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High-pressure inactivation of Bacillus cereus spores in the presence of argon
International Journal of Food Microbiology 72 (2002) 239 – 242 www.elsevier.com/locate/ijfoodmicro
Short communication
High-pressure inactivation of Bacillus cereus spores in the presence of argon Keiko Fujii a,*, Ayami Ohtani b, Junko Watanabe b, Hiro Ohgoshi b, Tomoyuki Fujii c, Kazuo Honma d a Faculty of Education, Yamagata University, Yamagata, Yamagata 990-8560, Japan Department of Food and Nutrition, Japan Women’s University, Bunkyo-ku, Tokyo 112-0015, Japan c Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan d Research Institute of QP Corporation, Fuchu, Tokyo 183-0034, Japan b
Received 18 June 2001; received in revised form 3 September 2001; accepted 10 September 2001
Abstract We investigated the high-pressure inactivation of Bacillus cereus spores in water containing argon. At the pressure of 600 MPa, addition of argon accelerated the inactivation of spores at 20 °C, but had no effect on the inactivation at 40 °C. The influence of added argon on inactivation of the spores was marked under conditions with a strong ‘water ordering’ effect. The pressure resistance of B. cereus spores was thus shown to be affected by ‘water ordering’. D 2002 Elsevier Science B.V. All rights reserved. Keywords: High pressure; Bacillus cereus; Water ordering; Inactivation; Argon
1. Introduction When a hydrocarbon or rare gas is dissolved in water under appropriately selected temperature and pressure conditions, highly ordered ‘‘iceberg-like’’ form around solute molecules in aqueous solution due to hydrophobic hydration (Davidson, 1973). In some cases, a crystalline clathrate hydrate is formed in this way. The phenomenon whereby the crystallinity of the liquid structure of water increases is known as ‘water ordering’. A clathrate hydrate containing argon as the guest molecule can remain stable, for example
*
Corresponding author. Fax: +81-23-628-4353. E-mail address: [email protected] (K. Fujii).
at 124 °C or below at less than 0.1 MPa (Barrer and Edge, 1967) or at 0.8 °C or below at less than 8.7 MPa (Marshall et al., 1964). Since high-pressure treatment has recently become applicable to biochemical and food processing (Knorr, 1993; Cheftel, 1995), we have considered the combined use of ‘water ordering’ with high pressure, achieved by dissolving a rare gas in the water contained in foods or bioproducts. This combined effect might make it possible to establish novel non-heating processing/pasteurizing techniques without altering the quality of bioproducts. While Bacillus cereus is a cause of food poisoning, Bacillus is used as a research model in biotechnology to study bacterial nutrition, cell wall formation, sporogenesis, gene regulation and plasmid expression,
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 7 0 0 - 0
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K. Fujii et al. / International Journal of Food Microbiology 72 (2002) 239–242
production of insecticides (Agaisse and Lereclus, 1995), immunogenesis in vaccine production (Medina and Guzman, 2001), and resistance to extreme conditions (Nicholson et al., 2000). Inactivation of spores by high pressure is a two-step process: spore germination followed by inactivation of the germinated forms (Clouston and Wills, 1969). The influence of physicochemical agents on the initiation of germination and inactivation of B. cereus spores under high pressure has been documented (Raso et al., 1998). Although pressure-induced germination of B. subtilis spores has been recently studied (Wuytack et al., 1998), the biological responses of microorganisms under high pressure are still unclear (Abee and Wouters, 1999). We investigated the behaviour of B. cereus spores inactivated by high pressure, and addressed it from the viewpoint of ‘water ordering’ under high pressure achieved by dissolution of a rare gas.
2.3. High-pressure treatment Each sample containing dissolved argon was treated under high pressure of 500 and 600 MPa at 20, 30 and 40 °C for 30 min with a Mitsubishi Heavy Industries high-pressure generator model MFP-7000. The time to reach final pressure desired was within 90 s. Decompression could be completed within 15 s. 2.4. Enumeration of spore survival After the pressure treatment, each sample was inoculated onto plates containing a nutrient agar medium (0.3% Difco Bacto Beef Extract/0.5% Difco Bacto peptone) at pH 6.8 and incubated at 30 °C for 48 h. The survival was calculated by dividing the viable count after pressurization by that before pressurization.
3. Results and discussion 2. Materials and methods 2.1. Preparation of spore suspension A culture (24 h, 30 °C) of B. cereus IAM 12605 in nutrient broth (Difco, Detroit, MI, USA) was spread on a plate of standard agar medium (Eiken Chemical, Tokyo, Japan) and incubated at 30 °C for 2 weeks. The culture was collected with a glass spatula and was suspended in 1/15 M phosphate buffer solution (pH 7.1). After harvesting, it was heated to 80 °C for 30 min to exterminate all vegetative cells. The obtained suspension containing spores (about 105 spores/ml) was referred to as the standard spore suspension.
Fig. 1 shows the effects of treatment temperature on the survival of B. cereus spores at 600 MPa. The argon-free sample showed a 2-log10 reduction of spore numbers after pressurization for 30 min at 40 °C, although inactivation was not observed at 20 °C. The argon-containing sample showed a 1-log10 reduction of spore numbers after pressurization for 30 min at 20
2.2. Dissolution of argon The standard spore suspension was pipetted in 20ml portions into aluminum pouches (3 cm in diameter, 22 cm in length; three-layered structure consisting of polyester, aluminum foil and special high-density polyethylene), and then argon was dissolved in the suspension by bubbling. The bubbling was carried out for 10 min, because a preliminary experiment had shown that this produces no change in the viable count.
Fig. 1. Effect of added argon on inactivation of B. cereus spores at a treatment pressure of 600 MPa.
K. Fujii et al. / International Journal of Food Microbiology 72 (2002) 239–242
Fig. 2. Effect of added argon on inactivation of B. cereus spores at a treatment temperature of 30 °C.
°C. Both the argon-free and argon-containing samples showed a 2-log10 reduction of spore numbers after pressurization for 30 min at 40 °C, thus showing no effect of added argon. The argon-containing sample showed lower survival than the argon-free sample at 20 °C, different from the case at 40 °C. Although addition of argon had no effect on the inactivation of spores at 40 °C, it effectively accelerated the inactivation at 20 °C. The results of the treatment at 500 MPa were almost the same as those obtained by treatment at 600 MPa (data not shown). Values were calculated by dividing the survival in the presence of argon by that in the absence of argon (i.e., relative survival), to summarize the effects of the added argon. A relative survival of less than 1 means that added argon accelerated the inactivation. At the treatment pressure of 600 MPa, the decrease in relative survival was 0.91, 0.31 and 0.068, at 40, 30 and 20 °C, respectively. Relative survival was close to 1 at 40 °C, showing that addition of argon scarcely affected the inactivation effect. The addition of argon enhanced the inactivation of the spores at 20 °C. Fig. 2 shows the effect of pressure on the survival of B. cereus spores at 30 °C. The survival in argonfree samples decreased with increasing pressure. The influence of added argon on inactivation of the spores at 400 MPa was superior to that achieved at 600 MPa. When a hydrocarbon is dissolved in water under high pressure, the solubility –pressure curve under constant
241
pressure generally shows a peak (Sawamura et al., 1989). The hydrophobic hydration is elevated with an increase in pressure at the low-pressure side ( < 100– 150 MPa). At the high-pressure side, in contrast, the hydrophobic-hydration shell around the hydrocarbon is destroyed due to the water-ordering deformation resulting from compression. The results of this study show that argon has an inactivation accelerating effect within the low-temperature and low-pressure region where hydrophobic hydration generally arises. It is anticipated that under a high pressure of 500 MPa or above, where proteins would be denatured, high-pressure denaturation is enhanced with increased pressure. However, hydrophobic hydration is weakened with increased pressure. Therefore, it is considered that the effects of added argon on spore inactivation would differ due to the overlapping effects of these two phenomena. Although its roles in the initiation of germination or inactivation are not obvious, hydrophobic hydration would affect bacterial spore pressure resistance. Finally, it is expected that if a novel technique involving a combination of ‘water ordering’ and high pressure could be established, it would be useful for the processing of bioproducts and foods.
Acknowledgements This study was supported in part by The SKYLARK Food Science Institute of Japan.
References Abee, T., Wouters, J.A., 1999. Microbial stress response in minimal processing. Int. J. Food Microbiol. 50, 65 – 91. Agaisse, H., Lereclus, D., 1995. How does Bacillus thuringiensis produce so much insecticidal crystal protein? J. Bacteriol. 177, 6027 – 6032. Barrer, R.M., Edge, A.V.J., 1967. Gas hydrates containing argon, krypton, and xenon: kinetics and energetics of formation and equilibriums. Proc. R. Soc., Ser. A 300, 1 – 24. Cheftel, J.C., 1995. High-pressure microbial inactivation and food preservation. Food Sci. Technol. Int. 1, 75 – 90. Clouston, J.G., Wills, P.A., 1969. Initiation of germination and inactivation of Bacillus pumilus spore by hydrostatic pressure. J. Bacteriol. 97, 684 – 690. Davidson, D.W., 1973. Clathrate hydrates. In: Franks, F. (Ed.), Water: A Comprehensive Treatise, vol. 2. Plenum, New York, pp. 115 – 234.
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K. Fujii et al. / International Journal of Food Microbiology 72 (2002) 239–242
Knorr, D., 1993. Effects of high-hydrostatic pressure processes on food safety and quality. Food Technol. 47, 156 – 161. Marshall, D.R., Saito, S., Kobayashi, R., 1964. Hydrates at high pressure: Part I. Methane – water, argon – water, and nitrogen – water systems. AIChE J. 10, 202 – 205. Medina, E., Guzman, C.A., 2001. Use of live bacterial vaccine vectors for antigen delivery: potential and limitations. Vaccine 19, 1573 – 1580. Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J., Setlow, P., 2000. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol. Mol. Biol. Rev. 64, 548 – 572.
Raso, J., Gongora-Nieto, M.M., Barbosa-Canovas, G.V., Swanson, B.G., 1998. Influence of several environmental factors on the initiation of germination and inactivation of Bacillus cereus by high hydrostatic pressure. Int. J. Food Microbiol. 44, 125 – 132. Sawamura, S., Kitamura, K., Taniguchi, Y., 1989. Effect of pressure on the solubilities of benzene and alkylbenzenes in water. J. Phys. Chem. 93, 4931 – 4935. Wuytack, E.Y., Boven, S., Michiels, C.W., 1998. Comparative study of pressure-induced germination of Bacillus subtilis spores at low and high pressures. Appl. Environ. Microbiol. 64, 3220 – 3224.
Short communication
High-pressure inactivation of Bacillus cereus spores in the presence of argon Keiko Fujii a,*, Ayami Ohtani b, Junko Watanabe b, Hiro Ohgoshi b, Tomoyuki Fujii c, Kazuo Honma d a Faculty of Education, Yamagata University, Yamagata, Yamagata 990-8560, Japan Department of Food and Nutrition, Japan Women’s University, Bunkyo-ku, Tokyo 112-0015, Japan c Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan d Research Institute of QP Corporation, Fuchu, Tokyo 183-0034, Japan b
Received 18 June 2001; received in revised form 3 September 2001; accepted 10 September 2001
Abstract We investigated the high-pressure inactivation of Bacillus cereus spores in water containing argon. At the pressure of 600 MPa, addition of argon accelerated the inactivation of spores at 20 °C, but had no effect on the inactivation at 40 °C. The influence of added argon on inactivation of the spores was marked under conditions with a strong ‘water ordering’ effect. The pressure resistance of B. cereus spores was thus shown to be affected by ‘water ordering’. D 2002 Elsevier Science B.V. All rights reserved. Keywords: High pressure; Bacillus cereus; Water ordering; Inactivation; Argon
1. Introduction When a hydrocarbon or rare gas is dissolved in water under appropriately selected temperature and pressure conditions, highly ordered ‘‘iceberg-like’’ form around solute molecules in aqueous solution due to hydrophobic hydration (Davidson, 1973). In some cases, a crystalline clathrate hydrate is formed in this way. The phenomenon whereby the crystallinity of the liquid structure of water increases is known as ‘water ordering’. A clathrate hydrate containing argon as the guest molecule can remain stable, for example
*
Corresponding author. Fax: +81-23-628-4353. E-mail address: [email protected] (K. Fujii).
at 124 °C or below at less than 0.1 MPa (Barrer and Edge, 1967) or at 0.8 °C or below at less than 8.7 MPa (Marshall et al., 1964). Since high-pressure treatment has recently become applicable to biochemical and food processing (Knorr, 1993; Cheftel, 1995), we have considered the combined use of ‘water ordering’ with high pressure, achieved by dissolving a rare gas in the water contained in foods or bioproducts. This combined effect might make it possible to establish novel non-heating processing/pasteurizing techniques without altering the quality of bioproducts. While Bacillus cereus is a cause of food poisoning, Bacillus is used as a research model in biotechnology to study bacterial nutrition, cell wall formation, sporogenesis, gene regulation and plasmid expression,
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 7 0 0 - 0
240
K. Fujii et al. / International Journal of Food Microbiology 72 (2002) 239–242
production of insecticides (Agaisse and Lereclus, 1995), immunogenesis in vaccine production (Medina and Guzman, 2001), and resistance to extreme conditions (Nicholson et al., 2000). Inactivation of spores by high pressure is a two-step process: spore germination followed by inactivation of the germinated forms (Clouston and Wills, 1969). The influence of physicochemical agents on the initiation of germination and inactivation of B. cereus spores under high pressure has been documented (Raso et al., 1998). Although pressure-induced germination of B. subtilis spores has been recently studied (Wuytack et al., 1998), the biological responses of microorganisms under high pressure are still unclear (Abee and Wouters, 1999). We investigated the behaviour of B. cereus spores inactivated by high pressure, and addressed it from the viewpoint of ‘water ordering’ under high pressure achieved by dissolution of a rare gas.
2.3. High-pressure treatment Each sample containing dissolved argon was treated under high pressure of 500 and 600 MPa at 20, 30 and 40 °C for 30 min with a Mitsubishi Heavy Industries high-pressure generator model MFP-7000. The time to reach final pressure desired was within 90 s. Decompression could be completed within 15 s. 2.4. Enumeration of spore survival After the pressure treatment, each sample was inoculated onto plates containing a nutrient agar medium (0.3% Difco Bacto Beef Extract/0.5% Difco Bacto peptone) at pH 6.8 and incubated at 30 °C for 48 h. The survival was calculated by dividing the viable count after pressurization by that before pressurization.
3. Results and discussion 2. Materials and methods 2.1. Preparation of spore suspension A culture (24 h, 30 °C) of B. cereus IAM 12605 in nutrient broth (Difco, Detroit, MI, USA) was spread on a plate of standard agar medium (Eiken Chemical, Tokyo, Japan) and incubated at 30 °C for 2 weeks. The culture was collected with a glass spatula and was suspended in 1/15 M phosphate buffer solution (pH 7.1). After harvesting, it was heated to 80 °C for 30 min to exterminate all vegetative cells. The obtained suspension containing spores (about 105 spores/ml) was referred to as the standard spore suspension.
Fig. 1 shows the effects of treatment temperature on the survival of B. cereus spores at 600 MPa. The argon-free sample showed a 2-log10 reduction of spore numbers after pressurization for 30 min at 40 °C, although inactivation was not observed at 20 °C. The argon-containing sample showed a 1-log10 reduction of spore numbers after pressurization for 30 min at 20
2.2. Dissolution of argon The standard spore suspension was pipetted in 20ml portions into aluminum pouches (3 cm in diameter, 22 cm in length; three-layered structure consisting of polyester, aluminum foil and special high-density polyethylene), and then argon was dissolved in the suspension by bubbling. The bubbling was carried out for 10 min, because a preliminary experiment had shown that this produces no change in the viable count.
Fig. 1. Effect of added argon on inactivation of B. cereus spores at a treatment pressure of 600 MPa.
K. Fujii et al. / International Journal of Food Microbiology 72 (2002) 239–242
Fig. 2. Effect of added argon on inactivation of B. cereus spores at a treatment temperature of 30 °C.
°C. Both the argon-free and argon-containing samples showed a 2-log10 reduction of spore numbers after pressurization for 30 min at 40 °C, thus showing no effect of added argon. The argon-containing sample showed lower survival than the argon-free sample at 20 °C, different from the case at 40 °C. Although addition of argon had no effect on the inactivation of spores at 40 °C, it effectively accelerated the inactivation at 20 °C. The results of the treatment at 500 MPa were almost the same as those obtained by treatment at 600 MPa (data not shown). Values were calculated by dividing the survival in the presence of argon by that in the absence of argon (i.e., relative survival), to summarize the effects of the added argon. A relative survival of less than 1 means that added argon accelerated the inactivation. At the treatment pressure of 600 MPa, the decrease in relative survival was 0.91, 0.31 and 0.068, at 40, 30 and 20 °C, respectively. Relative survival was close to 1 at 40 °C, showing that addition of argon scarcely affected the inactivation effect. The addition of argon enhanced the inactivation of the spores at 20 °C. Fig. 2 shows the effect of pressure on the survival of B. cereus spores at 30 °C. The survival in argonfree samples decreased with increasing pressure. The influence of added argon on inactivation of the spores at 400 MPa was superior to that achieved at 600 MPa. When a hydrocarbon is dissolved in water under high pressure, the solubility –pressure curve under constant
241
pressure generally shows a peak (Sawamura et al., 1989). The hydrophobic hydration is elevated with an increase in pressure at the low-pressure side ( < 100– 150 MPa). At the high-pressure side, in contrast, the hydrophobic-hydration shell around the hydrocarbon is destroyed due to the water-ordering deformation resulting from compression. The results of this study show that argon has an inactivation accelerating effect within the low-temperature and low-pressure region where hydrophobic hydration generally arises. It is anticipated that under a high pressure of 500 MPa or above, where proteins would be denatured, high-pressure denaturation is enhanced with increased pressure. However, hydrophobic hydration is weakened with increased pressure. Therefore, it is considered that the effects of added argon on spore inactivation would differ due to the overlapping effects of these two phenomena. Although its roles in the initiation of germination or inactivation are not obvious, hydrophobic hydration would affect bacterial spore pressure resistance. Finally, it is expected that if a novel technique involving a combination of ‘water ordering’ and high pressure could be established, it would be useful for the processing of bioproducts and foods.
Acknowledgements This study was supported in part by The SKYLARK Food Science Institute of Japan.
References Abee, T., Wouters, J.A., 1999. Microbial stress response in minimal processing. Int. J. Food Microbiol. 50, 65 – 91. Agaisse, H., Lereclus, D., 1995. How does Bacillus thuringiensis produce so much insecticidal crystal protein? J. Bacteriol. 177, 6027 – 6032. Barrer, R.M., Edge, A.V.J., 1967. Gas hydrates containing argon, krypton, and xenon: kinetics and energetics of formation and equilibriums. Proc. R. Soc., Ser. A 300, 1 – 24. Cheftel, J.C., 1995. High-pressure microbial inactivation and food preservation. Food Sci. Technol. Int. 1, 75 – 90. Clouston, J.G., Wills, P.A., 1969. Initiation of germination and inactivation of Bacillus pumilus spore by hydrostatic pressure. J. Bacteriol. 97, 684 – 690. Davidson, D.W., 1973. Clathrate hydrates. In: Franks, F. (Ed.), Water: A Comprehensive Treatise, vol. 2. Plenum, New York, pp. 115 – 234.
242
K. Fujii et al. / International Journal of Food Microbiology 72 (2002) 239–242
Knorr, D., 1993. Effects of high-hydrostatic pressure processes on food safety and quality. Food Technol. 47, 156 – 161. Marshall, D.R., Saito, S., Kobayashi, R., 1964. Hydrates at high pressure: Part I. Methane – water, argon – water, and nitrogen – water systems. AIChE J. 10, 202 – 205. Medina, E., Guzman, C.A., 2001. Use of live bacterial vaccine vectors for antigen delivery: potential and limitations. Vaccine 19, 1573 – 1580. Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J., Setlow, P., 2000. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol. Mol. Biol. Rev. 64, 548 – 572.
Raso, J., Gongora-Nieto, M.M., Barbosa-Canovas, G.V., Swanson, B.G., 1998. Influence of several environmental factors on the initiation of germination and inactivation of Bacillus cereus by high hydrostatic pressure. Int. J. Food Microbiol. 44, 125 – 132. Sawamura, S., Kitamura, K., Taniguchi, Y., 1989. Effect of pressure on the solubilities of benzene and alkylbenzenes in water. J. Phys. Chem. 93, 4931 – 4935. Wuytack, E.Y., Boven, S., Michiels, C.W., 1998. Comparative study of pressure-induced germination of Bacillus subtilis spores at low and high pressures. Appl. Environ. Microbiol. 64, 3220 – 3224.