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Inactivation of Bacillus subtilis spores by supercritical CO2 treatment
Innovative Food Science and Emerging Technologies 4 (2003) 161–165
Inactivation of Bacillus subtilis spores by supercritical CO2 treatment S. Spilimbergoa,*, A. Bertuccoa, F.M. Laurob, G. Bertolonib a University of Padova, Department of Chemical Engineering via Marzolo 9, 35131 Padova, Italy University of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Via Gabelli 63, 35131 Padova, Italy
b
Abstract Bacillus subtilis spores were suspended in saline solution (107 cfuyml) and treated by both conventional heating and CO2 batch treatment at an operating pressure in the range of 70–150 bar under identical temperature conditions. Temperatures tested were in the range of 36–75 8C. Survival curves indicated significantly higher lethality when spores were treated with supercritical CO2 (SC-CO2) rather than with heating alone. These results appear particularly evident at 60 8C, a temperature at which conventional heating gave no spore-inactivation after a treating time as long as 24 h, whereas a 6 h SC-CO2 treatment led to complete sterilization. At 75 8C spores were partially killed with conventional heating but a treatment of 2 with SC-CO2 hours assured total inactivation. It is concluded that spore-inactivation during SC-CO2 treatment was only in part due to thermal effect (at the higher temperature of 75 8C) and there was a significant additional effect caused by CO2 penetration inside the latent bacteria forms. 䊚 2002 Elsevier Science Ltd. All rights reserved. Keywords: Supercritical CO2; Spore-inactivation; Bacillus subtilis Industrial relevance: The present work confirms existing data and adds important insights into the possible inactivation of baterial spores via induction of germination by SC-CO2. This offers, besides high pressure assisted sterilisation, another possible technology to inactivate spores at mild thermal conditions. It also opens the possibility to reduce bacterial counts in conventional extraction processes with SC-CO2.
1. Introduction In food, as well as pharmaceuticals and polymeric materials, microbial-inactivation is a crucial parameter in the safety and shelf-life of the product. The methods commonly used for polymer treatment are steam sterilization, ethylene oxide exposure and g irradiation, while in the case of food processing the most widespread technique is U.H.T. (ultra high temperature) treatment. All these methods assure a satisfactory microbial inactivation, but have a number of side effects (Guidoin et al., 1985; Zhang, Bjursten, Freij-Larson, Kober & Wesslen, 1996; Konig, Ruffieux, Wintermantel & Blasser, 1997). For instance, in the case of thermally or hydrolytically labile polymers, autoclaving can destroy materials, while U.H.T. can cause changes in the organoleptic features of temperature-sensitive substances and the denaturation of vitamins and proteins. This is the reason why alternative and economically convenient *Corresponding author. Tel.: q39-049-8275491; fax: q39-0498275461. E-mail address: [email protected] (S. Spilimbergo).
methods have been studied in the recent years. Among all, we mention high-voltage pulsed electric field (Sitzmann, 1995; Barbosa-Canovas, Pierson, Zhang & Schaffner, 2000), ultra-sound (Sherba, Weigel & O’Brein, 1991; Sala, Burgos, Condon, Lopez & Raso, 1995) and high hydrostatic pressure treatments (Ludwig, Gross, Scigalla & Sojka, 1994; Schreck, & Ludwig, 1997). The use of supercritical CO2 (SC-CO2) has been recently investigated and continues to attract interest. A supercritical fluid is a fluid above its critical temperature (Tc) and critical pressure (Pc), that possesses unique properties such as high density and low viscosity. Carbon dioxide is a non-toxic and inexpensive gas and has been considered in the sterilization of biomaterials (Dillow, Dehghani, Hrkack, Foster & Langer, 1999), of bone allografts (Fages et al., 1998) and of food storages (Quirin, 1988); it was shown that CO2 is effective against bacteria, viruses and insects at different stages of development. Furthermore, since the critical values of pressure and temperature are relatively low (73.8 bar and 31 8C, respectively), the gas is relatively easy to handle under supercritical conditions.
1466-8564/03/$ - see front matter 䊚 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1466-8564(02)00089-9
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S. Spilimbergo et al. / Innovative Food Science and Emerging Technologies 4 (2003) 161–165
In previous work, promising results were found when CO2 was used to reduce the activity of a wide range of microorganisms (Spilimbergo, Elvassore & Bertucco, 2002b). On the other hand, inactivation of bacterial spores is crucial in the sterilization of food, polymeric and pharmaceutical materials. Spores are the most resistant form of bacteria: their structure is different and more complex when compared to that of vegetative cells. Dormant bacterial endospores are highly resistant to a number of physical and chemical treatments which are normally considered germicidal (Setlow, 1995). Spores owe some of their resistance to the presence of an outer proteinaceous layer termed spore coat (Gould, 1983; Driks, 1999). This coat protects a dormant spore from enzymes, such as lysozyme (Gould & Hitchins, 1963), from both mechanical (Gould, 1983) and chemical disruption, such as that obtained by hydrogen peroxide (Gould & Hitchins, 1963). Furthermore, spores are strongly dehydrated compared to living cells and are impermeable to most dyes. Spores are characterized by their capacity to survive under extreme conditions such as high and low temperature, high and low pH and high hydrostatic pressure, and can remain latent for many years (Cano & Borucki, 1995). Few works addressing spore inactivation by SC-CO2 can be found in literature. In 1997 Enamoto and coworkers (Enomoto, Nakamura, Hakoda & Amaya, 1997) reduced the survival ratio of a sample of bacterial spores of Bacillus megaterium (108 colony forming units per ml, cfuyml) in physiological solution, to approximately 101 cfuyml with a batch treatment of 30 h at 58 bar and 60 8C. In the same year Ishikawa et al. (1997) achieved a total microbial inactivation of Bacillus subtilis, B. polymyxa and B. cereus (all of them from an initial concentration of 106 cfuyml) with a semi-continuous apparatus, the so-called micro-bubble method, after a treatment of 1 h at 300 bar and a temperature of 50–60 8C. One year later, Ballestra and Cuq (1998), reduced the survival count of B. subtilis spores to approximately 103.5 cfuyml and Byssochlamys fulva ascospores to approximately 101 cfuyml, from an initial suspension of 107 and 105 cfuyml, respectively, with a batch treatment of 1 h, at an operating pressure of 50 bar and a temperature of 80 8C. In 2002, Spilimbergo found that the microbial activity reduction of B. subtilis spores was made possible by coupling the effect of higher temperature (75 8C) and longer treatment time (24 h), and compared this result to that on bacteria species (Spilimbergo, Elvassore & Bertucco, 2002a). From the quotations above it is concluded that the open literature is rather conflicting, foggy and full of gaps as regards spore inactivation by SC-CO2. In this digest, the aim of our work is to better investigate and clarify the lethal effect of SC-CO2 treatment on Bacillus subtilis spores, used as test microorganisms, to compare it with conventional heating, and
to study the kinetics of spore inactivation for both of these sterilization methods. 2. Materials and methods 2.1. Bacterial strains and media Bacillus subtilis var. niger ATCC 9372, a sporeforming Gram-positive bacterium, was selected as a test microorganisms as it is well known to be resistant to heat, radiation, most chemical disinfectants and antibiotics when compared to vegetative bacterial forms. Furthermore, B. subtilis var. niger together with B. stearthermophilus and B. pumilus are currently used as test microorganisms for steam, ethylene oxide, dry and radiation standard sterilization tests in microbiology. The spores were prepared by plating a stock culture on nutrient agar. After an incubation of 24 h at 37 8C the plates were left at room temperature for 4 days. Sporulation was monitored by phase-contrast microscopy. After an almost complete sporulation had occurred, the plates were flooded with 2–3 ml of saline solution (0.9% NaCl) and the surface was gently scraped with a plastic sterile rod. All the contents were recovered in a sterile bottle, vortexed and shocked at 80 8C for 10 min to kill the remaining vegetative cells in the sample. The suspension was then centrifuged for 10 min at 3000=g. The supernatant was discharged and the spore-pellet was re-suspended in saline solution, poured in sterile testtubes and stored at y20 8C until used. The spore concentration was approximately 109 cfuyml. 2.2. Counting Before and after each treatment viable counts of surviving spores were determined by the standard plating technique (Speck, 1976). In addition, as the treated sample is concerned, spore viability has been determined in diluted as well as in undiluted conditions. In order to further check that complete sterilization was achieved, small volumes (1–3 ml) of treated sample were inoculated in liquid medium brain heart infusion (BHI), incubated at 37 8C for 24 h, plated in brain heart agar (BHA), and colonies were counted. The results are expressed as % survival as well as log reduction, log (No yN), where No and N are the numbers of cfuyml before and after the treatment, respectively. All the values (reported in the Results section) are the averages among three different runs. 2.3. CO2 treatment A batch procedure was used for the SC-CO2 treatment. The sterilization chamber was the same as in a previous work (Spilimbergo et al., 2002a) but it was used in a batch mode.
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Table 1 Inactivation of Bacillus subtilis spores as a function of time during SC-CO2 batch treatments at 36 and 50 8C P (bar)
T (8C)
Time (h)
Log(No yN)**
Survival (%)
75 80 120 80 120
36 36 36 50 50
24 48 48 48 48
0.5 0.9 0.9 1 1
32 13 13 10 10
**
(No yN) is the ratio between the cfuyml before and after the treatment.
The sterile vessel was filled with 10 ml of sporesuspension previously prepared, diluted to 107 cfuyml, the screw plugs were sealed under sterile conditions and all the set-up was heated by an electrical resistance up to the set point value (from 36 to 75 8C). When the temperature reached the desired value, the CO2-pump was switched on, the inlet valve was opened and the outlet valve closed, letting CO2 bubble through the vessel. When the operating pressure was reached, the pump was switched off and the inlet valve was kept completely closed for all the treatment time. In order to control the temperature during the experiment, the highpressure vessel, which is thermally insulated, is equipped with a resistance temperature probe, Pt 100 V, located inside, a temperature on-off control, and an electric resistance. After carrying out the experiment, the outlet valve was heated to avoid freezing during the venting of CO2 and then opened until a complete discharge of pressure was attained. As regards the cycled pressure treatments, the sample (at a concentration of 107 cfuyml) was subjected to cycling variations in pressure, up to 150 bar, and the amplitude of pressure variation, DP, was changed between 80 and 110 bar. When the pressure reached its maximum value, the system was maintained under continuous flow of CO2 for a pre-set treatment time. In order to select the best operating conditions, different variables, such as the number of cycles per hour, the time at which the pressure was kept at its maximum value, the temperature and DP were varied during different runs of experiments. As temperature treatment is concerned, a tube containing 50 ml of physiological solution with 107 cfuyml
Fig. 1. Survival of Bacillus subtilis spores as a function of time, at 75 8C during SC-CO2 treatment (s) and conventional heating (h).
of B. subtilis spores was kept at the desired temperature (60 or 75 8C) by a thermostatic water-bath (Ocras Zambelli s.a.s, Torino, Italy). At fixed times a sample was taken from the tube in order to measure the survival kinetics. After any type of treatment, the same procedure was followed: the solution of the treated spore-suspension was shocked in a water-bath for 10 min in order to kill the vegetative cells and activate the spores, spread onto BHA plates and, after an incubation at 37 8C for 24 h, the surviving colonies were counted. 3. Results The result of the survival assays (Table 1) show that B. subtilis spores were resistant to SC-CO2 treatment. Even a prolonged treatment (48 h) with a pressure up to 120 bar and temperatures of 50 8C did not significantly affect the viable cell survival. Disappointing results were obtained even with cycled pressure treatments at 36 and 50 8C, carried out for different treatment times (Table 2). Therefore, the temperature was increased. Suspensions (107 cfuyml) were treated with SC-CO2 for 2–6 h at a pressure of 70 bar and a temperature of 75 8C. At the same time, the effect of temperature alone was observed under atmospheric conditions.
Table 2 Inactivation of Bacillus subtilis spores as a function of time during SC-CO2 pressure-cycled treatment at different temperatures and at different number of cycles per hour Cyclesyh
T (8C)
Time* (min)
DP (bar)
Treatment time (min)
Log(No yN)**
Survival (%)
20 3 2 6
36 50 50 50
1 15 20 4
80 110 110 110
30 60 60 60
2 0.8 1 1.1
1 16 10 8
*
Refers to the time at which the pressure is at its maximum value. (No yN) is the ratio between the cfuyml before and after the treatment.
**
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Fig. 2. Survival of Bacillus subtilis spores as a function of time, at 60 8C during SC-CO2 treatment (s) and conventional heating (h).
The results show that after as little as 2 h of SCCO2 treatment at 75 8C, there is a complete sterilization of the spore suspension (Fig. 1). This effect is mainly due to the action of CO2 since spores treated for the same time (2 h) at a temperature of 75 8C show a 100% survival, and slowly decline only after longer incubations (up to 6 h). Considering that high temperatures can be deleterious to the characteristics of the food or the pharmaceutical compound that has to be sterilized, CO2 treatment was carried out at the temperature of 60 8C and at an operating pressure of 90 bar. The results shown in Fig. 2 point out that temperature alone does not affect spore viability up to 24 h in atmospheric conditions. On the contrary, a treatment at the same temperature with SC-CO2 completely sterilizes the spore suspension after 6 h with a linear decrease in cfuyml counts during the first 5 h. 4. Discussion It is noteworthy that temperature has a primary role in the efficiency of CO2 microbial inactivation treatment. Neither cycled pressure treatments at the same temperatures (35–50 8C) nor batch treatments seem to be effective for spore inactivation even though the kills are higher for cycled treatments (Tables 1 and 2). In this situation, a sudden change of pressure is likely to induce partial activation of the dormant spores, leading to cell modifications which make them more sensitive to metabolic disturbances induced by CO2 within their structure. Our results show that, together with a temperature not lower than 60 8C, SC-CO2 treatment exhibits a clear killing effect against bacterial spores. In fact, after a treatment time of 2 h with SC-CO2 at 75 8C all the latent spores were inactivated, whereas a treating time of 6 h with a conventional heating was able to inactivate them only partially.
The results obtained at 60 8C clearly indicate that CO2 has a fundamental role in spore inactivation. No inactivation was detected during 24 h in the thermostatic bath, while after 6 h of SC-CO2 treatment at the same temperature all the spores were killed. Our data show that the presence of pressurized CO2 inside the sample enhanced the lethal thermal effect to bacterial spores. This effect is much more evident at 60 8C, when the temperature alone is not able to inactivate spores at all. With respect to the bacterial inactivation mechanism, many different hypotheses have already been discussed (Haas et al., 1989; Lin, Zhiying & Chen, 1993; Nakamura, Enomoto, Fukushima, Nagai & Hakoda, 1994; Enomoto, Nakamura and Hakoda, 1997; Dillow et al., 1999). We think that a synergistic effect of intracellular pH decrease and modification of membrane permeability is the most probable one (Spilimbergo et al., 2002a). The same hypothesis cannot be applied for spore inactivation, as the spore core is extremely dehydratated (Ishihara, Saito & Takano, 1999). Even though CO2 dissolves in water and forms carbonic acid, thus lowering the extracellular pH, the gas cannot penetrate inside these latent forms as no water is present within their structure. We think that the presence of CO2, coupled with a mild heating, can be sufficient to promote spore activation and germination. It is known that germination is activated by a sudden change in environmental condition such as temperature, acidification, pressure drop. It is likely that the effect of CO2 acidification, added to the effect of temperature, is sufficient to promote activation, so that spores recruit water and modify their structure thus becoming more sensitive to CO2 treatment. At this stage CO2 can easily penetrate through the membrane, causing an increase of its fluidity and permeability and destroying its essential domains (Isenschmid, Marison & von Stockar, 1995). At the same time, it is feasible that under pressure, an excess of CO2 passes through the altered membrane and lowers the internal pH by exceeding the buffer capacity of the cell pool enough to collapse the pH gradient and the proton motive force across the membrane (Hong and Pyun, 1999). 5. Conclusions The presence of SC-CO2, in association with mild heating, can yield B. subtilis spore-sterilization efficiency. With a treating time of 6 h at an operating pressure of 90 bar at 60 8C a complete B. subtilis inactivation can be achieved. The same conditions without the presence of supercritical fluid gave no inactivation of B. subtilis at all. Our data indicate the possibility to exploit such a method to sterilize foodstuffs, temperature-sensitive materials, such as pharmaceuticals or polymeric mate-
S. Spilimbergo et al. / Innovative Food Science and Emerging Technologies 4 (2003) 161–165
rials, at the industrial scale. Further investigation is needed to confirm this deactivation effect of CO2 toward spores in the presence of complex substrates and to better elucidate the causes of the enhanced sporicidal activity exerted by the CO2 treatment. These results can be qualitatively generalized to other spore-forming Gram-positive bacteria, as B. subtilis is currently used as test micro-organism in standard sterilization tests. Nevertheless, some quantitative differences in the results could arise because of peculiar intrinsic features of each single microorganism. References Ballestra, P., & Cuq, J. L. (1998). Influence of pressurized carbon dioxide on the thermal inactivation of bacterial and fungal spores. Lebensmittel-Wissenschaft und Technologie, 31, 84 –88. Barbosa-Canovas, G. V., Pierson, M. D., Zhang, Q. H., & Schaffner, D. W., 2000. Pulsed Electric Field. Journal of Food Science, Suppl. 65–79. Cano, R. J., & Borucki, M. K. (1995). Revival and identification of bacterial spores in 25- to 40-million-year-old domenican amber. Science, 268, 1060 –1064. Dillow, A. K., Dehghani, F., Hrkack, J. S., Foster, N. R, & Langer, R. (1999). Bacterial inactivation by using near- and supercritical carbon dioxide. Proceedings of the National Academy Science USA, 96, 10344 –10348. Driks, A. (1999). Bacillus subtilis spore coat. Microbiology and Molecular Biology Reviews, 63, 1 –20. Enomoto, A., Nakamura, K., Hakoda, M., & Amaya, N. (1997). Lethal effect of high-pressure carbon dioxide on a bacterial spore. Journal of Fermentation and Bioengineering, 83, 305 –307. Enomoto, A., Nakamura, K., & Hakoda, M. (1997). Inactivation of food microorganisms by high-pressure carbon dioxide treatment with or without explosive decompression. Bioscience Biotechnology Biochemistry, 61, 1133 –1137. Fages, J., Jean, E., Frayssinett, P., Poirier, B., Mathon, D., Autefage, A., & Larzul, D. (1998). Bone allografts and supercritical processing: effects on osteointegration and viral safety. The Journal of Supercritical Fluids, 13, 351 –356. Gould, G. W., & Hitchins, A. D. (1963). Sensitization of bacterial spores to lysozyme and to hydrogen peroxide with agents which rupture disulfide bounds. Journal of General Microbiology, 33, 413 –423. Gould, G. W. (1983). Mechanism of resistence and dormancy. In A. Hurst, G. W. Gould, (pp. 173 –209) The Bacterial Spores, vol. 2. London: Academic Press. Guidoin, R., Snyder, R., King, M., Martin, L., Botzko, K., Award, J., & Marios, M. (1985). A compound arterial prothesis: the importance of the sterilization procedure on the healing and stability of albuminated polyester grafts. Biomaterials, 6, 122 –128. Haas, G. J., Prescott, H. E, Dudley, E., Dik, R., Hintlian, C., & Keane, L. (1989). Inactivation of microorganisms by carbon dioxide under pressure. Journal of Food Safety, 9, 253 –265.
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Hong, S. I., & Pyun, Y. R. (1999). Inactivation kinetics of Lactobacillus plantarum by high pressure carbon dioxide. Journal of Food Science, 64, 728 –733. Konig, C., Ruffieux, K., Wintermantel, E., & Blasser, J. (1997). Autosterilization of biodegradable implants by injection molding process. Journal of Biomedical Materials Research, 38, 115 –119. Isenschmid, A., Marison, I. W., & von Stockar, U. (1995). The influence of pressure and temperature of compressed CO2 on the survival of yeast cells. Journal of Biotechnology Netherlands, 39, 229 –237. Ishihara, Y., Saito, H., & Takano, J. (1999). Differences in the surface membranes and water content between the vegetative cells and spores of Bacillus subtilis. Cell Biochemistry and Function, 17, 9 – 13. Ishikawa, H., Shimoda, M., Tamaya, K., Yonekura, A., Kawano, T., & Osajima, Y. (1997). Inactivation of bacillus spores by the supercritical carbon dioxide micro-bubble method. Bioscience Biotechnology Biochemistry, 61, 1022 –1023. Lin, H., Zhiying, Y., & Chen, L. F. (1993). Inactivation of Leuconostoc dextranicum with carbon dioxide under pressure. The Chemical Engineering Journal, 52, B29 –B34. Ludwig, H., Gross, P., Scigalla, W., & Sojka, B (1994). Pressure inactivation of microorganisms. High Pressure Research, 12, 193. Nakamura, K., Enomoto, A., Fukushima, H., Nagai, K, & Hakoda, M. (1994). Disruption of microbial cells by flash discharge of high-pressure carbon dioxide. Bioscience Biotechnology Biochemistry, 58, 1297 –1301. Quirin, K. W. (1988). Non-Extractive Applications. In K. W. Quirin, E. Stahl, D. Gerard, Dense gases fur extraction and refining (pp. 218 –223). Berlin: Springer. Sala, F. J., Burgos, J., Condon, S., Lopez, P., & Raso, J. (1995). Effect of heat and ultrasound on microorganisms and enzymes. In G. W. Gould, New methods of food preservation (pp. 176 –204). Glasgow: Blackie Acedemic & Professional. Schreck, C., & Ludwig, H. (1997). The inactivation of vegetative bacteria by pressure. In K. Heremans, High pressure Research in Biosciences and Biotechnology . Belgium: Press Leuven. Setlow, P. (1995). Mechanisms for prevention of damage to DNA in spores of Bacillus species. Annual Reviews Microbiology, 49, 29 – 54. Sherba, G., Weigel, R. M., & O’Brein, J. R. (1991). Quantitative assessment of the germicidal efficiency of ultrasonic energy. Applied and Environmental Microbiology, 57, 2079 –2084. Sitzmann, W. (1995). High-voltage pulse techniques for food preservation. In G. W. Gould, New methods of food preservation (pp. 236 –252). Glasgow: Blackie Acedemic & Professional. Speck, M. L. (1976). (pp. 341 –387) Compendium of Methods for the Microbiological Examination of Foods, vol. 107–131. DC: American Publish Health Association. Spilimbergo, S., Elvassore, N., & Bertucco, A. (2002). Microbial inactivation by high-pressure. The Journal of Supercritical Fluids, 22, 55 –63. Spilimbergo, S., Elvassore, N. & Bertucco, A., 2002b. The effects of supercritical CO2 on inactivation of microorganisms in a semicontinuous process. The Italian Journal of Food Science, in press. Zhang, Y., Bjursten, L., Freij-Larson, C., Kober, M., & Wesslen, B. (1996). Tissue response to commercial silicone and polyurethan elastomers after different sterilization procedures. Biomaterials, 17, 2265 –2272.
Inactivation of Bacillus subtilis spores by supercritical CO2 treatment S. Spilimbergoa,*, A. Bertuccoa, F.M. Laurob, G. Bertolonib a University of Padova, Department of Chemical Engineering via Marzolo 9, 35131 Padova, Italy University of Padova, Department of Histology, Microbiology and Medical Biotechnologies, Via Gabelli 63, 35131 Padova, Italy
b
Abstract Bacillus subtilis spores were suspended in saline solution (107 cfuyml) and treated by both conventional heating and CO2 batch treatment at an operating pressure in the range of 70–150 bar under identical temperature conditions. Temperatures tested were in the range of 36–75 8C. Survival curves indicated significantly higher lethality when spores were treated with supercritical CO2 (SC-CO2) rather than with heating alone. These results appear particularly evident at 60 8C, a temperature at which conventional heating gave no spore-inactivation after a treating time as long as 24 h, whereas a 6 h SC-CO2 treatment led to complete sterilization. At 75 8C spores were partially killed with conventional heating but a treatment of 2 with SC-CO2 hours assured total inactivation. It is concluded that spore-inactivation during SC-CO2 treatment was only in part due to thermal effect (at the higher temperature of 75 8C) and there was a significant additional effect caused by CO2 penetration inside the latent bacteria forms. 䊚 2002 Elsevier Science Ltd. All rights reserved. Keywords: Supercritical CO2; Spore-inactivation; Bacillus subtilis Industrial relevance: The present work confirms existing data and adds important insights into the possible inactivation of baterial spores via induction of germination by SC-CO2. This offers, besides high pressure assisted sterilisation, another possible technology to inactivate spores at mild thermal conditions. It also opens the possibility to reduce bacterial counts in conventional extraction processes with SC-CO2.
1. Introduction In food, as well as pharmaceuticals and polymeric materials, microbial-inactivation is a crucial parameter in the safety and shelf-life of the product. The methods commonly used for polymer treatment are steam sterilization, ethylene oxide exposure and g irradiation, while in the case of food processing the most widespread technique is U.H.T. (ultra high temperature) treatment. All these methods assure a satisfactory microbial inactivation, but have a number of side effects (Guidoin et al., 1985; Zhang, Bjursten, Freij-Larson, Kober & Wesslen, 1996; Konig, Ruffieux, Wintermantel & Blasser, 1997). For instance, in the case of thermally or hydrolytically labile polymers, autoclaving can destroy materials, while U.H.T. can cause changes in the organoleptic features of temperature-sensitive substances and the denaturation of vitamins and proteins. This is the reason why alternative and economically convenient *Corresponding author. Tel.: q39-049-8275491; fax: q39-0498275461. E-mail address: [email protected] (S. Spilimbergo).
methods have been studied in the recent years. Among all, we mention high-voltage pulsed electric field (Sitzmann, 1995; Barbosa-Canovas, Pierson, Zhang & Schaffner, 2000), ultra-sound (Sherba, Weigel & O’Brein, 1991; Sala, Burgos, Condon, Lopez & Raso, 1995) and high hydrostatic pressure treatments (Ludwig, Gross, Scigalla & Sojka, 1994; Schreck, & Ludwig, 1997). The use of supercritical CO2 (SC-CO2) has been recently investigated and continues to attract interest. A supercritical fluid is a fluid above its critical temperature (Tc) and critical pressure (Pc), that possesses unique properties such as high density and low viscosity. Carbon dioxide is a non-toxic and inexpensive gas and has been considered in the sterilization of biomaterials (Dillow, Dehghani, Hrkack, Foster & Langer, 1999), of bone allografts (Fages et al., 1998) and of food storages (Quirin, 1988); it was shown that CO2 is effective against bacteria, viruses and insects at different stages of development. Furthermore, since the critical values of pressure and temperature are relatively low (73.8 bar and 31 8C, respectively), the gas is relatively easy to handle under supercritical conditions.
1466-8564/03/$ - see front matter 䊚 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1466-8564(02)00089-9
162
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In previous work, promising results were found when CO2 was used to reduce the activity of a wide range of microorganisms (Spilimbergo, Elvassore & Bertucco, 2002b). On the other hand, inactivation of bacterial spores is crucial in the sterilization of food, polymeric and pharmaceutical materials. Spores are the most resistant form of bacteria: their structure is different and more complex when compared to that of vegetative cells. Dormant bacterial endospores are highly resistant to a number of physical and chemical treatments which are normally considered germicidal (Setlow, 1995). Spores owe some of their resistance to the presence of an outer proteinaceous layer termed spore coat (Gould, 1983; Driks, 1999). This coat protects a dormant spore from enzymes, such as lysozyme (Gould & Hitchins, 1963), from both mechanical (Gould, 1983) and chemical disruption, such as that obtained by hydrogen peroxide (Gould & Hitchins, 1963). Furthermore, spores are strongly dehydrated compared to living cells and are impermeable to most dyes. Spores are characterized by their capacity to survive under extreme conditions such as high and low temperature, high and low pH and high hydrostatic pressure, and can remain latent for many years (Cano & Borucki, 1995). Few works addressing spore inactivation by SC-CO2 can be found in literature. In 1997 Enamoto and coworkers (Enomoto, Nakamura, Hakoda & Amaya, 1997) reduced the survival ratio of a sample of bacterial spores of Bacillus megaterium (108 colony forming units per ml, cfuyml) in physiological solution, to approximately 101 cfuyml with a batch treatment of 30 h at 58 bar and 60 8C. In the same year Ishikawa et al. (1997) achieved a total microbial inactivation of Bacillus subtilis, B. polymyxa and B. cereus (all of them from an initial concentration of 106 cfuyml) with a semi-continuous apparatus, the so-called micro-bubble method, after a treatment of 1 h at 300 bar and a temperature of 50–60 8C. One year later, Ballestra and Cuq (1998), reduced the survival count of B. subtilis spores to approximately 103.5 cfuyml and Byssochlamys fulva ascospores to approximately 101 cfuyml, from an initial suspension of 107 and 105 cfuyml, respectively, with a batch treatment of 1 h, at an operating pressure of 50 bar and a temperature of 80 8C. In 2002, Spilimbergo found that the microbial activity reduction of B. subtilis spores was made possible by coupling the effect of higher temperature (75 8C) and longer treatment time (24 h), and compared this result to that on bacteria species (Spilimbergo, Elvassore & Bertucco, 2002a). From the quotations above it is concluded that the open literature is rather conflicting, foggy and full of gaps as regards spore inactivation by SC-CO2. In this digest, the aim of our work is to better investigate and clarify the lethal effect of SC-CO2 treatment on Bacillus subtilis spores, used as test microorganisms, to compare it with conventional heating, and
to study the kinetics of spore inactivation for both of these sterilization methods. 2. Materials and methods 2.1. Bacterial strains and media Bacillus subtilis var. niger ATCC 9372, a sporeforming Gram-positive bacterium, was selected as a test microorganisms as it is well known to be resistant to heat, radiation, most chemical disinfectants and antibiotics when compared to vegetative bacterial forms. Furthermore, B. subtilis var. niger together with B. stearthermophilus and B. pumilus are currently used as test microorganisms for steam, ethylene oxide, dry and radiation standard sterilization tests in microbiology. The spores were prepared by plating a stock culture on nutrient agar. After an incubation of 24 h at 37 8C the plates were left at room temperature for 4 days. Sporulation was monitored by phase-contrast microscopy. After an almost complete sporulation had occurred, the plates were flooded with 2–3 ml of saline solution (0.9% NaCl) and the surface was gently scraped with a plastic sterile rod. All the contents were recovered in a sterile bottle, vortexed and shocked at 80 8C for 10 min to kill the remaining vegetative cells in the sample. The suspension was then centrifuged for 10 min at 3000=g. The supernatant was discharged and the spore-pellet was re-suspended in saline solution, poured in sterile testtubes and stored at y20 8C until used. The spore concentration was approximately 109 cfuyml. 2.2. Counting Before and after each treatment viable counts of surviving spores were determined by the standard plating technique (Speck, 1976). In addition, as the treated sample is concerned, spore viability has been determined in diluted as well as in undiluted conditions. In order to further check that complete sterilization was achieved, small volumes (1–3 ml) of treated sample were inoculated in liquid medium brain heart infusion (BHI), incubated at 37 8C for 24 h, plated in brain heart agar (BHA), and colonies were counted. The results are expressed as % survival as well as log reduction, log (No yN), where No and N are the numbers of cfuyml before and after the treatment, respectively. All the values (reported in the Results section) are the averages among three different runs. 2.3. CO2 treatment A batch procedure was used for the SC-CO2 treatment. The sterilization chamber was the same as in a previous work (Spilimbergo et al., 2002a) but it was used in a batch mode.
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Table 1 Inactivation of Bacillus subtilis spores as a function of time during SC-CO2 batch treatments at 36 and 50 8C P (bar)
T (8C)
Time (h)
Log(No yN)**
Survival (%)
75 80 120 80 120
36 36 36 50 50
24 48 48 48 48
0.5 0.9 0.9 1 1
32 13 13 10 10
**
(No yN) is the ratio between the cfuyml before and after the treatment.
The sterile vessel was filled with 10 ml of sporesuspension previously prepared, diluted to 107 cfuyml, the screw plugs were sealed under sterile conditions and all the set-up was heated by an electrical resistance up to the set point value (from 36 to 75 8C). When the temperature reached the desired value, the CO2-pump was switched on, the inlet valve was opened and the outlet valve closed, letting CO2 bubble through the vessel. When the operating pressure was reached, the pump was switched off and the inlet valve was kept completely closed for all the treatment time. In order to control the temperature during the experiment, the highpressure vessel, which is thermally insulated, is equipped with a resistance temperature probe, Pt 100 V, located inside, a temperature on-off control, and an electric resistance. After carrying out the experiment, the outlet valve was heated to avoid freezing during the venting of CO2 and then opened until a complete discharge of pressure was attained. As regards the cycled pressure treatments, the sample (at a concentration of 107 cfuyml) was subjected to cycling variations in pressure, up to 150 bar, and the amplitude of pressure variation, DP, was changed between 80 and 110 bar. When the pressure reached its maximum value, the system was maintained under continuous flow of CO2 for a pre-set treatment time. In order to select the best operating conditions, different variables, such as the number of cycles per hour, the time at which the pressure was kept at its maximum value, the temperature and DP were varied during different runs of experiments. As temperature treatment is concerned, a tube containing 50 ml of physiological solution with 107 cfuyml
Fig. 1. Survival of Bacillus subtilis spores as a function of time, at 75 8C during SC-CO2 treatment (s) and conventional heating (h).
of B. subtilis spores was kept at the desired temperature (60 or 75 8C) by a thermostatic water-bath (Ocras Zambelli s.a.s, Torino, Italy). At fixed times a sample was taken from the tube in order to measure the survival kinetics. After any type of treatment, the same procedure was followed: the solution of the treated spore-suspension was shocked in a water-bath for 10 min in order to kill the vegetative cells and activate the spores, spread onto BHA plates and, after an incubation at 37 8C for 24 h, the surviving colonies were counted. 3. Results The result of the survival assays (Table 1) show that B. subtilis spores were resistant to SC-CO2 treatment. Even a prolonged treatment (48 h) with a pressure up to 120 bar and temperatures of 50 8C did not significantly affect the viable cell survival. Disappointing results were obtained even with cycled pressure treatments at 36 and 50 8C, carried out for different treatment times (Table 2). Therefore, the temperature was increased. Suspensions (107 cfuyml) were treated with SC-CO2 for 2–6 h at a pressure of 70 bar and a temperature of 75 8C. At the same time, the effect of temperature alone was observed under atmospheric conditions.
Table 2 Inactivation of Bacillus subtilis spores as a function of time during SC-CO2 pressure-cycled treatment at different temperatures and at different number of cycles per hour Cyclesyh
T (8C)
Time* (min)
DP (bar)
Treatment time (min)
Log(No yN)**
Survival (%)
20 3 2 6
36 50 50 50
1 15 20 4
80 110 110 110
30 60 60 60
2 0.8 1 1.1
1 16 10 8
*
Refers to the time at which the pressure is at its maximum value. (No yN) is the ratio between the cfuyml before and after the treatment.
**
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Fig. 2. Survival of Bacillus subtilis spores as a function of time, at 60 8C during SC-CO2 treatment (s) and conventional heating (h).
The results show that after as little as 2 h of SCCO2 treatment at 75 8C, there is a complete sterilization of the spore suspension (Fig. 1). This effect is mainly due to the action of CO2 since spores treated for the same time (2 h) at a temperature of 75 8C show a 100% survival, and slowly decline only after longer incubations (up to 6 h). Considering that high temperatures can be deleterious to the characteristics of the food or the pharmaceutical compound that has to be sterilized, CO2 treatment was carried out at the temperature of 60 8C and at an operating pressure of 90 bar. The results shown in Fig. 2 point out that temperature alone does not affect spore viability up to 24 h in atmospheric conditions. On the contrary, a treatment at the same temperature with SC-CO2 completely sterilizes the spore suspension after 6 h with a linear decrease in cfuyml counts during the first 5 h. 4. Discussion It is noteworthy that temperature has a primary role in the efficiency of CO2 microbial inactivation treatment. Neither cycled pressure treatments at the same temperatures (35–50 8C) nor batch treatments seem to be effective for spore inactivation even though the kills are higher for cycled treatments (Tables 1 and 2). In this situation, a sudden change of pressure is likely to induce partial activation of the dormant spores, leading to cell modifications which make them more sensitive to metabolic disturbances induced by CO2 within their structure. Our results show that, together with a temperature not lower than 60 8C, SC-CO2 treatment exhibits a clear killing effect against bacterial spores. In fact, after a treatment time of 2 h with SC-CO2 at 75 8C all the latent spores were inactivated, whereas a treating time of 6 h with a conventional heating was able to inactivate them only partially.
The results obtained at 60 8C clearly indicate that CO2 has a fundamental role in spore inactivation. No inactivation was detected during 24 h in the thermostatic bath, while after 6 h of SC-CO2 treatment at the same temperature all the spores were killed. Our data show that the presence of pressurized CO2 inside the sample enhanced the lethal thermal effect to bacterial spores. This effect is much more evident at 60 8C, when the temperature alone is not able to inactivate spores at all. With respect to the bacterial inactivation mechanism, many different hypotheses have already been discussed (Haas et al., 1989; Lin, Zhiying & Chen, 1993; Nakamura, Enomoto, Fukushima, Nagai & Hakoda, 1994; Enomoto, Nakamura and Hakoda, 1997; Dillow et al., 1999). We think that a synergistic effect of intracellular pH decrease and modification of membrane permeability is the most probable one (Spilimbergo et al., 2002a). The same hypothesis cannot be applied for spore inactivation, as the spore core is extremely dehydratated (Ishihara, Saito & Takano, 1999). Even though CO2 dissolves in water and forms carbonic acid, thus lowering the extracellular pH, the gas cannot penetrate inside these latent forms as no water is present within their structure. We think that the presence of CO2, coupled with a mild heating, can be sufficient to promote spore activation and germination. It is known that germination is activated by a sudden change in environmental condition such as temperature, acidification, pressure drop. It is likely that the effect of CO2 acidification, added to the effect of temperature, is sufficient to promote activation, so that spores recruit water and modify their structure thus becoming more sensitive to CO2 treatment. At this stage CO2 can easily penetrate through the membrane, causing an increase of its fluidity and permeability and destroying its essential domains (Isenschmid, Marison & von Stockar, 1995). At the same time, it is feasible that under pressure, an excess of CO2 passes through the altered membrane and lowers the internal pH by exceeding the buffer capacity of the cell pool enough to collapse the pH gradient and the proton motive force across the membrane (Hong and Pyun, 1999). 5. Conclusions The presence of SC-CO2, in association with mild heating, can yield B. subtilis spore-sterilization efficiency. With a treating time of 6 h at an operating pressure of 90 bar at 60 8C a complete B. subtilis inactivation can be achieved. The same conditions without the presence of supercritical fluid gave no inactivation of B. subtilis at all. Our data indicate the possibility to exploit such a method to sterilize foodstuffs, temperature-sensitive materials, such as pharmaceuticals or polymeric mate-
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