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Modeling the influence of electron beam irradiation on the heat resistance of Bacillus cereus spores
ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 23 (2006) 367–371 www.elsevier.com/locate/fm
Modeling the influence of electron beam irradiation on the heat resistance of Bacillus cereus spores M. Valeroa,, J.A. Sarrı´ asa, D. A´lvarezb, M.C. Salmero´na a
Departamento de Produccio´n Vegetal y Microbiologı´a, Escuela Po1ite´cnica Superior de Orihue1a, Universidad Miguel Herna´ndez - Campus de Orihue1a, Carretera de Benie1, km 3.2, 03312-Orihue1a, Alicante, Spain b Departamento de Tecnologı´a de los Alimentos, Nutricio´n y Bromatologı´a, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071-Murcia, Spain Received 14 February 2005; received in revised form 10 May 2005; accepted 10 May 2005 Available online 6 July 2005
Abstract The effect of electron beam irradiation (EBI) on Bacillus cereus spore heat resistance was investigated. Irradiation with accelerated electrons had an important heat-sensitizing effect on distilled-water spore suspensions. After irradiation doses of 1.3, 3.1, or 5.7 kGy followed by heating at 90 1C, calculated D90-values for strains Escuela Polite´cnica Superior de Orihuela (EPSO)-41WR and EPSO50UR were reduced more than 1.3, 2.4, and 4.6 times, respectively. Plots of calculated log DT-values versus irradiation doses (1.3, 3.1, and 5.7 kGy) yielded straight parallel lines for the 85–100 1C heating temperature range, which made it possible to develop an equation to predict the changes in heat sensitivity of B. cereus spores that occurred with changing irradiation dose. Radiationinduced heat-sensitivity was characterized by a zEBI-value which was determined as the irradiation dose that should be required to reduce the decimal reduction time (DT ) by one log10 cycle when log10 DT was plotted against irradiation treatment. A model is proposed to describe the influence of a pre-irradiation treatment with electron beams followed by heating on the heat resistance of B. cereus spores. This study also suggests the potential use of EBI followed by heating for food preservation. r 2005 Elsevier Ltd. All rights reserved. Keywords: Irradiation; Electron beams; Heat resistance; Radiation-induced heat-sensitivity; Bacillus cereus; Bacteria1 spores
1. Introduction Micro-organisms highly resistant to heat or radiation pose problems in food processing because of the severe treatment required to eliminate them. Such severe treatment may adversely affect the quality of the processed food as a result of the induced changes in organoleptic characteristics and loss of nutrients. Factors affecting the heat resistance of bacteria to food preservation processes include the type of bacteria and the nature of the suspension media. Spores and some vegetative cells are more resistant to heat in lipids Corresponding author. Tel.: +34 966749683; fax: +34 966749619.
E-mail addresses: [email protected] (M. Valero), [email protected] (D. A´lvarez). 0740-0020/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2005.05.006
than in aqueous suspension media. In both systems, however, a pre-irradiation treatment followed by heating has been found to have important synergistic effects on the destruction of bacterial spores (Farkas, 1990). Therefore, the heat resistance of micro-organisms can be reduced by substerilizing doses of electron beams, X-rays or g-rays. This heat-sensitizing effect of ionizing radiation has been demonstrated in aqueous media or foods for Bacillus cereus (Kan et al., 1957; De Lara et al., 2002), B. subtilis (Grecz et al., 1981; De Lara et al., 2002), Clostridium sporogenes (Kan et al., 1957; Shamsuzzaman, 1988; Shamsuzzaman et al., 1990), C. perfringens (Gombas and Gomez, 1978), C. botulinum (Kempe, 1955) and Listeria monocytogenes (Shamsuzzaman et al., 1992). Radiation-induced heat-sensitivity in C. sporogenes spores has been also reported in vegetable
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M. Valero et al. / Food Microbiology 23 (2006) 367–371
oils and various animal fats (Shamsuzzaman and Lucht, 1993). B. cereus has been recognized as a cause of foodpoisoning for more than 30 years. It causes diarrheal as well as emetic foodborne illness syndromes. This microorganism is a Gram-positive, rod-shaped, facultative aerobic spore former that is widely distributed in natural and commercial products due to the strong resistance of its spores to physical and chemical agents. Although there is information on the heat-sensitizing effects of electron beam irradiation (EBI) on spores of B. cereus strain INRA AVZ421 (De Lara et al., 2002) isolated from cooked chilled foods containing vegetables, the aim of this paper is to study the heat resistance of spores of two rice-isolated B. cereus, Escuela Polite´cnica Superior de Orihuela (EPSO)-41WR (white rice) and EPSO-50UR (unhusked rice), characterized by our laboratory (Sarrı´ as et al., 2002), after different irradiation treatments over the temperature range 85–100 1C. The influence of EBI on the heat resistance is also characterized by a zEBI-value and a model is proposed to describe the radiation-induced heat-sensitivity in B. cereus spores.
2. Material and methods 2.1. Bacterial strains, spore production and preparation of spore suspensions B. cereus strains EPSO-41WR and -50UR were used in this study. They were isolated from Spanish raw rice (WR and UR), confirmed by ISO 7932: 1993(F) procedure, and further characterized by API 50CH and API 20E strips, using APILAB Plus software V3.2.2 Version B 01.93 (bioMe´rieux, Marcy l’Etoile, France), and supplementary tests of motility, oxidase activity and enterotoxin production (Sarrı´ as et al., 2002). Spores were produced on fortified nutrient agar (FNA) (Mazas et al., 1995), at 30 1C for 4 days, harvested, centrifuged, resuspended in distilled water and stored at 4 1C until use as described by Valero et al. (2000). For each strain, viability of heat-shocked (80 1C for 10 min) spore suspensions was estimated by spread plating on plate count agar (PCA, Scharlau Chemie, S.A., Barcelona, Spain) at 30 1C for 24 h. 2.2. Irradiation treatments Irradiation was carried out at room temperature in a 10-MeV circular electron accelerator (Rhodotron) localized in the irradiation plant of IONMED ESTERILIZACIO´N, S.A. (Taranco´n, Cuenca, Spain) as described by Sarrı´ as et al. (2003). For irradiation treatment, distilled-water spore suspensions of the two B. cereus strains were dispensed in 1 ml volumes containing
between 106 and 107 spores ml1 into sterile 5-ml glass vials. Samples were exposed to electron beams at programmed irradiation doses of 1, 3, and 5 kGy. The absorbed irradiation doses were measured using cellulose triacetate (CTA) which were attached to the surface of the vials. The irradiation dosimetry revealed that the real doses applied were 1.3, 3.1, and 5.7 kGy. 2.3. Determination of spore heat resistance All heat treatments, applied before and after an irradiation treatment, were performed in 10 ml microhematocrite capillary tubes (Vitrex, Modulohm A/S, Herlev, Denmark) which ensure uniform heat transmission (Ferna´ndez et al., 1999). Capillary tubes were filled with irradiated and non-irradiated spore suspensions in sterilized water. These were centrally injected into glass capillary tubes as described by Valero et al. (2002). Three series of 6–8 capillary tubes were filled from each condition and strain assayed. They were submerged in a stirred oil bath with immersion circulator HAAKE DC5 (Gebru¨der HAAKE GmbH, Karlsruhe, Germany) at 85, 90, 95, or 100 1C constant temperature. Subsequently, tubes were removed at regular intervals and cooled in ice-water, washed in soap solution, rinsed in distilled water and immersed in ethanol. Spores surviving heat treatments were recovered by incubation on PCA at 30 1C for 24 h. All experiments were performed in triplicate. 2.4. DT and z-values calculation The log10 numbers of surviving spores were plotted against time and DT-values were calculated from the slope of the linear phase of the spore destruction as the time in minutes needed to decreased the population one log10 cycle (ICMSF, 1980). The analysis of variance for DT-values by irradiated and non-irradiated spores after heating at each temperature and a multiple comparison procedure of means (Fisher’s least significant difference, LSD) were performed using Statgraphicss Plus for Windows 3.0 (Statistical Graphic Corp. and Graphic Software Systems Inc., Rockville, Maryland, USA). z-Values were determined as the increase in temperature required to reduce the decimal reduction time (DT ) by one log10 cycle when log10 DT was plotted against temperature. To determine the radiation dose required to reduce DT by one log10 cycle, logarithms of the DT-value means were plotted against the corresponding irradiation dose. Data points were fitted by the method of least-squares, and the inverse negative of the slope of the regression line was taken as the zEBI-value that characterized the effect of irradiation on the heat resistance of B. cereus spores.
ARTICLE IN PRESS M. Valero et al. / Food Microbiology 23 (2006) 367–371
3. Results 3.1. Heat resistance Fig. 1 shows survival curves at 95 1C obtained before and after an irradiation treatment of 3.1 kGy. After irradiation, both B. cereus spore suspensions tested, EPSO-4lWR (Fig. 1A) and EPSO-50UR (Fig. 1B), showed significant decrease in heat resistance. The mean DT-values7S.D. at 85, 90, 95, and 100 1C determined for spores of the two strains exposed to different irradiation doses are shown in Table 1. The z-values obtained for spores of the two B. cereus strains in the temperature range and irradiation doses tested are listed in Table 2. For a given strain, these values are very similar regardless of the irradiation treatment applied. High determination coefficients (R2 ) were obtained for all cases. 3.2. Influence of irradiation on the thermal resistance of B. cereus spores Fig. 2 illustrates the influence of irradiation of distilled-water spore suspensions on the DT-values for 6
Log CFU mL-1
5 4 3 2 1 0 0
2
4
6
8
10
12
Time (min)
(A)
7
Log CFU mL-1
6 5 4 3 2 1 0 0 (B)
2
4
6 Time (min)
8
10
12
Fig. 1. Survival curves of spores of B. cereus strains (A) EPSO-41WR and (B) EPSO-50UR heated at 95 1C in distilled water before (open symbols, &) and after (closed symbols, m) an irradiation treatment of 3.1 kGy.
369
B. cereus spores. The plots of log DT-values versus irradiation doses at heating temperature yielded straight lines for the range studied. As Fig. 2A and B shows, all straight lines are basically parallel. At a given temperature, the relation between the DT-values and the irradiation doses shows that the DT-values decrease when the irradiation doses increase. The effect of irradiation on the heat resistance of spores could be characterized and quantified by a zEBI-value which leads to a 10-fold reduction of DT-value. For each bacterial strain and heating temperature the zEBI-value was determined and shown in Table 3.
4. Discussion DT and z-values obtained in this study over the temperature range 85–100 1C (Tables 1 and 2) are in agreement with those published previously by us (Sarrı´ as et al., 2002). Strain EPSO-41WR, with D90values ranging from 20.38 to 23.26 min, was much more heat resistant than the other strain assayed. D10-values have been used to characterize radiation resistance of B. cereus spore suspensions for irradiation doses between 0 and 7 kGy (Sarrı´ as et al., 2002). Variation in radiation resistance observed among the two isolates tested here was lower than that found in heat resistance. In fact, no statistically significant differences in radiation resistance were observed between strains EPSO-41WR and EPSO-50UR of B. cereus. D10-values between 2.48 and 2.75 kGy, with an average of 2.62 kGy, were ca1culated for both strains. D10-values in the range of 1–4 kGy are typical values for bacterial spores, inc1uding B. cereus spores, exposed to radiation (Thayer and Boyd, 1994; Monk et al., 1995; van Gerwen et al., 1999; De Lara et al., 2002). The lack of correlation between heat resistance and radiation resistance of spore suspensions is understandable because the mechanisms involved in the inactivation of bacterial spores by ionizing radiation are very different from those involved in their heat destruction. The most sensitive targets for heat destruction of bacterial spores seem to be core enzymes (Palop et al., 1998) or spore membranes (Setlow, 1995), while inactivation by radiations is mainly based on DNA damage and radiolisis (Farkas, 1989). On the other hand, a pre-irradiation treatment followed by heating was effective in reducing heat resistance of B. cereus spores (Fig. 1). When spore suspensions of strain EPSO-41WR were exposed to 1.3, 3.1, or 5.7 kGy, the average D90-values were reduced 1.3, 2.5, and 4.6 times, respectively (Table 1). After the same irradiation treatments, the D90-value reductions estimated for the strain EPSO-50UR were very similar, namely 1.4-, 2.6-, and 4.9-fold. For each strain and
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Table 1 Mean DT-values (min) of three experiments 7S.D. obtained for spores of two B. cereus strains isolated from raw rice, exposed to different irradiation doses, and heated at several temperatures Strains
Dose (kGy)
D85
D90
D95
D100
EPSO-41WR
0.0 1.3 3.1 5.7
NT NT NT NT
22.0571.49a 16.7870.16b 8.9070.11c 4.7670.27d
5.0270.19a 4.2270.07b 2.1570.11c 1.0970.08d
0.9970.09a 0.7270.02b 0.4870.03c 0.2070.02d
EPSO-50UR
0.0 1.3 3.1 5.7
22.2470.18a 16.3470.28b 9.2270.11c 4.5570.19d
7.4570.07a 5.5070.21b 2.9070.14c 1.5370.06d
2.2870.11a 1.5670.09b 0.8970.04c 0.4670.02d
0.7070.02 NT NT NT
NT, not tested. Values within columns followed by different letter are significantly different (Pp0:05). Table 2 z-Values for spores of two B. cereus strains isolated from raw rice, exposed to different irradiation doses, and heated in distilled water
1.5
Coefficient R2
0.5
EPSO-41WR
0.0 1.3 3.1 5.7
7.42 7.31 7.88 7.26
0.999 0.995 0.999 0.998
0.0 1.3 3.1 5.7
9.96 9.80 9.85 10.05
0.999 0.998 0.999 0.999
EPSO-50UR
irradiation dose, the DT-value reductions were closely related with independence of the heating temperature. The D90-value reductions by irradiation for the strains EPSO-41WR and EPSO-50UR reported here are slightly lower than those published by De Lara et al. (2002) for the B. cereus strain INRA AVZ421. In this case, D90-value was reduced more than three times for spore suspensions in double distilled water. The plots of log DT-values versus irradiation dose yielded straight lines for both strains and each heating temperature (Fig. 2), but in the irradiation range studied, none of them cover one log10 reduction cycle. The high values of calculated zEBI show this clearly (Table 3). These zEBI-values are higher than those irradiation doses applied in this and earlier studies (Sarrı´ as et al., 2003). Doses over 7.5 kGy cause decontamination of husked rice as well as browning and deterioration of its flavor and texture. The relationship between the calculated log D90values of EPSO-41WR spores and the dose of electron beams, e.g., was fitted according to the following linear regression: D90 ¼ 1:35189 0:12004 kGy
ðR2 ¼ 0:993Þ.
In the 85–100 1C heating temperature range studied, the average of the slopes of the lines obtained was 0.1209870.000846 for EPSO-41WR spores, and
Log DT
Dose (kGy)
0 -0.5 -1 0
1
2
0
1
2
(A)
Log DT
z-Value (1C)
Strains
1
(B)
3 Doses (kGy)
4
5
6
3
4
5
6
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 Doses (kGy)
Fig. 2. Log DT (min) versus dose of electron beams (kGy). Determination of zEBI-values for the B. cereus strains (A) EPSO41WR and (B) EPSO-50UR at different temperatures: 85 (\); 90 (’); 95 (J); and 100 1C (m).
0.1228770.000478 for EPSO-50UR spores. As a whole, the average of the slopes was 0.1219370.001204. Therefore, if a DT-value of a B. cereus distilled-water spore suspension at a given temperature after an irradiation dose is known, the DT-value for the same suspension at the same temperature after any other irradiation dose can be deduced by the following equation: DT1 ¼ DT2 100:12ðkGy2kGy1Þ , where DT1 and DT2 are DT-values of spores at the heating temperature ‘T’ after an irradiation treatment ‘kGy1’ or ‘kGy2’, respectively. This equation yields
ARTICLE IN PRESS M. Valero et al. / Food Microbiology 23 (2006) 367–371 Table 3 zEBI-Values for spores of two B. cereus strains isolated from raw rice, exposed to different irradiation doses, and heated at several temperatures Strains
Temperature (1C)
zEBI-Value (kGy)
Coefficient R2
EPSO-41WR
90 95 100
8.33 8.22 8.25
0.993 0.985 0.990
EPSO-50UR
85 90 95
8.15 8.10 8.16
0.998 0.994 0.997
good results in all the irradiation range studied by us (0–6 kGy). We have shown that EBI is able to sensitize to heat spores of two enterotoxigenic B. cereus strains isolated from Spanish rice and the effect of irradiation on the thermal resistance of spores has been characterized by specific zEBI-values (zEBI is the irradiation dose which leads to a 10-fold reduction of DT-value). Evaluation of a greater number of strains representing the two types of foodborne illness is required to confirm the findings, although previous results of Kan et al. (1957) and De Lara et al. (2002) support the heat-sensitizing effect of radiation on the thermal resistance of B. cereus spores. Furthermore, this study indicates the potential use of a treatment combining low electron beam doses and mild heating to inactivate the spore population present in foods. It is well established that the heat resistance of bacterial spores is influenced by environmental conditions during sporulation, the nature and the pH of the heating medium, and the recovery conditions. When heating is used in combination with irradiation, then the critical process factors of this method (dose and dose rate, temperature, pH, O2, dilution effects, etc.) must be taken into account.
Acknowledgements This work has been carried out with financial support from FEDER and it is an integral part of the I+D Project IFD97-1005-C04 coordinated by Centro de Edafologı´ a y Biologı´ a Aplicada del Segura (CEBAS). The authors are grateful to Manuel Giner Pastor for his valuable technical assistance. References De Lara, J., Ferna´ndez, P.S., Periago, P.M., Palop, A., 2002. Irradiation of spores of Bacillus cereus and Bacillus subtilis with electron beams. Innovative Food Sci. Emerging Technol. 3, 379–384. Farkas, J., 1989. Microbiological safety of irradiated foods. Int. J. Food Microbiol. 9, 1–15.
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Farkas, J., 1990. Combination of irradiation with mild heat treatment. Food Control 1, 223–229. Ferna´ndez, A., Salmero´n, M.C., Ferna´ndez, P.S., Martı´ nez, A., 1999. Application of a frequency distribution model to describe the heating inactivation of two strains of Bacillus cereus. Trends Food Sci. Technol. 10, 158–162. Gombas, D.E., Gomez, R.F., 1978. Sensitization of Clostridium perfringens spores to heat by gamma radiation. Appl. Environ. Microbiol. 36, 403–407. Grecz, N., Bruzser, G., Amin, I., 1981. Effect of radiation and heat on bacterial spore DNA. In: Combination Processes in Food Irradiation. Proceedings of a Symposium, Colombo, November 24–28, 1980. International Atomic Energy Agency, Vienna, pp. 3–20. ICMSF, 1980. Microbiological Ecology of Foods. Factors Affecting Life and Death of Microorganisms, vol. 1. Academic Press, San Diego. Kan, B.A., Goldblith, A., Proctor, B.E., 1957. Complementary effect of heat and ionizing radiation. Food Res. 22, 509–518. Kempe, L., 1955. Combined effects of heat and radiation in food sterilization. Appl. Microbiol. 3, 346–352. Mazas, M., Gonza´lez, I., Lo´pez, M., Gonza´lez, J., Martı´ n Sarmiento, R., 1995. Effects of sporulation media and strain on thermal resistance of Bacillus cereus spores. Int. J. Food Sci. Technol. 30, 71–78. Monk, J.D., Beuchat, L.R., Doyle, M.P., 1995. Irradiation inactivation of foodborne microorganisms. J. Food Prot. 58, 197–208. Palop, A., Rutherford, G.C., Marquis, R.E., 1998. Inactivation of enzymes within spores of Bacillus megaterium ATCC 19213 by hydroperoxides. Can. J. Microbiol. 44, 465–470. Sarrı´ as, J.A., Valero, M., Salmero´n, M.C., 2002. Enumeration, isolation and characterization of Bacillus cereus strains from Spanish raw rice. Food Microbiol. 19, 589–595. Sarrı´ as, J.A., Valero, M., Salmero´n, M.C., 2003. Elimination of Bacillus cereus contamination in raw rice by electron beam irradiation. Food Microbiol. 20, 327–332. Setlow, P., 1995. Mechanisms for the prevention of damage to DNA in spores of Bacillus species. Ann. Rev. Microbiol. 49, 29–54. Shamsuzzaman, K., 1988. Effects of combined heat and radiation on the survival of Clostridium sporogenes. Radiat. Phys. Chem. 31, 187–193. Shamsuzzaman, K., Lucht, L., 1993. Resistance of Clostridium sporogenes spores to radiation and heat in various nonaqueous suspension media. J. Food Prot. 56, 10–12. Shamsuzzaman, K., Payne, B., Cole, L., Borsa, J., 1990. Radiationinduced heat sensitivity and its persistence in Clostridium sporogenes spores in various media. Can. Inst. Food Sci. Technol. J. 23, 114–120. Shamsuzzaman, K., Chuaqui-Offermanns, N., Lucht, L., McDougall, T., Borsa, J., 1992. Microbiological and other characteristics of chicken breast meat following electron-beam and sous-vide treatment. J. Food Prot. 55, 528–533. Thayer, D.W., Boyd, G., 1994. Control of enterotoxic Bacillus cereus on poultry or red meats and in beef gravy by gamma radiation. J. Food Prot. 57, 758–764. Valero, M., Leontidis, S., Ferna´ndez, P.S., Martı´ nez, A., Salmero´n, M.C., 2000. Growth of Bacillus cereus in natural and acidified carrot substrates over the temperature range 5–30 1C. Food Microbiol. 17, 605–612. Valero, M., Herna´ndez-Herrero, L.A., Ferna´ndez, P.S., Salmero´n, M.C., 2002. Characterization of Bacillus cereus isolates from fresh vegetables and refrigerated minimally processed foods by biochemical and physiological tests. Food Microbiol. 19, 491–499. van Gerwen, S.J.C., Rombouts, F.M., van’t Riet, K., Zwietering, M.H., 1999. A data analysis of the irradiation parameter D10 for bacteria and spores under various conditions. J. Food Prot. 62, 1024–1032.
Modeling the influence of electron beam irradiation on the heat resistance of Bacillus cereus spores M. Valeroa,, J.A. Sarrı´ asa, D. A´lvarezb, M.C. Salmero´na a
Departamento de Produccio´n Vegetal y Microbiologı´a, Escuela Po1ite´cnica Superior de Orihue1a, Universidad Miguel Herna´ndez - Campus de Orihue1a, Carretera de Benie1, km 3.2, 03312-Orihue1a, Alicante, Spain b Departamento de Tecnologı´a de los Alimentos, Nutricio´n y Bromatologı´a, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071-Murcia, Spain Received 14 February 2005; received in revised form 10 May 2005; accepted 10 May 2005 Available online 6 July 2005
Abstract The effect of electron beam irradiation (EBI) on Bacillus cereus spore heat resistance was investigated. Irradiation with accelerated electrons had an important heat-sensitizing effect on distilled-water spore suspensions. After irradiation doses of 1.3, 3.1, or 5.7 kGy followed by heating at 90 1C, calculated D90-values for strains Escuela Polite´cnica Superior de Orihuela (EPSO)-41WR and EPSO50UR were reduced more than 1.3, 2.4, and 4.6 times, respectively. Plots of calculated log DT-values versus irradiation doses (1.3, 3.1, and 5.7 kGy) yielded straight parallel lines for the 85–100 1C heating temperature range, which made it possible to develop an equation to predict the changes in heat sensitivity of B. cereus spores that occurred with changing irradiation dose. Radiationinduced heat-sensitivity was characterized by a zEBI-value which was determined as the irradiation dose that should be required to reduce the decimal reduction time (DT ) by one log10 cycle when log10 DT was plotted against irradiation treatment. A model is proposed to describe the influence of a pre-irradiation treatment with electron beams followed by heating on the heat resistance of B. cereus spores. This study also suggests the potential use of EBI followed by heating for food preservation. r 2005 Elsevier Ltd. All rights reserved. Keywords: Irradiation; Electron beams; Heat resistance; Radiation-induced heat-sensitivity; Bacillus cereus; Bacteria1 spores
1. Introduction Micro-organisms highly resistant to heat or radiation pose problems in food processing because of the severe treatment required to eliminate them. Such severe treatment may adversely affect the quality of the processed food as a result of the induced changes in organoleptic characteristics and loss of nutrients. Factors affecting the heat resistance of bacteria to food preservation processes include the type of bacteria and the nature of the suspension media. Spores and some vegetative cells are more resistant to heat in lipids Corresponding author. Tel.: +34 966749683; fax: +34 966749619.
E-mail addresses: [email protected] (M. Valero), [email protected] (D. A´lvarez). 0740-0020/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2005.05.006
than in aqueous suspension media. In both systems, however, a pre-irradiation treatment followed by heating has been found to have important synergistic effects on the destruction of bacterial spores (Farkas, 1990). Therefore, the heat resistance of micro-organisms can be reduced by substerilizing doses of electron beams, X-rays or g-rays. This heat-sensitizing effect of ionizing radiation has been demonstrated in aqueous media or foods for Bacillus cereus (Kan et al., 1957; De Lara et al., 2002), B. subtilis (Grecz et al., 1981; De Lara et al., 2002), Clostridium sporogenes (Kan et al., 1957; Shamsuzzaman, 1988; Shamsuzzaman et al., 1990), C. perfringens (Gombas and Gomez, 1978), C. botulinum (Kempe, 1955) and Listeria monocytogenes (Shamsuzzaman et al., 1992). Radiation-induced heat-sensitivity in C. sporogenes spores has been also reported in vegetable
ARTICLE IN PRESS 368
M. Valero et al. / Food Microbiology 23 (2006) 367–371
oils and various animal fats (Shamsuzzaman and Lucht, 1993). B. cereus has been recognized as a cause of foodpoisoning for more than 30 years. It causes diarrheal as well as emetic foodborne illness syndromes. This microorganism is a Gram-positive, rod-shaped, facultative aerobic spore former that is widely distributed in natural and commercial products due to the strong resistance of its spores to physical and chemical agents. Although there is information on the heat-sensitizing effects of electron beam irradiation (EBI) on spores of B. cereus strain INRA AVZ421 (De Lara et al., 2002) isolated from cooked chilled foods containing vegetables, the aim of this paper is to study the heat resistance of spores of two rice-isolated B. cereus, Escuela Polite´cnica Superior de Orihuela (EPSO)-41WR (white rice) and EPSO-50UR (unhusked rice), characterized by our laboratory (Sarrı´ as et al., 2002), after different irradiation treatments over the temperature range 85–100 1C. The influence of EBI on the heat resistance is also characterized by a zEBI-value and a model is proposed to describe the radiation-induced heat-sensitivity in B. cereus spores.
2. Material and methods 2.1. Bacterial strains, spore production and preparation of spore suspensions B. cereus strains EPSO-41WR and -50UR were used in this study. They were isolated from Spanish raw rice (WR and UR), confirmed by ISO 7932: 1993(F) procedure, and further characterized by API 50CH and API 20E strips, using APILAB Plus software V3.2.2 Version B 01.93 (bioMe´rieux, Marcy l’Etoile, France), and supplementary tests of motility, oxidase activity and enterotoxin production (Sarrı´ as et al., 2002). Spores were produced on fortified nutrient agar (FNA) (Mazas et al., 1995), at 30 1C for 4 days, harvested, centrifuged, resuspended in distilled water and stored at 4 1C until use as described by Valero et al. (2000). For each strain, viability of heat-shocked (80 1C for 10 min) spore suspensions was estimated by spread plating on plate count agar (PCA, Scharlau Chemie, S.A., Barcelona, Spain) at 30 1C for 24 h. 2.2. Irradiation treatments Irradiation was carried out at room temperature in a 10-MeV circular electron accelerator (Rhodotron) localized in the irradiation plant of IONMED ESTERILIZACIO´N, S.A. (Taranco´n, Cuenca, Spain) as described by Sarrı´ as et al. (2003). For irradiation treatment, distilled-water spore suspensions of the two B. cereus strains were dispensed in 1 ml volumes containing
between 106 and 107 spores ml1 into sterile 5-ml glass vials. Samples were exposed to electron beams at programmed irradiation doses of 1, 3, and 5 kGy. The absorbed irradiation doses were measured using cellulose triacetate (CTA) which were attached to the surface of the vials. The irradiation dosimetry revealed that the real doses applied were 1.3, 3.1, and 5.7 kGy. 2.3. Determination of spore heat resistance All heat treatments, applied before and after an irradiation treatment, were performed in 10 ml microhematocrite capillary tubes (Vitrex, Modulohm A/S, Herlev, Denmark) which ensure uniform heat transmission (Ferna´ndez et al., 1999). Capillary tubes were filled with irradiated and non-irradiated spore suspensions in sterilized water. These were centrally injected into glass capillary tubes as described by Valero et al. (2002). Three series of 6–8 capillary tubes were filled from each condition and strain assayed. They were submerged in a stirred oil bath with immersion circulator HAAKE DC5 (Gebru¨der HAAKE GmbH, Karlsruhe, Germany) at 85, 90, 95, or 100 1C constant temperature. Subsequently, tubes were removed at regular intervals and cooled in ice-water, washed in soap solution, rinsed in distilled water and immersed in ethanol. Spores surviving heat treatments were recovered by incubation on PCA at 30 1C for 24 h. All experiments were performed in triplicate. 2.4. DT and z-values calculation The log10 numbers of surviving spores were plotted against time and DT-values were calculated from the slope of the linear phase of the spore destruction as the time in minutes needed to decreased the population one log10 cycle (ICMSF, 1980). The analysis of variance for DT-values by irradiated and non-irradiated spores after heating at each temperature and a multiple comparison procedure of means (Fisher’s least significant difference, LSD) were performed using Statgraphicss Plus for Windows 3.0 (Statistical Graphic Corp. and Graphic Software Systems Inc., Rockville, Maryland, USA). z-Values were determined as the increase in temperature required to reduce the decimal reduction time (DT ) by one log10 cycle when log10 DT was plotted against temperature. To determine the radiation dose required to reduce DT by one log10 cycle, logarithms of the DT-value means were plotted against the corresponding irradiation dose. Data points were fitted by the method of least-squares, and the inverse negative of the slope of the regression line was taken as the zEBI-value that characterized the effect of irradiation on the heat resistance of B. cereus spores.
ARTICLE IN PRESS M. Valero et al. / Food Microbiology 23 (2006) 367–371
3. Results 3.1. Heat resistance Fig. 1 shows survival curves at 95 1C obtained before and after an irradiation treatment of 3.1 kGy. After irradiation, both B. cereus spore suspensions tested, EPSO-4lWR (Fig. 1A) and EPSO-50UR (Fig. 1B), showed significant decrease in heat resistance. The mean DT-values7S.D. at 85, 90, 95, and 100 1C determined for spores of the two strains exposed to different irradiation doses are shown in Table 1. The z-values obtained for spores of the two B. cereus strains in the temperature range and irradiation doses tested are listed in Table 2. For a given strain, these values are very similar regardless of the irradiation treatment applied. High determination coefficients (R2 ) were obtained for all cases. 3.2. Influence of irradiation on the thermal resistance of B. cereus spores Fig. 2 illustrates the influence of irradiation of distilled-water spore suspensions on the DT-values for 6
Log CFU mL-1
5 4 3 2 1 0 0
2
4
6
8
10
12
Time (min)
(A)
7
Log CFU mL-1
6 5 4 3 2 1 0 0 (B)
2
4
6 Time (min)
8
10
12
Fig. 1. Survival curves of spores of B. cereus strains (A) EPSO-41WR and (B) EPSO-50UR heated at 95 1C in distilled water before (open symbols, &) and after (closed symbols, m) an irradiation treatment of 3.1 kGy.
369
B. cereus spores. The plots of log DT-values versus irradiation doses at heating temperature yielded straight lines for the range studied. As Fig. 2A and B shows, all straight lines are basically parallel. At a given temperature, the relation between the DT-values and the irradiation doses shows that the DT-values decrease when the irradiation doses increase. The effect of irradiation on the heat resistance of spores could be characterized and quantified by a zEBI-value which leads to a 10-fold reduction of DT-value. For each bacterial strain and heating temperature the zEBI-value was determined and shown in Table 3.
4. Discussion DT and z-values obtained in this study over the temperature range 85–100 1C (Tables 1 and 2) are in agreement with those published previously by us (Sarrı´ as et al., 2002). Strain EPSO-41WR, with D90values ranging from 20.38 to 23.26 min, was much more heat resistant than the other strain assayed. D10-values have been used to characterize radiation resistance of B. cereus spore suspensions for irradiation doses between 0 and 7 kGy (Sarrı´ as et al., 2002). Variation in radiation resistance observed among the two isolates tested here was lower than that found in heat resistance. In fact, no statistically significant differences in radiation resistance were observed between strains EPSO-41WR and EPSO-50UR of B. cereus. D10-values between 2.48 and 2.75 kGy, with an average of 2.62 kGy, were ca1culated for both strains. D10-values in the range of 1–4 kGy are typical values for bacterial spores, inc1uding B. cereus spores, exposed to radiation (Thayer and Boyd, 1994; Monk et al., 1995; van Gerwen et al., 1999; De Lara et al., 2002). The lack of correlation between heat resistance and radiation resistance of spore suspensions is understandable because the mechanisms involved in the inactivation of bacterial spores by ionizing radiation are very different from those involved in their heat destruction. The most sensitive targets for heat destruction of bacterial spores seem to be core enzymes (Palop et al., 1998) or spore membranes (Setlow, 1995), while inactivation by radiations is mainly based on DNA damage and radiolisis (Farkas, 1989). On the other hand, a pre-irradiation treatment followed by heating was effective in reducing heat resistance of B. cereus spores (Fig. 1). When spore suspensions of strain EPSO-41WR were exposed to 1.3, 3.1, or 5.7 kGy, the average D90-values were reduced 1.3, 2.5, and 4.6 times, respectively (Table 1). After the same irradiation treatments, the D90-value reductions estimated for the strain EPSO-50UR were very similar, namely 1.4-, 2.6-, and 4.9-fold. For each strain and
ARTICLE IN PRESS M. Valero et al. / Food Microbiology 23 (2006) 367–371
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Table 1 Mean DT-values (min) of three experiments 7S.D. obtained for spores of two B. cereus strains isolated from raw rice, exposed to different irradiation doses, and heated at several temperatures Strains
Dose (kGy)
D85
D90
D95
D100
EPSO-41WR
0.0 1.3 3.1 5.7
NT NT NT NT
22.0571.49a 16.7870.16b 8.9070.11c 4.7670.27d
5.0270.19a 4.2270.07b 2.1570.11c 1.0970.08d
0.9970.09a 0.7270.02b 0.4870.03c 0.2070.02d
EPSO-50UR
0.0 1.3 3.1 5.7
22.2470.18a 16.3470.28b 9.2270.11c 4.5570.19d
7.4570.07a 5.5070.21b 2.9070.14c 1.5370.06d
2.2870.11a 1.5670.09b 0.8970.04c 0.4670.02d
0.7070.02 NT NT NT
NT, not tested. Values within columns followed by different letter are significantly different (Pp0:05). Table 2 z-Values for spores of two B. cereus strains isolated from raw rice, exposed to different irradiation doses, and heated in distilled water
1.5
Coefficient R2
0.5
EPSO-41WR
0.0 1.3 3.1 5.7
7.42 7.31 7.88 7.26
0.999 0.995 0.999 0.998
0.0 1.3 3.1 5.7
9.96 9.80 9.85 10.05
0.999 0.998 0.999 0.999
EPSO-50UR
irradiation dose, the DT-value reductions were closely related with independence of the heating temperature. The D90-value reductions by irradiation for the strains EPSO-41WR and EPSO-50UR reported here are slightly lower than those published by De Lara et al. (2002) for the B. cereus strain INRA AVZ421. In this case, D90-value was reduced more than three times for spore suspensions in double distilled water. The plots of log DT-values versus irradiation dose yielded straight lines for both strains and each heating temperature (Fig. 2), but in the irradiation range studied, none of them cover one log10 reduction cycle. The high values of calculated zEBI show this clearly (Table 3). These zEBI-values are higher than those irradiation doses applied in this and earlier studies (Sarrı´ as et al., 2003). Doses over 7.5 kGy cause decontamination of husked rice as well as browning and deterioration of its flavor and texture. The relationship between the calculated log D90values of EPSO-41WR spores and the dose of electron beams, e.g., was fitted according to the following linear regression: D90 ¼ 1:35189 0:12004 kGy
ðR2 ¼ 0:993Þ.
In the 85–100 1C heating temperature range studied, the average of the slopes of the lines obtained was 0.1209870.000846 for EPSO-41WR spores, and
Log DT
Dose (kGy)
0 -0.5 -1 0
1
2
0
1
2
(A)
Log DT
z-Value (1C)
Strains
1
(B)
3 Doses (kGy)
4
5
6
3
4
5
6
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 Doses (kGy)
Fig. 2. Log DT (min) versus dose of electron beams (kGy). Determination of zEBI-values for the B. cereus strains (A) EPSO41WR and (B) EPSO-50UR at different temperatures: 85 (\); 90 (’); 95 (J); and 100 1C (m).
0.1228770.000478 for EPSO-50UR spores. As a whole, the average of the slopes was 0.1219370.001204. Therefore, if a DT-value of a B. cereus distilled-water spore suspension at a given temperature after an irradiation dose is known, the DT-value for the same suspension at the same temperature after any other irradiation dose can be deduced by the following equation: DT1 ¼ DT2 100:12ðkGy2kGy1Þ , where DT1 and DT2 are DT-values of spores at the heating temperature ‘T’ after an irradiation treatment ‘kGy1’ or ‘kGy2’, respectively. This equation yields
ARTICLE IN PRESS M. Valero et al. / Food Microbiology 23 (2006) 367–371 Table 3 zEBI-Values for spores of two B. cereus strains isolated from raw rice, exposed to different irradiation doses, and heated at several temperatures Strains
Temperature (1C)
zEBI-Value (kGy)
Coefficient R2
EPSO-41WR
90 95 100
8.33 8.22 8.25
0.993 0.985 0.990
EPSO-50UR
85 90 95
8.15 8.10 8.16
0.998 0.994 0.997
good results in all the irradiation range studied by us (0–6 kGy). We have shown that EBI is able to sensitize to heat spores of two enterotoxigenic B. cereus strains isolated from Spanish rice and the effect of irradiation on the thermal resistance of spores has been characterized by specific zEBI-values (zEBI is the irradiation dose which leads to a 10-fold reduction of DT-value). Evaluation of a greater number of strains representing the two types of foodborne illness is required to confirm the findings, although previous results of Kan et al. (1957) and De Lara et al. (2002) support the heat-sensitizing effect of radiation on the thermal resistance of B. cereus spores. Furthermore, this study indicates the potential use of a treatment combining low electron beam doses and mild heating to inactivate the spore population present in foods. It is well established that the heat resistance of bacterial spores is influenced by environmental conditions during sporulation, the nature and the pH of the heating medium, and the recovery conditions. When heating is used in combination with irradiation, then the critical process factors of this method (dose and dose rate, temperature, pH, O2, dilution effects, etc.) must be taken into account.
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