the rate of inactivation is slower at the initial stage and increased sharply at the later stage. The dissolution of CO2 into the aqueous solution and then diffusion into cells controls the duration and inactivation rate at the early stage. An increase of pressure and temperature enhances the antimicrobial effect of CO2 under pressure. Inactivation is also affected by agitation and release of pressure. Ethanol is an effective and novel aid to intensify inactivation of microorganisms in CO2. E. coli cells after CO2 treatment are subjected to injury on membranes so as to lose salt tolerance and induce a leakage of intracellular materials. The changes of cell structure and inactivation of several enzymes are observed after treatment. But the most important inactivation mechanism may be the lowering of pH due to the penetration of CO2, which leads to the inhibition of certain metabolic and regulating processes. 1. Introduction Carbon dioxide plays an important physiological role in organisms. It can control the respiration of fresh fruits and vegetables and also conserve water in them. It has been shown that CO2 stimulates or inhibits microbial growth and reproduction since many years ago.1 Antimicrobial effect not only on the aerobic but also on the anaerobic under CO2 of elevated pressure at a normal temperature was found out by Kamihira et al. in 1987, which differs from the inhibition effect on aerobic bacteria at a normal atmosphere.2 Although hydrostatic pressurization beyond 100 MPa pressure has the ability to inactivate microbes, the antimicrobial effect with CO2 at elevated pressure is also quite effective even under 10 MPa. Sterilization of bioactive products is one of the most important operations to many foods and pharmaceuticals. Thermal processing is a conventional and efficient method for sterilization. However, it will destroy thermally unstable substances. As a consequence, several novel methods have been developed including irradiation, ultraviolet rays, microwaves, pulsed electric field, superheated steam, ultrafiltration, etc. These techniques have different sterilization effects with some disadvantages inevitably. Sterilization with CO2 at elevated pressure is a nonthermal process that would be suitable for the system including proteins and enzymes, as well as for heat-sensitive foods. No residual impurity or poisonous materials would be preserved after treatment of CO2. Therefore, it might be a good substitute for the thermal sterilization method when heat-labile materials are to be treated. Previous studies have been reported since nearly 20 years ago, but most of them were on inactivation of kinds of microbes and kinetic analysis. Lin et al.3,4 studied antimicrobial effect of CO2 on Saccaromyces cereVisiae and Leuconostoc dextranicum. They found that several factors influenced inactivation and declared extraction of lipids out of cells might play a good role in the antimicrobial process. Pressure inactivation kinetics of CO2 on kinds of microbes such as Listeria monocytogenes, Salmonella typhimurium, and E. coli were also investigated.5-10 * To whom correspondence should be addressed. Tel.: +86-57187951982. Fax: +86-571-87951015. E-mail: [email protected].

CO2 was almost unavailable to inactivation of bacterial spores at a normal temperature but effective at temperatures above 80 °C.11 The changes of enzymatic activity in E. coli were studied presuming that the death of E. coli might be related to the inactivation of key enzymes.12 Hong and Pyun13 investigated membrane damage and enzyme inactivation of Lactobacillus plantarum under elevated pressure CO2. Several hypotheses have been suggested, but few researchers could explain the mechanism for cell inactivation clearly so far. The objective of this paper is to investigate the inactivation of microorganisms comprehensively. In order to understand the mechanism of inactivation of cells, E. coli was used as a representative model and studied particularly. First we conducted the experiments on the rate of inactivation of microorganisms and influencing factors such as treatment time, pressure, temperature, operating conditions, and entrainer role. Then, studies of E. coli on salt tolerance, environmental pH, UV absorbing, leakage of proteins, cells structure, and constitution of enzymatic activities were done to explore the mechanism. A hypothesis of mechanism would be derived from the phenomena of inactivation and the changes of cells. In addition, several representative species of microorganisms were also selected to test and evaluate the antimicrobial effect of CO2. 2. Materials and Methods 2.1. Materials. 2.1.1. Microorganisms. Besides E. coli, other four strains were used in the experiment, i.e., Lactobacillus breVis, Saccaromyces cereVisiae, Bacillus subtilis, and Absidia coerulea. All of them were preserved on agar slant at 4 °C by the Institute of Bioengineering in Zhejiang University. 2.1.2. Experimental Equipment. The apparatus as shown in Figure 1 was designed by our lab. The cylindrical pressure vessel made of stainless steel with the internal volume of 43 mL was used for containing E. coli and pressured CO2. Temperature accuracy in the vessel could be controlled within (0.5 °C by a thermostat. Usually in the experiment, the vessel was filled with 25 mL of solution and a nozzle of CO2 was immerged into the solution. A small magnetic bead was placed in the vessel to facilitate mass transfer. The temperature of the CO2 was adjusted by heat exchanger before being introduced

10.1021/ie0702330 CCC: $37.00 © 2007 American Chemical Society Published on Web 08/03/2007

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Figure 1. Schematic diagram of experimental apparatus for CO2 treatment at the elevated pressure: 1, CO2 cylinder; 2, plunger pump; 3, heat exchanger; 4, pressure vessel; 5, thermostat; 6, magnetic stirrer; 7, manometer; 8, feed tank; 9, outlet; 10, valve.

into the vessel. The purity of CO2 supplied by the gas station of Zhejiang University was 99%. 2.2. Methods. 2.2.1. Culture of Microorganisms. E. coli and B. subtilis were grown in a medium containing 0.3% beef extract, 1% peptone, and 0.5% NaCl with the volume of 100 mL. After being incubated in 37 °C for 18-20 h, the cells were harvested by centrifugation at 8000 rpm for 10 min and then resuspended in 200 mL of sterile solution with 0.85% NaCl and 0.1% peptone (pH ) 7.5), respectively. The B. subtilis cells were at a state of anabiosis. L. breVis was grown in 100 mL of GYP medium with 1% glucose, 1% yeast extract, 0.5% peptone, and 0.2% sodium acetate (pH ) 7.0) at 30 °C for 48 h. Then L. breVis cells were centrifuged and resuspended in the solution alike. S. cereVisiae was incubated in 200 mL of medium with 2% glucose, 2% peptone, and 1% yeast extract (pH ) 6.5) at 30 °C for 28 h and then centrifuged and resuspended. A. coerulea was cultivated on agar slant with 30% potato juice and 2% glucose (pH ) 6.8) at 28 °C for 7 days to form conidia. Then the conidia were collected and suspended in 200 mL of sterile solution. 2.2.2. CO2 Treatment of Cells Suspension. CO2 was introduced into the pressure vessel to blow the air off, and then 25 mL of cells suspension was added in the pressure vessel. The thermostat was turned up to the set temperature (25, 35, or 45 °C), and simultaneously CO2 was also heated up. The cells were agitated by the magic stirrer at 180 rpm. When the temperature was equilibrated, CO2 was injected into the vessel to reach the desired pressure. After the cells were exposed to the pressure for the desired period time, the sample of the suspension was discharged directly from the outlet to a sterile tube. 2.2.3. Viable Cell Counts. Each sample was serially diluted with sterile water. The viable cell counts were measured by plating 0.1 mL of the sample on the agar medium and incubating the plates at an adaptive temperature. The number of viable cells was determined by counting colonies grown on each agar plate. The microbial survival rate was expressed by the ratio of the viable cell counts after CO2 treatment (N) to initial cell counts (N0), and we used log10 of the survival rate to account for the effect of inactivation. 2.2.4. Measurement of Cells Size. E. coli was used as 0.3 mL of each sample treated under CO2 of 7.8 MPa at 35 °C, and the untreated sample was suspended in 9 mL of phosphate buffer (pH ) 7.5); then the mean diameter of E. coli cells was determined with Zetasizer Nano Z-S (Malvern, U.K.).

Figure 2. Effect of pressure on inactivation of E. coli cells with highpressure CO2 treatment at 35 °C under agitation and initial pH 7.5 (9, 7.8 MPa; 1, 4.9 MPa; b, 2.0 MPa).

2.2.5. Observation of Cells Structure. The scanned electron microscopy (SEM) and transmission electron microscopy (TEM) were performed on E. coli treated under 7.8 MPa of CO2 at 35 °C for 60 min. The cells were placed on a filter paper and fixed with glutaraldehyde solution and osmic acid solution and washed by phosphate buffer. Then the samples were dried by ethanol, isoamyl acetate, and supercritical carbon dioxide and observed with SEM (XL30ESEM, Philips, Holand). Furthermore, the samples needed to be dried with acetone and then cut into thin slices. These sections were dyed and then observed with TEM (JEM-1230, JEOL, Japan). 2.2.6. UV-Absorbing Substances. E. coli cells suspended in 0.85% NaCl solution were treated under 7.8 MPa of CO2 pressure at 35 °C and then centrifuged at 8000 rpm for 10 min. Absorbance of the supernatant was measured at 260 and 280 nm, respectively, with a spectrophotometer (Ultrospec 3300 pro, GE Healthcare). 2.2.7. Amount of Leakage Proteins. The amount of protein in the supernatant was determined by the method of Coomassie brilliant blue R-250 dye.14 It is a precise measurement for the concentration of proteins above 1 µg. 2.2.8. Enzymatic Activity. The enzymatic activities of E. coli cells treated and untreated were determined by APIZYM system (Biomerieux, Marcy-l’Etoile, France). APIZYM is a semiquantitative method designed for the assay of enzymatic activities. It allowed the systematic and rapid determination of 19 parallel enzymatic reactions using small sample quantities with the amount ranging from 0 to 40 nM. The samples were centrifuged at 8000 rpm for 10 min and then resuspended in an appropriate volume for the APIZYM test. 3. Results and Discussion 3.1. Inactivation of E. coli Cells under CO2 at Elevated Pressure. 3.1.1. Effect of Pressure on Inactivation. The CO2 pressure ranging from 1 to 10 MPa was studied, which was also the spectrum in most high-pressure CO2 processing. In this experiment, 2, 4.9, and 7.8 MPa pressure were operated. The pressure used is much lower than sterilization of ultrahighpressure processing ranging from 100 to 300 MPa, which can unfold and inactivate proteins. The pressured E. coli cells were inactivated remarkably by carbon dioxide at the elevated pressure, as shown in Figure 2. The number of viable cells would get about a decrease of 8 log10 for 60 min under pressure of 7.8 MPa CO2 at 35 °C. The differences between inactivation rates of E. coli in aqueous solution under 7.8, 4.9, and 2.0 MPa

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Figure 3. Effect of temperature on inactivation of E. coli cells under 7.8 MPa of CO2 with agitation and initial pH 7.5 (9, 45 °C; 1, 35 °C; b, 25 °C).

at 35 °C were statistically significant. Inactivation was accelerated by increasing CO2 pressure ranging from 2.0 to 7.8 MPa. It took about 10 min to kill 90% of viable cells at 7.8 MPa, while it took about 20 min at 4.9 MPa and 35 min at 2.0 MPa, correspondingly. The higher the pressure, the shorter was the exposure time required to inactivate the same level of cells. Figure 2 also showed that there were two distinct phases in the process. The rate of inactivation was slower at the initial period and increased sharply in first-order kinetics at the later stage. The duration of the first stage and the inactivation rate of the second stage were sensitive to pressure variation. These phenomena were probably involved in the dissolving rate and solubility of CO2. Generally pressure controlled both dissolving rate and solubility of CO2. High pressure enhanced CO2 dissolution into the aqueous solution and then diffusion into cells through the cellular membrane and, thus, resulted in higher solubility. There were two phases with two distinct inactivation rates in the process, implying that the action of CO2 diffusing into aqueous solution and cells dominated the time and inactivation rate at the initial period, which was a limiting step. When the concentration of CO2 inside cells reached a critical level, it would exert a lethal effect on cells and cause a high inactivation rate of cells, which was also supposed by Lin et al.3,4 So, an increase of pressure intensified the antimicrobial effect of CO2 under pressure. 3.1.2. Effect of Temperature on Inactivation. Temperature effects diffusion and solubility of CO2 and also would affect the inactivation rate of E. coli. Survival rates of E. coli at the temperatures of 25, 35, and 45 °C under 7.8 MPa were compared and shown in Figure 3. An increase of temperature shortened the duration of the first stage and led to an effective inactivation of cells. It took 5 min at 35 °C and ∼10 min at 25 °C accordingly. An increase of temperature stimulated the mass diffusion in aqueous solution and cells but decreased the solubility.15 It was stated that higher temperature increased the fluidity of the cellular membrane16 to make penetration of CO2 easily. However, taking into account the three factors altogether, higher temperature apparently increases the inactivation rate of E. coli as a whole. 3.1.3. Effect of Operating Conditions on Inactivation. Agitation was one of the important factors for the inactivation effect of CO2. Without agitation, the rates of inactivation decreased remarkably to about half of the rates with agitation at 180 rpm (Figure 4). In a way, the diffusion rate of CO2 was the key factor dominating the antimicrobial effect of CO2 at

Figure 4. Effect of agitation and releasing pressure on inactivation of E. coli cells under 7.8 MPa of CO2 pressure at 35 °C (9, with agitation and flash release of pressure; 1, with agitation and slow release of pressure; b, without agitation and flash release of pressure).

the elevated pressure because establishing the mass transfer equilibrium between CO2 and aqueous solution needed a longer time,17 and agitation facilitated dissolution of CO2 into the aqueous solution and then diffusion into cells. Figure 4 also presented the rate of inactivation of E. coli with flash or slow release of pressure. In comparison with the rate of inactivation with flash release of pressure, the rate of inactivation with slow release of pressure displayed about 0.5 logs of decrease. These results may be attributed to the fierce expansion of cells and extraction of lipids or other substances from cells or cell membrane with the great unbalanced pressure between the inside and the outside of cells.3 But this explanation would not be the intrinsic inactivation mechanism because the differences of inactivation between the two modes of pressure release were not distinct enough. Another supported example was the obvious antimicrobial effect of gaseous and liquid CO2 in Figures 2 and 3, while the powerful extraction of lipid substances would usually happen only in supercritical regions. 3.1.4. Effect of Ethanol as an Entrainer on Inactivation. An entrainer has been used to improve the properties of CO2 for a very long time. Ethanol was selected because it is almost harmless to people if it remains in a small quantity in food. Ethanol has the ability to increase the solubility of CO2 and alter the conformation of proteins to some extent.18 Ethanol can also inactivate cells and is lethal to cells, but the effect of inactivation lies on the content of the ethanol. The inactivation of E. coli is quite limited, and >10% of viable cells remain when only 10% or 20% of ethanol was added, respectively, even for 60 min, as indicated in Figure 5. That means low content of ethanol itself is not appropriate for sterilization. Inactivation of CO2 with a low content of ethanol has also been studied by Kamihira et al., and the improvement of antimicrobial effect was quite limited.2 However, in Figure 6, the antimicrobial effect of CO2 was strengthened inconceivably by adding ethanol at a weight ratio of 10% and 5% only for a short period, which greatly differed from the results reported by Kamihira et al. The effect of inactivation with 10% ethanol under 4.9 MPa of CO2 for 5 min exceeded the effect without ethanol under 7.8 MPa for 60 min, and it worked even with 2% ethanol addition for 5 min. Kamihira et al. attributed the improvement to the modification of CO2 properties by ethanol, whereas the increase of CO2 solubility induced by 10% ethanol was only about 15%,19 and it was impossible to enact the effect of inactivation so greatly and quickly. The influences of ethanol

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Figure 5. Inactivation of E. coli with 10% and 20% ethanol (w/w) at 35 °C for 15 and 60 min (diagonal-lined bar, 10% ethanol; wavy-lined bar, 20% ethanol). Figure 7. Inactivation of E. coli cells under 7.8 MPa of N2 with agitation and initial pH 3.3 and CO2 at 35 °C with agitation and initial pH 7.5 (9, treatment with CO2; 1, treatment with N2). Table 2. Effect of Ethanol as an Entrainer on Inactivation of Microorganisms under CO2 at Elevated Pressure at 35 °C for 5 min inactivation rate (log10 of survival ratio)

Figure 6. Effect of ethanol as the entrainer on inactivation of E. coli under CO2 at elevated pressure at 35 °C for 5 min (1, no EtOH; b, 2% EtOH; 2, 5% EtOH; 9, 10% EtOH). Table 1. Inactivation of Microorganisms under 7.8 MPa of CO2 Pressure at 35 °C for 30 and 60 min inactivation rate (log10 of survival ratio) microorganism

treated for 30 min

treated for 60 min

E. coli L. breVis S. cereVisiae B. subtilis A. coerulea

-4.3 -3.7 -4.6 -0.3 -2.1

-7.8 -7.3 -8.8 -0.7 -5.2

to microbial cells act as denaturalizing and precipitating protein in cells, inhibiting certain enzymes such as dehydrogenase and oxidase and destroying some substances on cell walls and membranes20 to help CO2 enter into the cells more quickly. It was the synergistic effects that CO2 and ethanol exhibited. Alternatively, adding ethanol made the sterilization of pressured CO2 quicker and more efficient. 3.1.5. Inactivation of Other Microorganisms under Elevated Pressured CO2. The antimicrobial effects of CO2 to several species of microorganisms were studied and summarized in Table 1. Both bacteria, i.e., E. coli (Gram-) and L. breVis (Gram+), without spores are sensitive to elevated pressured CO2 at ambient temperature, and the decease of the number of viable cells is more than 10-7 after treatment for 1 h. The inactivation of S. cereVisiae and A. coerulea (conidia) is also efficient in the presence of elevated pressured CO2. However, elevated pressured CO2 remains nearly inefficient to B. subtilis. The greater amount of polymer in the cell wall (Gram+) is not the main reason why low efficacy happened when B. subtilis was

microorganism

adding 5% ethanol

adding 10% ethanol

E. coli L. breVis S. cereVisiae B. subtilis A. coerulea

-8.4 -7.5 -7.4 -0.3 -4.4

no growth no growth no growth -0.6 -8.2

treated, because L. breVis (also Gram+) can be inactivated efficiently. What’s more, the structure and components of the cell walls do not have the ability to prevent CO2 from permeating into the cells. It is presumable that the cells transformed to spores under the hard condition of elevated CO2 pressure and spores have the capacity to resist the sterile effect of CO2, like its capacity to resist the sterile effect of thermal processing. It is concluded that elevated pressured CO2 is a good method to inactivate microorganisms at an ambient temperature, with the exception of bacteria with spores. Ethanol is also a good aid to inactivate species of microorganisms besides E. coli as shown in Table 2. All of the microorganisms studied except for B. subtilis have a great improvement of inactivation rate in the presence of ethanol with the content of 5% and 10% under elevated pressured CO2. These results illustrate that it is efficient to comprehensively apply ethanol to sterilization under elevated pressured CO2. 3.2. Remarks on Inactivation Mechanism. 3.2.1. Mechanical Effect of Pressure and Influence of pH in Environment. Mechanical impact of high pressure may be lethal to cells. So nitrogen was used in place of CO2 to study the effect of pressure solely. The experiment was performed at the same pressure and temperature and also with initial pH 3.3, which was nearly the pH under 7.8 MPa of CO2.18 More than 80% of cells were found to have survived after treatment with N2 for 60 min, which was extraordinarily different from the power of elevated pressured CO2 (see Figure 7). That clearly indicated that the mechanical impact of pressure was not the cause of inactivation of E. coli under elevated pressured CO2. The visible reason why N2 did not work may be attributed to the poor solubility in aqueous solution and its lack of ability to be close to the cells. Aqueous solution of sodium bicarbonate is an alkaline buffer under CO2 at elevated pressure, and it would react with carbonic acid formed from the dissolved CO2. Thus, the pH in aqueous solution of NaHCO3 was not lowered under CO2 treatment,

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Figure 8. Inactivation of E. coli cells in aqueous solution with NaHCO3 and without NaHCO3 with initial pH 7.5 under 7.8 MPa of CO2 pressure at 35 °C with agitation (9, with 0.25 M NaHCO3; 1, without NaHCO3).

Figure 9. Mean diameter changes of E. coli treated under 7.8 MPa of CO2 pressure at 35 °C.

which remained above 7. The viable cells were suspended in 0.25 M aqueous solution of NaHCO3and treated under elevated pressured CO2 to compare the process without NaHCO3 (Figure 8). These results showed that the environmental pH of the cells suspension during the treatment was not directly related with the inactivation of cells. Viable cells were capable of resisting low environmental pH. Furthermore, the inactivation in aqueous solution of NaHCO3 was a little more effective than that without NaHCO3. The gaseous CO2 ejected into solution was constituted by CO2, H2CO3, HCO3-, and CO32-. When CO2 entered the aqueous solution, it would dissociate into HCO-3 and CO32first and enter into cells later. Hence, CO2 would penetrate cells more quickly in aqueous solution of NaHCO3 where HCO3and CO32- already existed and did not need to further dissociate. These results substantiated the conclusion aforementioned that the diffusion rate of CO2 into cells was the key factor dominating the antimicrobial effect of CO2 at the elevated pressure. 3.2.2. Integrity of Treated Cells. The mean diameters of E. coli cells treated under CO2 of 7.8 MPa at 35 °C were determined, and the results were shown in Figure 9. The mean diameters almost did not change with the increase of treatment time. It could be inferred that cells treated under CO2 did not break down and the integrity of the whole cells was maintained. 3.2.3. Injury of Cell Membrane. In this procedure, two kinds of agar media were prepared. One was a complete culture medium containing 0.3% beef extract, 1% peptone, and 0.5%

Figure 10. Survival of E. coli exposed to 7.8 MPa CO2 pressure treatment at 35 °C on agar of complete medium and agar of selective medium (1, agar medium with 0.5% NaCl; 9, agar medium with 4% NaCl).

Figure 11. UV absorbance of E. coli leakage into extracellular suspension under 7.8 MPa pressure CO2 at 35 °C (9, 260 nm; 1, 280 nm).

NaCl, and the other was a selective culture medium containing 0.3% beef extract, 1% peptone, and 4% NaCl. The selective medium is only used to count the number of cells with intact membranes, because cells with injured membranes lose salt tolerance and are unable to form colonies on selective medium containing a high level of sodium chloride. As shown in Figure 10, the number of survivors treated with CO2 of 7.8 MPa at 35 °C was decreased 1 or 2 logs more on the selective medium than that on the complete medium. It also suggested that >90% of the survivors on complete medium were subjected to injury on the cell membrane. With cellular injury of membrane, some cell components such as nucleic acid, proteins, amino acids, and lipids may leak out and be found in the extracellular solution. UV-absorbing measurement is an easy and accessible method to determine the increase of leakage of intracellular materials because all of these substances have a UV-absorbance peak. Absorbance at 260 and 280 nm increased rapidly in the earlier 20 min and then gradually leveled off, respectively (Figure 11). By comparing with the phenomena that >99% of viable cells were inactivated during the earlier 20 min under 7.8 MPa of CO2 pressure at 35 °C, it can be seen that the inactivation of E. coli may be due to the injury of the cell membrane. These results were consistent with the viewpoint of membrane injury. In order to determine the extent of membrane injury in the inactivation of cells, the amounts of proteins leaking out of the

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Figure 12. Amounts of proteins leaking out of cells under 7.8 MPa of CO2 pressure at 35 °C.

cells were measured. The treatment time was prolonged to 16 h, and the results were shown in Figure 12. The level of protein leaking out was very low for the first 1 h, which implied that the cytoplasm did not leak out in a great deal during that period and the injury to the membrane was not serious. The protein concentration increased greatly after 2 h and reached to 0.035 mg/mL for the treatment of 16 h, which could be the outcome of membrane damage such as disruption and rupture. It was reported in the same way that severe membrane damage of yeast cells required a much longer duration under elevated pressured

CO2.21 So the injury of cell membrane would not be the primary cause of cell death. 3.2.4. Variations in Cell Structure. The SEM observation of E. coli cells treated under 7.8 MPa of CO2 at 35 °C was shown in Figure 13. Intact cells remained and no distinct cracks were observed. It also implied that the cell wall was intact and did not deform; otherwise, cells would not keep their original shape without the intact cell wall. The TEM observation showed great differences between E. coli cells untreated and treated (Figure 14). It was distinctly seen that there was a higher density of cytoplasm in the cells untreated, while the density was reduced in the cells treated for 1 h. After treatment, the intracellular substances assembled and vacant space existed between the wall and cytoplasm. So the leakage of substances and the injury of membrane should not be ignored. 3.2.5. Influence on Enzymatic Activity. Some enzymatic activities of E. coli untreated and treated with 7.8 MPa of CO2 pressure at 35 °C were assessed by the use of the APIZYM system. This system is a semiquantitative method designed for the quick determination of enzymatic activity in a complex sample. Seven kinds of constitutive enzymes were identified in the untreated cells (Table 3). Esterase, leucine arylamidase, β-galactosidase, and β-glucuronidase had already lost their entire activities with CO2 treatment for 10 min, and acid phosphatase and naphthol-AS-BI-phosphohydrolase had a significant decrease of activities, too. The observed selective inactivation of enzymatic activities for that short time’s treatment would result

Figure 13. SEM of E. coli cells untreated (A) and treated (B) with 7.8 MPa of CO2 pressure at 35 °C for 1 h.

Figure 14. TEM of E. coli cells untreated (A) and treated (B) with 7.8 MPa of CO2 pressure at 35 °C for 1 h.

Ind. Eng. Chem. Res., Vol. 46, No. 19, 2007 6351 Table 3. Variations of Enzymatic Activity of E. coli Treated with 7.8 MPa of CO2 Pressure at 35 °C activity mark enzyme

untreated

treated for 10 min

treated for 60 min

alkaline phosphatase esterase (C4) leucine arylamidase acid phosphatase naphthol-AS-BI-phosphohydrol ase β-galactosidase β-glucuronidase

5 1 5 5 5 2 4

5 0 0 3 2 0 0

5 0 0 2 1 0 0

from the drop of pH of the microorganisms during the treatment. CO2 was able to penetrate into cells and lead to a notable acidification in cells, which would precipitate the enzymes with an acidic isoelectric point like β-galactosidase. Besides, hydrolyzation and inactivation of some kinds of enzymes at elevated pressure of CO2 had also been reported.18,22,23 It was possible that certain metabolic and regulating processes were inhibited by inactivation of key enzymes. The hypothesis of protein precipitation at low pH accorded with the great synergistic effect of CO2 and ethanol because ethanol acted as a good precipitating aid to the isoelectric precipitation of proteins under pressure.18 Another possible reaction may be related to the effect of carbonate, which could precipitate intracellular ions such as calcium and magnesium in cells and cell membranes. Certain types of calcium- and magnesium-sensitive proteins would be accessible to precipitation by carbonic acid,4 which may be parts of the essential proteins involving intracellular regulation. However, it was uncertain whether the inactivation of some enzymes or proteins was the primary cause of cell death. 3.2.6. Inactivation Mechanism. Carbon dioxide dissolves in aqueous solution with a good solubility under elevated pressure. It is assumed that a large amount of CO2 molecules diffuse into cells through the membrane and injure the membrane structure in a way so that a leakage of some intracellular components takes place. What’s more, carbonic acid is formed with CO2 diffusion, and then pH in cells is lowered simultaneously. It is well-known that microbes can live in an extracellular environment of low pH but the intracellular pH tends to be alkaline, and the intracellular substances such as DNA, ATP, and some kinds of enzymes are susceptible to lower pH. Thus, when the amount of carbonic acid exceeds the buffer capacity of cytoplasm in cells, the metabolic and regulating processes may be inhibited owing to the too-low level of pH in the intracellular environment. H+ mediated hydrolysis may also occur. This idea is supported by the example that the inactivation of dry cells is much less inefficient2,7 because of the small amount of carbonic acid formed in the dry state. The injury of the membrane would also facilitate the entering of CO2 and acid into the environment of low pH and result in a dynamic equilibrium. Determination of intracellular pH can be realized by fluorescent probe24 or weak acid probe,25 but these two methods are both difficult to operate on-line. 4. Conclusions It is clear that microorganisms without spores are inactivated remarkably by carbon dioxide at the elevated pressure. The process can be divided into two phases based on the different rates of inactivation: the rate of inactivation is slower at the initial period and increases sharply at the later stage. The dissolving of CO2 into the aqueous solution and then diffusion into cells controls the time and inactivation rate at the early

stage. An increase of pressure and temperature enhances the antimicrobial effect of CO2 under pressure. Inactivation is also affected by agitation and release of pressure. Ethanol is an effective aid of CO2 to inactivate species of microorganisms. With these processes, it is convincing that CO2 of elevated pressure would be a good method for sterilization at normal temperature. E. coli cells as a representative model after CO2 treatment are subjected to injury on the membrane so as to lose salt tolerance and get a leakage of intracellular materials. The inactivation of several constitutive enzymes plays an important role in the death of cells. But the most important cause may be the lowering of pH due to the penetration of CO2, which leads to the inhibition of certain metabolic and regulating processes. Acknowledgment The authors would like to thank National Natural Science Foundation of China for the financial support (20676118). Literature Cited (1) Molin, G.; Ternstron, A. Effect of Packaging under Carbon Dioxide, Nitrogen or Air on the Microbial Flora of Stored at 4 °C. J. Appl. Bacteriol. 1979, 47, 197-208. (2) Kamihira, M.; Taniguchi, M.; Kobayashi, T. Sterilization of Microorganisms with Supercritical Carbon Dioxide. Agric. Biol. Chem. 1987, 51, 407-412. (3) Lin, H. M.; Yang, Z. Y.; Chen, L. F. Inactivation of Saccharomyces cereVisiae by Supercritical and Subcritical Carbon Dioxide. Biotechnol. Prog. 1992, 8, 458-461. (4) Lin, H. M.; Yang, Z. Y.; Chen, L. F. Inactivation of Leuconostoc dextranicum with Carbon Dioxide under Pressure. Chem. Eng. J. 1993, 52, B29-B34. (5) Hong, S. I.; Park, W. S.; Pyun Y. R. Inactivation of Lactobacillus sp. from Kimchi by High Pressure Carbon Dioxide. Lebensm.-Wiss. Technol. 1997, 30, 681-685. (6) Debs-Louka, E.; Louka, N.; Abraham, G.; Chabot, V.; Allaf, K. Effect of Compressed Carbon Dioxide on Microbial Cell Viability. Appl. EnViron. Microbiol. 1999, 65, 626-631. (7) Angela, D.; Fariba, D.; Jeffrey, H.; Neil, F.; Robert, L. Bacterial Inactivation by Using Near- and Supercritical Carbon Dioxide. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10344-10348. (8) Erkmen, O.; Karaman, H. Kinetic Studies on the High Pressure Carbon Dioxide Inactivation of Salmonella typhimurium. J. Food Eng. 2001, 50, 25-28. (9) Erkmen, O. Kinetic Analysis of Listeria monocytogenes Inactivation by High Pressure Carbon Dioxide. J. Food Eng. 2001, 47, 7-10. (10) Karaman, H.; Erkmen, O. High Carbon Dioxide Pressure Inactivation Kinetics of Escherichia coli Broth. Food Microbiol. 2001, 18, 11-16. (11) Ballestra, P.; Cuq, J. L. Influence of Pressurized Carbon Dioxide on the Thermal Inactivation of Bacterial and Fungal Spores. Lebensm.Wiss. Technol. 1998, 31, 84-88. (12) Ballestra, P.; Abreu da Silva, A.; Cuq, J. L. Inactivation of Escherichia coli by Carbon Dioxide under Pressure. J. Food Sci. 1996, 61, 829-831. (13) Hong, S. I.; Pyun, Y. R. Membrane Damage and Enzyme Inactivation of Lactobacillus plantarum by High Pressure Treatment. Int. J. Food Microbiol. 2001, 63, 19-28. (14) Yu, B. B. Experiment Instruction on Biochemistry; Tsinghua University Press: Beijing, China, 2004. (15) Vorholz, J.; Harismiadis, V. I.; Panagiotopoulos, A. Z.; Maurera, B. R.; Molecular Simulation of the Solubility of Carbon Dioxide in Aqueous Solutions of Sodium Chloride. Fluid Phase Equilib. 2004, 226, 237-250. (16) Shen, P. Microbiology; Higher Education Press: Beijing, China, 2000. (17) Zhu, Z. Q. Supercritical Fluid Technology: Principles and Application; Chemical Industry Press: Beijing, China, 2000. (18) Qi, X. M.; Yao, S. J.; Guan, Y. X. Novel Isoelectric Precipitation of Proteins in Pressurized Carbon Dioxide-Water-Ethanol System. Biotechnol. Prog. 2004, 20, 1176-1182. (19) Yao, S. J.; Guan, Y. X.; Zhu, Z. Q. Investigation of Phase Equilibrium for Ternary Systems Containing Ethanol, Water and Carbon Dioxide at Elevated Pressures. Fluid Phase Equilib. 1994, 99, 249-259.

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(20) Li, B S.; Ruan, Z. Non-Thermal Sterilizing Technology and Application; Chemical Industry Press: Beijing, China, 2004. (21) Lin, H. M.; Chan, E. C.; Chen, C.; Chen, L. F. Disintegration of Yeast Cells by Pressurized Carbon Dioxide. Biotechnol. Prog. 1991, 7, 201204. (22) Qi, X. M.; Yao, S. J.; Guan, Y. X.; Zhu, Z. Q. Study on Stability of Alkaline Proteinase in the Carbon Dioxide-Water-Ethanol System at Elevated Pressure. J. Chem. Eng. Chin. UniV. 2003, 17, 515-520. (23) Yao, S. J.; Guan, Y. X.; He, W. Z.; Qi, X. M.; Zhu, Z. Q. Activities of Several Enzymes in Ethanol + Water at Elevated Pressure of Carbon Dioxide. J. Chem. Eng. Data 2004, 49, 1333-1339.

(24) Slavlk, J. Intracellular pH of Yeast Cells Measured with Fluorescent Probes. FEBS Lett. 1982, 140, 22-26. (25) Henriques, M.; Quinas, C.; Loureiro-Dias, M. C. Extrusion of Benzoic Acid in Saccharomyces cereVisiae by an Energy-Dependent Mechanism. Microbiology 1997, 143, 1877-1883.

ReceiVed for reView February 10, 2007 ReVised manuscript receiVed June 30, 2007 Accepted July 9, 2007 IE0702330