Inactivation of Bacillus Subtilis Spores with

European Journal of Scientific Research ISSN 1450-216X Vol.40 No.1 (2010), pp.6 -14 © EuroJournals Publishing, Inc. 2010 http://www.eurojournals.com/ejsr.htm

Inactivation of Bacillus Subtilis Spores with Pressurized CO2 and Influence of O2, N2O and CH3CH2OH on its Sporicidal Activity Oulé Mathias Faculty of Science, University College of Saint-Boniface 200 Cathédrale Ave, Winnipeg MB, Canada R2H 0H7 E-mail: [email protected] Tano Kablan UFR of Food Science and Technology, Abobo Adjamé University, Abidjan Cote d’Ivoire, 02 BP 801 Abidjan 02 E-mail: [email protected] Arul Joseph Department of Food and Nutrition Science Pavillon Paul-Comtois Laval University Québec, Canada. G1K 7P4 E-mail: [email protected] Abstract Bacillus Subtilis spores were treated with vapour CO2 (V-CO2), liquid CO2 (L-CO2) and supercritical CO2 (SC-CO2) in the presence of 3%O2, 5%N2O and 2%CH3CH2OH as sensitizers. Exposure of the spores to CO2 under pressures ranging from 2.5 to 25 MPa and at temperatures between 25ºC and 70ºC was performed in a double-walled reactor of 1 L capacity equipped with a magnetic stirring system. The effect of CO2 varied with its physical state. V-CO2 and L-CO2 did not reduce the number of spores even in the presence of sensitizers. However, SC-CO2 provoked irreversible inactivation of the spores. Adding 2%CH3CH2OH and 5%N2O to the CO2 increased the inactivation of the spores by 100% and 30% respectively. The presence of 3%O2 in V-CO2, L-CO2 or SC-CO2 did not influence the effect of the treatments on the spores. Keywords: Bacillus subtilis, spores, CO2 under pressure, sensitizers and sporicidal activity

1. Introduction Bacterial spores are of considerable interest due to their remarkable resistance to physical agents and regular antiseptics. Their tolerance to heat and the fact that they can survive a number of years in a dried state are of great importance in medicine as well as in food preservation. Several studies have been done on bacterial vegetative cells and spore inactivation using pressurized CO2. One of the interesting properties of CO2 is that its critical coordinates are low (7.4

Inactivation of Bacillus Subtilis Spores with Pressurized CO2 and Influence of O2, N2O and CH3CH2OH on its Sporicidal Activity

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MPa and 31.06ºC). At this point, the CO2 gas with a density of 0.47g/ml is impossible to liquefy regardless of the increase in pressure. Above the critical point (pressures > 7.4 MPa and temperatures > 31.06ºC), CO2 in the system is in its supercritical state (Enviroguide, 1984). Because of its antimicrobial properties, supercritical CO2 (SC-CO2) can be used in food preservation. Its microbicidal effect on bacterial vegetative cells has been the focus of many studies (Oulé et al., 2006; Hong and Pyun, 1999; Kamihira et al., 1987; Nakamura et al., 1994; Jones and Greenfield, 1982; Hong et al., 1997; Isenschrnid et al. 1995; Ballestra et al., 1996; Lin et al., 1992; Lin et al., 1994). However, research on the effect of pure CO2 on bacterial spores is limited (Madigan et al., 2002; Driks, 1999). Ballestra and Cuq (1998) reported that no antimicrobial effect was observed during the treatment of B. subtilis spores with pressurized CO2 under 5 MPa at temperatures below 80°C. However, CO2 under 5MPa increased the inactivation of spores at temperature higher than 80ºC. Spilimbergo et al.(2003), also combined heat and SC-CO2 during Bacillus spores inactivation. They concluded that spore inactivation during treatment was only in part due to the thermal effect at the higher temperature of 75 °C and that there was a significant additional effect caused by CO2 penetration inside the latent bacterial forms. Zhang et al., (2006) demonstrated the killing power of SC-CO2 on B. pumilus spores in the presence of trace of additives. They reported that H2O2 is an effective additive, while alcohol and water did not give complete killing. The inactivation of Clostridium sporogenes spores by pressurized CO2 has been studied extensively. Kamhira et al. (1987) treated Aspergillus niger conidia and B. subtilis and B. stearothermophilus spores with pressurized CO2, using 2%CH3CH2OH and 0.5%acid as entrainers. They noticed no effect from the treatment with pure CO2 on spores. The addition of entrainers increased the sterilising power of pressurized CO2 on conidia and spores. Haas et al. (1989) reported the inactivation of C. sporogenes spores by CO2 at a pressure of 6.8 MPa and at ambient temperatures. Ishikawa et al. (1997) reported a 3-log reduction of various Bacillus species after a 30min. treatment with CO2 at a pressure of 30 MPa and at 40ºC. At 45, 50 and 55ºC and under the same pressure, they reduced by 6-log the population of B. polymixa, B. cereus and B. subtilis spores respectively. Other studies on the effect of pressurized CO2 against bacterial spores including Bacillus cereus (Dillow et al., 1999), Bacillus megaterium (Enomoto et al., 1997a, b), Geobacillus stearothermophilus (Watanabe et al., 2003), have been done. The objective of this work was to inactivate B. subtilis spores with pressurized CO2 in the presence of 2%CH3CH2OH, 5%N2O and 3%O2 as sensitizers to increase the sterilizing activity.

2. Materials and Methods 2.1. Microorganism and Culture Media The microorganism used in the course of this work was Bacillus subtilis ATCC 6051obtained from the Department of Biochemistry of Laval University (Quebec, Canada). The culture medium was nutrient broth supplemented with yeast extract (0.3%; w/v) and glucose (0.5%; w/v). The bacterial count was achieved on nutrient agar supplemented in the same way. 2.2. Spores Preparation The Bacillus subtilis ATCC 6051 spores used were prepared using the standard method described by Kamihira et al. (1987). Nutrient-agar dishes were inoculated with a series of dilutions of a nutrient broth B. subtilis culture and incubated at 37ºC for 2 days. The cells were collected by and suspended in distilled water and centrifuged for 10 min. at 12,000 x g. They were suspended in 10 ml of distilled water and incubated at 70ºC for 10 minutes to eliminate vegetative cells. The spores were then washed by centrifugation in sterile distilled water for 10 min. at 12,000 x g before treatment.

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Oulé Mathias, Tano Kablan and Arul Joseph

2.3. Apparatus Figure 1 presents the apparatus used for the completion of this work. This apparatus was equipped with an INC Model 29501-3 reactor (Newport Scientific, Jessup, Md., USA) possessing a stainless steel double wall and having a 1 L capacity, an INC Model 46-1387 compressor (Newport Scientific) to allow pressure increases in the reactor, a Model EK16 pump (American Lewa, Hollistan, Mass., USA) to allow the injection of substances into the reactor during pressurization, many transmission valves, a Dyna/Mag Model MM-016 magnetic agitator (Pressure Products Industries, Warminster, Penn., USA), and a CO2 cylinder directly attached to the reactor. A constant temperature was maintained by a Model RMS 20 water bath (MGW Lauda, Königshofen, Germany) joined to the double wall in which the water circulated, controlled by a thermocouple (O.F. Ekland, Cape Coral, Fla., USA) placed inside the reactor and a temperature indicator. The pressure in the reactor was controlled by a manometer and maintained constant with regulation valves. The system was also equipped with piping which permitted the circulation of CO2 from the gas cylinder to the reactor and to the sampler. Samples were collected in detachable 20 mL metallic tubes placed in close proximity to a flame. The contents were then transferred aseptically to sterile 50 mL Erlenmeyer t1asks. The metallic tubes were rinsed with sterile distilled water between samplings, and were washed in distilled water and sterilized in the autoclave after each experiment. Figure 1: Apparatus used for Treatments

Ag P

Va

T

TC

Fi C Va

Re Po CO2 Va

va

H2O Va

Va

Va

Va

H2O S

WB Re, reactor; Ag, agitation; Po, pomp; C, compressor; WB, water bath; P; pressure indicator; T, temperature indicator; S, sampling; Fi, filter; Va, valves.


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2.4. Treatment and Analysis Spores were exposed to V-CO2 under 2.5 MPa at 25ºC, to L-CO2 (12 MPa at 25ºC) and to SC-CO2 (25 MPa at 40, 55 and 70ºC) in the reactor described above. Spore suspensions (250 ml) having approximately an initial count of 106 CFU/ml, were aseptically placed in the reactor. The blends of compressed gases (CO2 + 5%N2O and CO2 + 3%O2), obtained from the Praxair company (Vanier, Québec, Canada), were used to treat spores suspensions. With the pump described above, 2%CH3CH2OH was injected into the reactor during CO2 pressurization. The number of viable cells after treatment was determined by plating 0.1 ml of the samples on nutrient agar incubated at 30°C for 2 days. Microbial survival was expressed as the log10 of the ratio between the number of viable cells after treatment and the number of cells initially present (log10[N/No]). The results are statistically examined as mean values by one-way analysis of variance. Reported D-values and lags (L) corresponding to the time during which the number of cells remained constant before inactivation are averages of three experiments.

3. Results and Discussion 3.1. Inactivation of Bacillus subtilis spores with CO2 under a Pressure of 25 Mpa As shown in the first 2 columns of Table 1, the spores strongly resisted to the treatments in conditions of pressure and temperature where CO2 was in its liquid or vapour phase. Even after 120 min. of treatment in the presence of sensitizers, V-CO2 and L-CO2 did not have any effect on spores, and their number remained constant. Bacterial spores are known to be resistant to common antimicrobial physicochemical agents. The resistance of spores to CO2 treatment at temperatures lower than 50ºC has also been observed, but has not yet been clearly elucidated (Ballestra and. Cuq, 1998; Spilimbergo et al., 2003). However, it is possible to predict that their resistance is due to two predominant factors: firstly, their remarkable impermeability, owing to the relatively thick layers covering the central area, and secondly, the dehydrated state of their components. In fact, it has been established that CO2 penetrates cells by diffusion across the membrane, CO2 being rather soluble in phospholipids (Oulé et al., 2006). The cytoplasmic membrane of spores is very thin and its phospholipid-content is low. Moreover, this membrane is surrounded by concentric layers rich in peptidoglycan and basic proteins. Diffusion of CO2 in spores to cause their inactivation is consequently rather limited, and explains their resistance to the L-CO2 and V-CO2 treatments. On the other hand, the dehydrated state of the spores should contribute largely to their resistance since the antimicrobial effect of CO2 requires water (Kamihira et al., 1987; Nakamura et al., 1994). Moreover, the solubilization of the basic proteins would increase the sensitivity of spores, rendering them more susceptible to heat in an acidic medium. However, the acidification by carbonic acid formation was not significant in the treatment with CO2. Carbonic acid represented only 0.16% of the aqueous CO2 (Daniels et al., 1985) and it is a relatively weak acid (pK = 6.3) with low dissociation (95% being in the form of non-dissociated form) at the experimental pH (pH5). Consequently, it could not have caused a significant drop in pH to affect the basic proteins and render spores more susceptible to the treatment.

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Table 1:

100%CO2

3%O2

5%N2O

2%ETOH

Influence of CO2 physical state and of the sensitizers on sporicidal effect of pressurized CO2.

L D R L D R F L D R F L D R F

V-CO2 (2.5MPa/25ºC) -

L-CO2 (12MPa/25ºC) -

SC-CO2 (25MPa/40ºC) 20 ± 0.0 20 ± 0.0 2 ±0.44 20 ± 0.5 20 ± 0.5 0 ± 0.0 1 ± 0.0 15 ± 0.0 15 ± 2.24 2.7 ± 0.26 1.33 ± 0.06 5 ± 0.0 10 ± 2.5 6 ± 0.0 2 ± 1.13

SC-CO2 (25MPa/55ºC) 10 ± 0.0 14 ± 1.0 4 ± 0.0 10 ± 0.0 12.7 ± 1.86 0 ± 0.0 1 ± 0.0 10 ± 0.0 10 ± 0.0 5.3 ± 0.81 1.4 ± 0.14 0 ± 0.0 6.3 ± 0.81 6 ± 0.0 2 ± 0.15

SC-CO2 (25MPa/60ºC) 5 ± 0.0 10 ± 0.0 4.9 ± 0.26 5 ± 0.53 10 ± 0.0 0 ± 0.0 1 ± 0.0 5 ± 0.0 7.6 ± 1.13 6 ± 0.0 1.31 ± 0.17 0 ± 0.0 5 ± 0.0 6 ± 0.0 2 ± 0.0

SC-CO2 (25MPa/70ºC) 0 ± 0.0 7 ± 0.18 6 ± 0.0 0 ± 0.0 7.1 ± 0.79 0 ± 0.0 1 ± 0.0 0± 0.0 5.4 ± 0.53 6 ± 0.0 1.29 ± 0.11 0 ± 0.0 3.6 ± 0.3 6 ± 0.0 2 ± 0.9

L = Latency time in minutes, D = D Value in minutes, R = Reduction of spores population expressed in log, f = Modification Factor = DCO2/Dsensitizer

However, in the supercritical state, CO2 caused the inactivation of spores and the presence of sensitizers increased this sterilizing power. The inactivation of the spores by CO2 without sensitizers under a pressure of 25 MPa increased with temperature (Fig.2). The D value and the latency time (L) decreased as the temperature increased (Table 1). At 40, 55, 60 and 70ºC, the latency time corresponded to 20, 10, 5 and 0 min. respectively; D-values corresponded respectively to 20, 14, 10 and 7 min. and there was a reduction of 2.7, 3.9, 4.9 and 6-log after 60 min. of the treatment. Under a pressure of 25 MPa and at 70ºC, all the spores were destroyed after 35 min. of the treatment, indicating that in highly supercritical conditions, CO2 can cause irreversible damage to spores. Our results confirmed those of Ishikawa et al. (1997) who reported a 3-log reduction of Bacillus spore populations after 30 min. of treatment with CO2 under a pressure of 30 MPa at 40ºC. At 45, 50 and 55ºC under the same pressure, they have obtained a 6-log reduction of the spore populations of B. Polymiaxa, B. cereus and B. subtilis respectively. The increase in temperature can cause a drop in the pH since water's dissociation constant (Kw) rises with the pressure and the temperature. The drop in pH can thus affect the basic proteins and accentuate the effect of the treatment on the spores. At 25 MPa and 70ºC, Kw and pKw of water correspond to 1.8113x10-13 and 12.742 respectively with a 0.63 unit drop in pH (Marshall and Frank, 1981). However, the pH drop alone could not explain the remarkable effect of the treatment on spores. Sporicidal activity of the treatment would principally be due to the pressure of CO2. Ballestra and Cuq (1998) reported that spores were first activated then killed thermally, SC-CO2 and water aiding in thermal activation and spore destruction at 5 MPa and 80 to 90ºC. Zhang et al., (2006) also indicated that the treatment of B. pumulis spores with CO2 and water under 27.5 MPa at 80ºC may activate spores prior to killing. The question is: if spores are first activated, then killed under pressurised CO2, why does the liquid CO2 not kill them? They should have been activated and killed by the liquid CO2 seeing as the bactericidal effect of liquid CO2 on E. coli cells was demonstrated by Oulé et al. (2006). The resistance of spores to liquid CO2 is due to the fact that their membrane is surrounded by many concentric layers, rich in peptidoglycan and basic proteins. The penetration of liquid CO2 in the spores is highly limited. However, in supercritical conditions, CO2 could cause a drop in the pH levels which would cause the inactivation of basic proteins. Also, due to the lower diffusion coefficient, the supercritical CO2 could penetrate the spores and carry out its sporicidal activity.


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Figure 2: Inactivation of Bacillus subtilis spores with CO2 under a pressure of 25 Mpa

3.2. Influence of Sensitizers on Sporicidal Activity of SC-CO2 The pressure applied during the treatment was 25 MPa (Fig. 3). The presence of O2 in the CO2 did not enhance the effect of the treatment on the spores. Latency times and D-values remained almost identical (Fig. 3). The suggestion that hydrostatic pressure can sensitize all microorganisms to O2 toxicity (ZoBell and Hittle, 1967) has not been verified here. Marquis and Thom (1992) have also observed that, in presence of O2 concentrations of 8 to 48 μg/ml, pressures increased up to 40 MPa did not increase the inactivation of E. coli and S. cerevisiae. On the other land, the addition of 5%N2O to CO2 permitted the reduction of the D-value, indicating that its presence accelerated spores inactivation. It generated an increase in the bactericidal activity of CO2 of about 30%. At 40ºC, D-value decreased from 20 min. (CO2 without sensitizers) to 15 min., which corresponds to an increase by a factor of 1.3 and a 30% increase in the bactericidal activity of CO2. The reduction of the number of viable spores intensified from 2-log (CO2 without sensitizers) to 2.7-log after 60 min. of treatment (Table 1). At 55ºC, the presence of N2O caused a 5.3-log reduction rather than the 4-log reduction by the CO2 without sensitizers, and a decrease in D-value from 13 to 10 min., corresponding to a 30% increase in the effect of the treatment. At 70ºC, the presence of N2O produced a 6-log reduction of the number of viable spores and a decrease of D-value from 7 min. (CO2 without sensitizers) to 5.4 min., which corresponds to a 30% increase in the effect of the treatment. For each experimental temperature, N2O increased the bactericidal activity of pressurized CO2 by about 30%, indicating that temperature did not influence the effect of the treatment. The mechanism by which N2O inhibits microorganism growth is still not completely understood. The best known effect of N2O is its narcotic effect on human beings at atmospheric pressure. It is an anesthetic gas which causes microtubules rupture in eukaryotic cells. It is also a strong oxidizing agent capable of provoking cell wall oxidization by liberation of free radicals . (N2O + H2O → N2 + OH- + OH ) and subsequently microbial inhibition. Moreover, these free radicals can affect lipid, nucleic acid and protein integrity (Marquis and Thom, 1992). Thom and Marquis (1984) have observed that N2O inhibits E. coli growth by provoking damage due to a process of

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cellular oxidation. The inhibition of microbial growth was probably not linked to its narcotic (except perhaps in the case of spore germination) or its anaesthetic action. Marquis and Thom (1992) have also observed inhibition of E. coli growth when N2O pressures range from 1 to 3 MPa and inactivation of other bacteria at 5 MPa. N2O provokes stress in microorganisms by acting on soxR and soxS, two genes governing the synthesis of stress proteins which fight against oxidation stress (Pelmont, 1993). Laskaris and Marquis (1992) have also demonstrated that pressurized N2O inhibits bacterial spore germination and induces, in the presence of O2, complete destruction of E. coli cells. Figure 3: Inactivation of Bacillus subtilis spores with CO2 under a pressure of 25 MPa at 40ºC in presence of sensitisers

The injection of 2%CH3CH2OH with CO2 under 25 MPa, compared to O2 or N2O, caused a high degree of spore inactivation. The reduction in latency time, in D-valour and in the number of spores was accentuated by its presence. At 40ºC, latency time and D-value passed respectively from 20 min. and 20 min. in 100% CO2 to 5 min. and 10 min in the presence of 2%CH3CH2OH; at 55ºC, they passed respectively from 10 min. and 13 min. in 100% CO2 to 0 min. and 6.3 min. in the presence of 2%CH3CH2OH, and at 60ºC, from 5 min. and 10 min. to 0min. and 6 min. At each temperature, all spores were inactivated after 60 min. of the treatment. CO2 with 2%CH3CH2OH provoked a reduction by half of the D-values, indicating that the level of inactivation was twice as high as when the cells were exposed to CO2 without sensitizers and under the same conditions. This corresponds to a 100% increase of the effect of CO2. Kamihira et al.(1987) have reported that CH3CH2OH possesses a potential capacity to reinforce the bactericidal effect of CO2. The increase in bactericidal activity in the presence of CH3CH2OH can probably be attributed to a synergistic action between CO2 and CH3CH2OH. As CO2 is a small hydrophobic molecule able to cross the membrane, CH3CH2OH is an amphilic molecule, hence capable of bonding with membrane proteins to cause alterations in proteinlipid interactions. This would contribute to membrane destabilization. CH3CH2OH can also cross the membrane and contribute to the denaturation of intracellular enzymes. The bactericidal effect of CO2 with CH3CH2OH could be due to its ability to modify the physical properties of supercritical CO2 by reducing its density and its viscosity, and by increasing its diffusion coefficient and its solubilizing power (Brunner and Peter, 1982). Consequently, by altering the physical properties of CO2, CH3CH2OH probably accelerated its penetration in the cells thereby reducing D-values and latency times.

4. Conclusion Bacterial spores possess various mechanisms aids in their resistance to physical agents and antiseptics. Their inactivation is still problematic and leads to difficulties in food conservation and nutrition. To


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destroy them effectively, sterilizing the sample for 10 minutes at 120ºC with humid heat (autoclave) proves a very effective measure. It is however impossible to consider this type of treatment for certain foods because of their heat-sensitive qualities. This study showed that the effect of CO2 varied with its physical state. V-CO2 and L-CO2 did not reduce the number of spores even in the presence of sensitizers. However, SC-CO2 provoked irreversible inactivation of the spores. Adding 2%CH3CH2OH and 5%N2O to the CO2 increased the inactivation of the spores by 100% and 30% respectively. Sterilization with highly supercritical CO2 and the exploration of methods to reinforce its bactericidal activity could be an effective technique used to destroy bacterial spores.

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