- Email: [email protected]
Inactivation of Alternaria brassicicola spores by supercritical carbon dioxide with ethanol entrainer
Journal of Microbiological Methods 88 (2012) 185–187
Contents lists available at SciVerse ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Inactivation of Alternaria brassicicola spores by supercritical carbon dioxide with ethanol entrainer Hyong Seok Park a, Hee Jung Choi b, Kyoung Heon Kim a,⁎ a b
School of Life Sciences & Biotechnology, Korea University, Seoul 136-713, Republic of Korea Department of Internal Medicine, Division of Infectious Diseases, Ewha Womans University School of Medicine, Seoul 158-710, Republic of Korea
a r t i c l e
i n f o
Article history: Received 26 June 2011 Received in revised form 21 October 2011 Accepted 1 November 2011 Available online 7 November 2011
a b s t r a c t Supercritical carbon dioxide (SC–CO2) was used to inactivate fungal spores of Alternaria brassicicola. The inactivation conditions were optimized using response surface methodology (RSM). When the SC–CO2–entrainer (ethanol) system was applied to fungal spores, the treatment time required for the complete inactivation of fungal spores was substantially reduced. © 2011 Elsevier B.V. All rights reserved.
Keywords: Supercritical carbon dioxide Inactivation Fungal spore Entrainer
Alternaria species are pathogenic fungi known to cause dark leaf spot in crucifers (Chen et al., 2005). These are also known to cause many human diseases including bronchial asthma (Bush and Prochnau, 2004) and allergic rhinitis (Stark et al., 2005). Supercritical carbon dioxide has a relatively low critical point (73.8 bar and 31.1 °C). SC–CO2 was found to cause both chemical and physical damage to microorganisms (Kim et al., 2008). However, only a few studies have been conducted to investigate the ability of SC–CO2 to inactivate spores of only one fungal species, Aspergillus niger (Kamihira et al., 1987; Shimoda et al., 2002). In this study, the inactivation of plant pathogenic fungal spores of Alternaria brassicicola using SC–CO2 was optimized by response surface methodology (RSM), and a SC–CO2–entrainer system, which was SC–CO2 modified with ethanol before delivery, was exploited for the enhancement of inactivating fungal spores. For SC–CO2 inactivation, the initial spore concentration of Alternaria brassicicola (KACC 40036) was set at approximately 1 × 10 7 colonyforming units (CFU)/mL in suspension. Following the previously described SC–CO2 treatment procedures (Kim et al., 2007), a loosely capped sterile tube containing 2 mL of spore suspension was loaded into the treatment reactor with an internal volume of 100 mL. Liquid CO2 (purity of 99.5%; Daehan Specialty Gases, Seoul, Korea) was then pumped into the treatment reactor. To investigate the effect of the ethanol entrainer, 16 g of ethanol (purity > 99.8%, Sigma, St. Louis, MO, USA) was pumped into the treatment reactor as an entrainer using a metering pump. The reduction of CFU/mL in the SC–CO2
⁎ Corresponding author. Tel.: + 82 2 3290 3028; fax: + 82 2 925 1970. E-mail address: [email protected] (K.H. Kim). 0167-7012/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2011.11.005
treated spores was then expressed by the ratio of the log10 CFU/mL of untreated spores to that of SC–CO2 treated spores. To optimize the inactivating conditions of spores of A. brassicicola by SC–CO2 treatment, experiments were designed using the Box-Behenken method (Myers and Montgomery, 1995). The log10 reduction in the concentration of a spore suspension was the dependent variable, and pressure (150, 200, and 250 bar), temperature (38, 42, and 46 °C), and treatment time (5, 15, and 25 min) were the independent variables. As the levels of all three independent variables increased, the log10 reduction in the concentration of spores increased. When spores were treated with SC–CO2 at the high level temperature (46 °C), complete inactivation (that is a log10 reduction of 7) was obtained at the medium treatment time and the low treatment pressure (15 min and 150 bar, respectively). When temperature was reduced to the low level (38 °C), the complete inactivation of spores did not occur, regardless of the treatment time and pressure. The analysis of variance (ANOVA) of the model equation of RSM revealed high significances (p b 0.01) for all regression parameters (linear, quadratic, cross product, and total model), and their regression coefficient (R 2) was 0.97. The predicted optimal conditions, which were found by the ridge analysis, were 196 bar, 46 °C, and 17 min, with a predicted log10 reduction of 6.37. As shown in Fig. 1a, the contour plot generated at a fixed temperature of 46 °C indicated that the log10 reduction in fungal spore concentration increased as the treatment time or pressure increased. The density of SC–CO2 is directly related to the dissolving power, and this value increases as pressure increases at a constant temperature (Lucien and Foster, 1999). The high dissolving power of SC–CO2 owing to the increased density of CO2 promotes the inactivation of
186
H.S. Park et al. / Journal of Microbiological Methods 88 (2012) 185–187
1999). However, in this study, other favorable effects caused by the higher temperature, such as a higher chemical reaction rate, increased diffusivity of CO2 (Kim et al., 2008), and increased fluidity of the cell membrane itself (Erkmen, 2001), may have increased the effectiveness with which SC–CO2 inactivated fungal spores. As shown in Fig. 1c, at a fixed treatment time of 25 min, the log10 reduction in the concentration of viable spores increased as the temperature and pressure increased. As shown in Fig. 2, SC–CO2 treatment at the low levels of pressure and temperature (150 bar and 38 °C, respectively), which produced the poorest reduction in spore concentration when SC–CO2 alone was used, were selected to investigate the effect of ethanol entrainer. In the presence of ethanol entrainer, the 7 log reduction was obtained after 45 min in this study. However, without ethanol entrainer, much longer treatment time of 90 min was required for the 7 log reduction to occur at the same pressure and temperature. Although membrane integrity in bacterial spores is known to be disrupted by ethanol itself (Setlow et al., 2002), the change in polarity and other physical and chemical properties of SC–CO2 induced by ethanol entrainer may have contributed to the enhanced lethality of SC–CO2 (Lucien and Foster, 1999). Meanwhile, at 150 bar and 38 °C, the mole fractions of the ternary system of CO2, ethanol, and water at equilibrium were 0.9792, 0.0162, and 0.0046, respectively (Yao et al., 1994). Therefore, the actual amount of ethanol dissolved in the SC–CO2 phase in the present study was estimated to be only 42 mg in 100 mL of the internal volume of the treatment vessel. Also, it was assumed that there was no direct contact between the remaining liquid ethanol and spores since the suspensioncontaining tube was capped to minimize the direct entrainment of liquid ethanol into the tube. In conclusion, the SC–CO2–ethanol entrainer system reduced the treatment time for the complete inactivation of fungal spores by half in comparison with using SC–CO2 without ethanol entrainer. The analysis of the mechanism of fungal spore inactivation and also the application of the present method to real systems such as seeds contaminated with fungi may be needed as future work.
Acknowledgments This work was supported by the Advanced Biomass R&D Center of Korea Grant (2010-0029734) funded by the Ministry of Education, Science and Technology, Korea. The facility support provided by the Institute of Biomedical Science and Food Safety, Korea University, is also acknowledged.
Fig. 1. Response surface plot of SC–CO2 inactivation of A. brassicicola spores. (a) Effect of pressure and treatment time with a fixed temperature of 46 °C. (b) Effect of temperature and treatment time at a fixed pressure of 250 bar. (c) Effect of pressure and temperature at a fixed treatment time of 25 min.
Log10 reduction of CFU/mL
8 Ethanol No ethanol
6
4
2
0
microorganisms through extraction or denaturing of intracellular materials (Kim et al., 2007, 2008). At a constant pressure of 250 bar, the log10 reduction of spores increased when treatment temperature and time increased (Fig. 1b). As the temperature increases at a fixed pressure, the decreased SC–CO2 density can result in the reduced dissolving power of SC–CO2 (Lucien and Foster,
0
15
30
45
60
90
120
150
Treatment Time (min) Fig. 2. Effect of ethanol as the entrainer of SC–CO2 on the inactivation of A. brassicicola spores at 150 bar and 38 °C. A total of 16 g of ethanol (> 99.8% purity) was pumped into a treatment reactor with a 100 mL internal volume.
H.S. Park et al. / Journal of Microbiological Methods 88 (2012) 185–187
References Bush, R.K., Prochnau, J.J., 2004. Alternaria-induced asthma. J. Allergy Clin. Immunol. 113, 227–234. Chen, L.Y., Price, T.V., Silvapulle, M.J., 2005. Dark leaf spot (Alternaria brassicicola) on Chinese cabbage: spatial patterns. Aust. J. Agric. Res. 56, 699–714. Erkmen, O., 2001. Effects of high-pressure carbon dioxide on Escherichia coli in nutrient broth and milk. Int. J. Food Microbiol. 65, 131–135. Kamihira, M., Taniguchi, M., Kobayashi, T., 1987. Sterilization of microorganisms with supercritical carbon dioxide. Agric. Biol. Chem. 51, 407–412. Kim, S.R., Rhee, M.S., Kim, B.C., Lee, H., Kim, K.H., 2007. Modeling of the inactivation of Salmonella typhimurium by supercritical carbon dioxide in physiological saline and phosphate-buffered saline. J. Microbiol. Methods 70, 132–141. Kim, S.R., Park, H.J., Yim, D.S., Kim, H.T., Choi, I.-G., Kim, K.H., 2008. Analysis of survival rates and cellular fatty acid profiles of Listeria monocytogenes treated with supercritical carbon dioxide under the influence of cosolvents. J. Microbiol. Methods 75, 47–54.
187
Lucien, F.P., Foster, N.R., 1999. Phase behavior and solubility. In: Jessop, P.G., Leitner, W. (Eds.), Chemical Synthesis Using Supercritical Fluids. Wiley-VCH, Weinheim, pp. 37–53. Myers, R.H., Montgomery, D.C., 1995. Response Surface Methodology. John Wiley & Sons Inc., New York. Setlow, B., Loshon, C.A., Genest, P.C., Cowan, A.E., Setlow, C., Setlow, P., 2002. Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol. J. Appl. Microbiol. 92, 362–375. Shimoda, M., Kago, H., Kojima, N., Miyake, M., Osajima, Y., Hayakawa, I., 2002. Accelerated death kinetics of Aspergillus niger spores under high-pressure carbonation. Appl. Environ. Microbiol. 68, 4162–4167. Stark, P.C., Celedón, J.C., Chew, G.L., Ryan, L.M., Burge, H.A., Muilenberg, M.L., Gold, D.R., 2005. Fungal levels in the home and allergic rhinitis by 5 years of age. Environ. Health Perspect. 113, 1405–1409. Yao, S., Guan, Y., Zhu, Z., 1994. Investigation of phase equilibrium for ternary systems containing ethanol, water and carbon dioxide at elevated pressures. Fluid Phase Equilib. 99, 249–259.
Contents lists available at SciVerse ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Inactivation of Alternaria brassicicola spores by supercritical carbon dioxide with ethanol entrainer Hyong Seok Park a, Hee Jung Choi b, Kyoung Heon Kim a,⁎ a b
School of Life Sciences & Biotechnology, Korea University, Seoul 136-713, Republic of Korea Department of Internal Medicine, Division of Infectious Diseases, Ewha Womans University School of Medicine, Seoul 158-710, Republic of Korea
a r t i c l e
i n f o
Article history: Received 26 June 2011 Received in revised form 21 October 2011 Accepted 1 November 2011 Available online 7 November 2011
a b s t r a c t Supercritical carbon dioxide (SC–CO2) was used to inactivate fungal spores of Alternaria brassicicola. The inactivation conditions were optimized using response surface methodology (RSM). When the SC–CO2–entrainer (ethanol) system was applied to fungal spores, the treatment time required for the complete inactivation of fungal spores was substantially reduced. © 2011 Elsevier B.V. All rights reserved.
Keywords: Supercritical carbon dioxide Inactivation Fungal spore Entrainer
Alternaria species are pathogenic fungi known to cause dark leaf spot in crucifers (Chen et al., 2005). These are also known to cause many human diseases including bronchial asthma (Bush and Prochnau, 2004) and allergic rhinitis (Stark et al., 2005). Supercritical carbon dioxide has a relatively low critical point (73.8 bar and 31.1 °C). SC–CO2 was found to cause both chemical and physical damage to microorganisms (Kim et al., 2008). However, only a few studies have been conducted to investigate the ability of SC–CO2 to inactivate spores of only one fungal species, Aspergillus niger (Kamihira et al., 1987; Shimoda et al., 2002). In this study, the inactivation of plant pathogenic fungal spores of Alternaria brassicicola using SC–CO2 was optimized by response surface methodology (RSM), and a SC–CO2–entrainer system, which was SC–CO2 modified with ethanol before delivery, was exploited for the enhancement of inactivating fungal spores. For SC–CO2 inactivation, the initial spore concentration of Alternaria brassicicola (KACC 40036) was set at approximately 1 × 10 7 colonyforming units (CFU)/mL in suspension. Following the previously described SC–CO2 treatment procedures (Kim et al., 2007), a loosely capped sterile tube containing 2 mL of spore suspension was loaded into the treatment reactor with an internal volume of 100 mL. Liquid CO2 (purity of 99.5%; Daehan Specialty Gases, Seoul, Korea) was then pumped into the treatment reactor. To investigate the effect of the ethanol entrainer, 16 g of ethanol (purity > 99.8%, Sigma, St. Louis, MO, USA) was pumped into the treatment reactor as an entrainer using a metering pump. The reduction of CFU/mL in the SC–CO2
⁎ Corresponding author. Tel.: + 82 2 3290 3028; fax: + 82 2 925 1970. E-mail address: [email protected] (K.H. Kim). 0167-7012/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2011.11.005
treated spores was then expressed by the ratio of the log10 CFU/mL of untreated spores to that of SC–CO2 treated spores. To optimize the inactivating conditions of spores of A. brassicicola by SC–CO2 treatment, experiments were designed using the Box-Behenken method (Myers and Montgomery, 1995). The log10 reduction in the concentration of a spore suspension was the dependent variable, and pressure (150, 200, and 250 bar), temperature (38, 42, and 46 °C), and treatment time (5, 15, and 25 min) were the independent variables. As the levels of all three independent variables increased, the log10 reduction in the concentration of spores increased. When spores were treated with SC–CO2 at the high level temperature (46 °C), complete inactivation (that is a log10 reduction of 7) was obtained at the medium treatment time and the low treatment pressure (15 min and 150 bar, respectively). When temperature was reduced to the low level (38 °C), the complete inactivation of spores did not occur, regardless of the treatment time and pressure. The analysis of variance (ANOVA) of the model equation of RSM revealed high significances (p b 0.01) for all regression parameters (linear, quadratic, cross product, and total model), and their regression coefficient (R 2) was 0.97. The predicted optimal conditions, which were found by the ridge analysis, were 196 bar, 46 °C, and 17 min, with a predicted log10 reduction of 6.37. As shown in Fig. 1a, the contour plot generated at a fixed temperature of 46 °C indicated that the log10 reduction in fungal spore concentration increased as the treatment time or pressure increased. The density of SC–CO2 is directly related to the dissolving power, and this value increases as pressure increases at a constant temperature (Lucien and Foster, 1999). The high dissolving power of SC–CO2 owing to the increased density of CO2 promotes the inactivation of
186
H.S. Park et al. / Journal of Microbiological Methods 88 (2012) 185–187
1999). However, in this study, other favorable effects caused by the higher temperature, such as a higher chemical reaction rate, increased diffusivity of CO2 (Kim et al., 2008), and increased fluidity of the cell membrane itself (Erkmen, 2001), may have increased the effectiveness with which SC–CO2 inactivated fungal spores. As shown in Fig. 1c, at a fixed treatment time of 25 min, the log10 reduction in the concentration of viable spores increased as the temperature and pressure increased. As shown in Fig. 2, SC–CO2 treatment at the low levels of pressure and temperature (150 bar and 38 °C, respectively), which produced the poorest reduction in spore concentration when SC–CO2 alone was used, were selected to investigate the effect of ethanol entrainer. In the presence of ethanol entrainer, the 7 log reduction was obtained after 45 min in this study. However, without ethanol entrainer, much longer treatment time of 90 min was required for the 7 log reduction to occur at the same pressure and temperature. Although membrane integrity in bacterial spores is known to be disrupted by ethanol itself (Setlow et al., 2002), the change in polarity and other physical and chemical properties of SC–CO2 induced by ethanol entrainer may have contributed to the enhanced lethality of SC–CO2 (Lucien and Foster, 1999). Meanwhile, at 150 bar and 38 °C, the mole fractions of the ternary system of CO2, ethanol, and water at equilibrium were 0.9792, 0.0162, and 0.0046, respectively (Yao et al., 1994). Therefore, the actual amount of ethanol dissolved in the SC–CO2 phase in the present study was estimated to be only 42 mg in 100 mL of the internal volume of the treatment vessel. Also, it was assumed that there was no direct contact between the remaining liquid ethanol and spores since the suspensioncontaining tube was capped to minimize the direct entrainment of liquid ethanol into the tube. In conclusion, the SC–CO2–ethanol entrainer system reduced the treatment time for the complete inactivation of fungal spores by half in comparison with using SC–CO2 without ethanol entrainer. The analysis of the mechanism of fungal spore inactivation and also the application of the present method to real systems such as seeds contaminated with fungi may be needed as future work.
Acknowledgments This work was supported by the Advanced Biomass R&D Center of Korea Grant (2010-0029734) funded by the Ministry of Education, Science and Technology, Korea. The facility support provided by the Institute of Biomedical Science and Food Safety, Korea University, is also acknowledged.
Fig. 1. Response surface plot of SC–CO2 inactivation of A. brassicicola spores. (a) Effect of pressure and treatment time with a fixed temperature of 46 °C. (b) Effect of temperature and treatment time at a fixed pressure of 250 bar. (c) Effect of pressure and temperature at a fixed treatment time of 25 min.
Log10 reduction of CFU/mL
8 Ethanol No ethanol
6
4
2
0
microorganisms through extraction or denaturing of intracellular materials (Kim et al., 2007, 2008). At a constant pressure of 250 bar, the log10 reduction of spores increased when treatment temperature and time increased (Fig. 1b). As the temperature increases at a fixed pressure, the decreased SC–CO2 density can result in the reduced dissolving power of SC–CO2 (Lucien and Foster,
0
15
30
45
60
90
120
150
Treatment Time (min) Fig. 2. Effect of ethanol as the entrainer of SC–CO2 on the inactivation of A. brassicicola spores at 150 bar and 38 °C. A total of 16 g of ethanol (> 99.8% purity) was pumped into a treatment reactor with a 100 mL internal volume.
H.S. Park et al. / Journal of Microbiological Methods 88 (2012) 185–187
References Bush, R.K., Prochnau, J.J., 2004. Alternaria-induced asthma. J. Allergy Clin. Immunol. 113, 227–234. Chen, L.Y., Price, T.V., Silvapulle, M.J., 2005. Dark leaf spot (Alternaria brassicicola) on Chinese cabbage: spatial patterns. Aust. J. Agric. Res. 56, 699–714. Erkmen, O., 2001. Effects of high-pressure carbon dioxide on Escherichia coli in nutrient broth and milk. Int. J. Food Microbiol. 65, 131–135. Kamihira, M., Taniguchi, M., Kobayashi, T., 1987. Sterilization of microorganisms with supercritical carbon dioxide. Agric. Biol. Chem. 51, 407–412. Kim, S.R., Rhee, M.S., Kim, B.C., Lee, H., Kim, K.H., 2007. Modeling of the inactivation of Salmonella typhimurium by supercritical carbon dioxide in physiological saline and phosphate-buffered saline. J. Microbiol. Methods 70, 132–141. Kim, S.R., Park, H.J., Yim, D.S., Kim, H.T., Choi, I.-G., Kim, K.H., 2008. Analysis of survival rates and cellular fatty acid profiles of Listeria monocytogenes treated with supercritical carbon dioxide under the influence of cosolvents. J. Microbiol. Methods 75, 47–54.
187
Lucien, F.P., Foster, N.R., 1999. Phase behavior and solubility. In: Jessop, P.G., Leitner, W. (Eds.), Chemical Synthesis Using Supercritical Fluids. Wiley-VCH, Weinheim, pp. 37–53. Myers, R.H., Montgomery, D.C., 1995. Response Surface Methodology. John Wiley & Sons Inc., New York. Setlow, B., Loshon, C.A., Genest, P.C., Cowan, A.E., Setlow, C., Setlow, P., 2002. Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol. J. Appl. Microbiol. 92, 362–375. Shimoda, M., Kago, H., Kojima, N., Miyake, M., Osajima, Y., Hayakawa, I., 2002. Accelerated death kinetics of Aspergillus niger spores under high-pressure carbonation. Appl. Environ. Microbiol. 68, 4162–4167. Stark, P.C., Celedón, J.C., Chew, G.L., Ryan, L.M., Burge, H.A., Muilenberg, M.L., Gold, D.R., 2005. Fungal levels in the home and allergic rhinitis by 5 years of age. Environ. Health Perspect. 113, 1405–1409. Yao, S., Guan, Y., Zhu, Z., 1994. Investigation of phase equilibrium for ternary systems containing ethanol, water and carbon dioxide at elevated pressures. Fluid Phase Equilib. 99, 249–259.