- Email: [email protected]
Thermal Death Kinetics of Spores of Bacillus sporothermodurans Isolated from UHT Milk
Int. Dairy Journal 8 (1998) 851—855 1999 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain PII : S0958-6946(98)00129-0 0958-6946/99/$ — see front matter
Thermal Death Kinetics of Spores of Bacillus sporothermodurans Isolated from UHT Milk Ingrid A. Huemer*, Nicolette Klijn, Henri W. J. Vogelsang and Leo P. M. Langeveld Netherlands Institute for Dairy Research (NIZO), P.O. Box 20, 6710 BA Ede, The Netherlands (Received 7 August 1998; accepted 26 November 1998) ABSTRACT In recent years reports have been published on non-sterility problems in UHT milk caused by the survival of very heat-resistant spores, which have been identified as belonging to the species Bacillus sporothermodurans. In order to solve the problems in dairy practice more information is needed on the thermal death kinetics of these spores. The heat resistance of spores of three Bacillus sporothermodurans strains isolated from non-sterile UHT milk was determined in the temperature range of 110—145°C and was compared with the heat resistance of Bacillus stearothermophilus spores. For the low temperatures (110—125°C) the heating was carried out in tubes. For the higher temperatures (130—145°C) a direct UHT sterilizer was used. D values of 3.4—7.9 s (B. stearothermophilus D "0.9 s) indicate an exceptionally high heat resistance of spores of B. sporothermodurans under UHT time (TDT) curves show different slopes for B. sporothermodurans and B. stearothermophilus, with conditions. Thermal death z"13.1—14.2°C and 9.1°C, respectively. To our knowledge there are no observations of other spores in the literature for which such high D values and z values have been demonstrated. 1999 Published by Elsevier Science Ltd. All rights reserved Keywords: heat resistance; Bacillus sporothermodurans; spores TDT curve
INTRODUCTION
Michielis (1988), who found a ‘heat-induced resistance’ after sublethal heat treatment of spores. All these observations, as well as so-far unknown mechanisms contributing to the heat-resistance properties of bacterial spores, indicate the difficulties in obtaining comparable kinetic data. The objective of the present investigation was to obtain more knowledge on the heat resistance of B. sporothermodurans spores in milk in order to have reliable data for design of optimal thermal process conditions. A second aim was to obtain information about the time-temperature range fit for the specific selection of B. sporothermodurans spores from other heat-resistant spores in raw milk.
The occurrence of heat-resistant mesophilic sporeforming bacteria (HRS) causing non-sterility problems in UHT milk products has been reported during recent years by several authors (Foschino et al., 1990; Kessler et al., 1994; Hammer et al., 1995). Molecular investigations of the 16S rRNA of these sporeformers showed that a new Bacillus species is involved (Klijn et al., 1994), which has been recently described by Petterson et al. (1996) as Bacillus sporothermodurans. Data concerning the phenotypic characterization of B. sporothermodurans have been published (Petterson et al., 1996; Klijn et al., 1997), but besides the practical observations no information on the heat resistance of these spores has been published. This is essential information for dairy practice in order to solve the problems in relation to the survival of spores in the UHT process. Microbial heat resistance is affected by various environmental factors, such as sporulation temperature (ElBisi and Ordal, 1956; Cook and Gilbert, 1968; Beaman and Gerhardt, 1986), pH and composition of the sporulation medium (Amaha and Ordal, 1957; Yokoya and York, 1965; Busta, 1967; Davies, 1975; Mayou and Jezeski, 1977; Stadhouders et al., 1980; Mazas et al., 1995) and the heating menstruum (Cook and Gilbert, 1968; Shehata et al., 1977; Mayou and Jezeski, 1977; Cook and Gilbert, 1968; Condo´n and Sala, 1992; Sala et al., 1994; Lo´pez et al., 1996). Another phenomenon influencing the heat resistance of spores was investigated by Etoa and
MATERIALS AND METHODS Bacterial strains The following strains were used for heat-resistance studies: Bacillus sporothermodurans J16 (obtained from the Federal Dairy Research Center, Kiel, Germany; NIZO B1106), Bacillus sporothermodurans B93-20-12 (NIZO B1107, isolated from UHT milk), Bacillus sporothermodurans MB921 (NIZO B1109, isolated from UHT milk) and Bacillus stearothermophilus C953 (NIZO B469). For the isolation of strains B93-20-12 and MB921 from UHT milk, the milk samples were heated for 30 or 90 min at 100°C. All strains were grown on ONA>-medium consisting of 25 g L\ Nutrient Broth (Oxoid, Hampshire, UK), 15 g L\ Bacto Agar (Oxoid), 1 mg L\ Vitamin B12
*Corresponding author. 851
852
I. A. Huemer et al.
(Sigma, St. Louis, Mo.), 8.4 mg L\ MnSO ) H O and 1 g L\ CaCl ) 2H O, pH 6.8. Preparation of spore suspensions The strains (one colony) were spread on the surface of large ONA> plates (diameter 14 cm, 100 mL medium) and were incubated for seven days at 37°C (B. sporothermodurans) or 55°C (B. stearothermophilus). For obtaining the spore suspension B. sporothermodurans J16 the ONA> plates were spread from a colony grown from the original stock culture of B. sporothermodurans J16. For obtaining the spore suspension J16 the ONA> plates were spread from a colony obtained after 10 successive transfers and cultures of strain J16. Sporulation was checked microscopically. About 0.1%—1% of the B. sporothermodurans and B. stearothermophilus cells had sporulated. To harvest spores, the dense carpet of bacteria was removed with 5 mL of cold, sterile peptone physiological salt solution (1 g L\ Peptone, Oxoid; 8.5 g L\ NaCl) transferred to a sterile vessel and heated at 80°C for 13 min in order to kill vegetative cells. The spore content was then between 3;10 and 3;10 mL\. The spore suspensions were stored at 4°C until required. Stocks of the spore suspensions are kept at !135°C. Heat resistance studies Sterile skim milk was inoculated with the spores about 1 h before heat treatment and was stored at 5°C. For the determination of the heat resistance at low temperatures (110—125°C) the dilution rate was 1 : 100. Samples were heated in sterile glass tubes (inner diameter 7.0 mm, wall thickness 1 mm). The tubes were filled with 2 mL of sample, closed with a sterile rubber stopper and clasped in a rack, which was submerged in a silicone oil bath set at the desired temperature. The temperature equilibration time was measured in a separate tube provided with a thermocouple and heated in the same way. A time—temperature profile is shown in Fig. 1. To reach 120°C in the sample, 2.8 min were required. Next to the temperature equilibration time, the samples were heated at different temperatures for different time
periods. After heat treatment the rack with tubes was transferred to a water-ice bath for cooling. Heat resistance studies at UHT temperatures (130—145°C) were carried out by using a direct steam injection-vacuum sterilizer with a capacity of 60 L h\ (VTIS, Alfa-Laval, Lund, Sweden). Portions of 1 : 10,000 inoculated skim milk were heated for different time-temperature combinations. The heated milk was cooled in the equipment to about 15°C and was filled aseptically into sterile bottles which were kept at 5°C until further treatment. The concentration of spores in the milk samples was determined before and after heat treatment within about 20 h. For the tube method zero time (time with initial spore concentration) was considered the time at which the contents of the tubes reached the desired temperature. In case of the UHT treatment the initial number of spores was determined after heating the untreated sample for 13 min at 80°C to kill possible contaminants in the milk which could have been caused by the not fully aseptic handling of the sample before the sterilisation procedure. After appropriate dilution in peptone physiological salt solution, survivors were enumerated by plating on ONA> with incubation for 48 h at 37°C for the detection of Bacillus sporothermodurans and 55°C for Bacillus stearothermophilus. The arithmetic mean of the results from duplicate plates was taken as the number of survivors in each dilution. After each experiment the identity of the isolates were checked by RAPD-analyses (Klijn et al., 1997) in order to eliminate possible contaminations. Computer-assisted calculation of kinetic data Activation energy (E ) and pre-exponential factor (k ) were calculated with the computer program KINEST (Vogelsang and de Jong, 1996), using the end concentrations (N), the initial concentrations (N ), the measured heating time (h) and the measured temperature (¹) as given data, according to the following first-order reaction: ln
N "k e\# 02h. N
The decimal reduction times (D values) were calculated from the number of decimal reductions of spores (log(N /N)) in dependence on the heating time, whereby only the linear part of the measured survivor curves was included in the calculations. The best values of E and k were computed by minimizing the difference between experimental and calculated log(D) values. Next, z values were calculated using the activation energy (E ) estimated with KINEST and the average temperature (¹) of the investigated temperature range according to the equation of de Jong (de Jong, 1996): ln(10)R¹ z" . E !ln(10)R¹ Comparison between measured and theoretical kinetic data Fig. 1. Time—temperature profile in a tube with skimmilk heated in an oil bath at 120°C (䉭) and measured destruction curve of Bacillus sporothermodurans J16 spores (䉱).
Based on the kinetic data E and ln k calculated from measurements of the temperature range 110—120°C and
853
Heat resistance of Bacillus sporothermodurans spores
135—145°C for B. sporothermodurans J16 and the measured time-temperature profiles in the tubes, the theoretical heat inactivation of B. sporothermodurans J16 spores was calculated for the temperature range 120—135°C with a computer model based on the abovementioned equations. Then measurements were performed within this temperature range both with the tube method and with the direct UHT sterilizer. In this way the theoretical spore destruction was compared with the measured destruction in both heating methods, in order to estimate the reliability of the data obtained in the tube methods at high temperatures.
RESULTS AND DISCUSSION Effect of subcultivation on the heat resistance Table 1 shows the D values obtained for two spore suspensions of J16 that differed in the number of culture passages applied before preparation of the spore suspension. The D values obtained for the spore suspension of the more original culture of J16 were about twice as high as the D values of the spore suspension of a culture which was obtained after several culture steps (J16 ). In order to maintain the natural heat resistance properties of the spores under laboratory conditions it seems to be necessary to avoid continous subculturing of the original isolates. Heat resistance Thermal death time (TDT) curves, representing the logarithm of the decimal reduction time (D values) in seconds plotted against temperature, are shown for two of the investigated strains in Fig. 2. Linear relationships of TDT curves obtained above temperatures of 121°C have been questioned by numerous authors (Busta, 1967; Burton et al., 1977; David and Merson, 1990). In this work straight lines were obtained in all cases for the complete temperature interval studied, with linear correlation coefficients higher than 0.99. This finding is in agreement with data published by Esselen and Pflug (1956) and Gaze and Brown (1988), who reported no variation in the z value. In Table 2 the reaction kinetic data of the heat inactivation of spores of all investigated strains are collected. The activation energy for the heat inactivation of
Table 1. The Effect of Subcultivation on the Thermal Resistance of B. sporothermodurans Spores D values (s) 2
Spore suspensions of B. sporothermodurans J16
¹ (°C) 110 J16 J16
120
1084 79 1428 135
130
135
140
23.9 6.7 42.2 14.3
1.6 5.0
J16 : A Spore suspension prepared after 10 culture passages. J16 : B Spore suspension prepared from original stock culture.
Fig. 2. Thermal death time curves of B. stearothermophilus spores (䉬) and B. sporothermodurans spores J16 (䉭); best fit lines through experimental data.
Table 2. Reaction Kinetic Data for the Heat Inactivation of Bacillus Spores in Skimmilk Strains
z (°C)
E (kJ mol\)
ln k (s\)
B. B. B. B.
9.1 13.1 14.2 13.2
345.7 241.9 224.4 240.0
101.5 69.6 64.0 69.3
stearothermophilus C953 sporothermodurans J16 sporothermodurans B93-20-12 sporothermodurans MB921
B. stearothermophilus spores calculated in this work, E "345.7 kJ mol\, is in good agreement with that found by Peri et al. (1988), E "345.4 kJ mol\, for about the same temperature range. Reported decimal reduction times of D "246 s (Behringer and Kessler, 1992) and D "210 s (Mayou and Jezeski, 1977) for the heat inactivation of B. stearothermophilus spores in skim milk, and those found by other researchers as gathered by Holdsworth (1992), compare well with our findings, D "191 s. At low-temperatures (Fig. 2) spores of B. sporothermodurans strains were slightly less heat resistant than spores of B. stearothermophilus, and about equally resistant at about 120°C. From the review of Holdsworth (1992) it can be seen that the heat resistance of spores of B. sporothermodurans at about 120°C may be very close to that of spores of, e.g. Bacillus subtilis (up to 30 s). So, it is not possible to specifically select for B. sporothermodurans spores by laboratory heating experiments at that temperature or lower temperatures. All investigated B. sporothermodurans spores showed a considerably higher heat resistance at UHT temperatures, with D values ranging from 3.4 to 7.9 s, com pared with B. stearothermophilus with a D value of 0.9 s (see Fig. 2). This is caused by the high z value. The similar high z values (z"13.1—14.2°C) as well as the similar relatively low values of the activation energies calculated (E "224.4—241.9 kJ mol\) indicate a common exceptionally high heat resistance in the UHT temperature range of the B. sporothermodurans spores
854
I. A. Huemer et al.
Table 3. Comparison between Measured and Calculated D Values for B. sporothermodurans J16 2 D values (s) 2
¹ (°C)
120 125 130 135
Measured in tubes
Measured with UHT pilot
Calculated
150 69 48 30
n.d. 85 43 15
199 84 31 13
n.d.: not determined.
investigated in this work. This is uncommon for aerobic spores. In the survey by Holdsworth (1992) high z values for other aerobic spores are mentioned. However on checking the literature concerned, the promised article had not been published (Fox and Pflug, 1968), the results at high temperatures had been gathered by tube experiments (Navani et al., 1970) or the z value had been estimated only up to 128°C (Segner et al., 1963). Comparison between measurement and theoretical kinetic data The linear correlation coefficient for the best fit between the measured data in the temperature range 110—145°C and mathematical model for B. sporothermodurans J16 spores is 0.992. At temperatures above 125°C the measurements obtained with the tube method had a deviation compared to the calculated values based on the model, whereas in the measurements with the UHT sterilizer the expected values were obtained. It can therefore be concluded that the tube method results in reliable measurements at temperatures below 125°C. At higher temperatures the Dvalues obtained in the tube method are higher than those based on the caculated model and the UHT measurements (Table 3). This might be caused by the heating-up time, but real-time monitoring of the temperature did not support this hypothesis. Apparently, there is at a certain point a difference between batch heating in tubes and the UHT process. Therefore, it was concluded that measurement at temperatures higher than 125°C cannot be carried out with the tube method described, but should be performed with a UHT sterilizer.
is not possible to specifically select for B. sporothermodurans spores. Only the unique heat resistance in the temperature range of 130—140°C might be used as a selective tool for the isolation of B. sporothermodurans from raw milk. D values of 3.4—7.9 s indicate an exceptionally high heat resistance of spores of B. sporothermodurans under UHT conditions (for B. stearothermophilus, D "0.9 s). TDT curves show different slopes for spores of B. sporothermodurans and B. stearothermophilus, with z" 13.1—14.2°C and 9.1°C, respectively. To the best of our knowledge there are no observations regarding other aerobic spores in the literature for which such high D values and z values have been demonstrated. Based on the data presented, solutions to inactivate these spores in UHT milk processing can be designed.
NOMENCLATURE D 2
e E k N N R ¹ h z
decimal reduction time; time (s) required for the number of spores to be reduced by a factor of ten at a given temperature ¹; the index ¹ is expressed in °C base of natural logarithms activation energy (kJ mol\) pre-exponential factor (s\) initial concentration of spores (cfu mL\) end concentration of spores (cfu mL\) universal gas constant 8.314 (J mol\ K\) absolute temperature (K) heating time (s) temperature change (°C) required to cause a tenfold change in D 2
ACKNOWLEDGEMENTS The authors thank Arjen A. Wagendorp for the genetic characterization of the isolates and Klaas Leffring for the carefully carried out UHT experiments. Part of the research work presented here was supported by Tetra Pak Research, Stuttgart, Germany (Dr Fritz Lembke). We thank Stork CRO, Nieuw Vennep, The Netherlands, for kindly providing equipment for the tube heating method.
REFERENCES Conclusions The observations presented in this article show that non-sterility in UHT milk can be caused by the survival of spores of B. sporothermodurans present in the milk prior to the heat treatment. B. sporothermodurans spores have an exceptionally high heat resistance at UHT temperatures, which is definitely different from the kinetic pattern of B. stearothermophilus spores. However, due to the specific kinetic characteristics of heat inactivation in the temperature range 110—120°C, the spores of B. sporothermodurans have heat resistance similar to or even lower than those of B. stearothermophilus, and their heat resistance is even very close to that of spores of B. subtilis. This means that within the temperature range that can be applied by common heating equipment in a laboratory, it
Amaha, M. and Ordal, J. Z. (1957) Effect of divalent cations in the sporulation medium on the thermal death rate of Bacillus coagulans var. thermoacidurans. Journal of Bacteriology 74, 596—604. Beaman, T. C. and Gerhardt, P. (1986) Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization and thermal adaption. Applied and Environmental Microbiology 52, 1242—1246. Behringer, R. and Kessler, H. G. (1992) Thermal destruction kinetics of spores of selected Bacillus strains in skimmilk and skimmilk concentrate. International Dairy Journal 2, 233—242. Burton, H., Perkin, A. G., Davies, F. L. and Underwood, H. M. (1977) Thermal death kinetics of Bacillus stearothermophilus spores at ultra high temperatures. III. Relationship between data from capillary tube experiments and from UHT sterilizers. Journal of Food ¹echnology 12, 149—161.
Heat resistance of Bacillus sporothermodurans spores Busta, F. F. (1967) Thermal inactivation characteristics of bacterial spores at ultra high temperatures. Applied Microbiology 15, 640—645. Cook, A. M. and Gilbert, R. J. (1968) Factors affecting the heat resistance of Bacillus stearothermophilus spores. II. The effect of sporulating conditions and nature of the heating medium. Journal of Food ¹echnology 3, 295—302. Condo´n, S. and Sala, F. J. (1992) Heat resistance of Bacillus subtilis in buffer and foods of different pH. Journal of Food Protection 55, 605—608. David, J. R. D. and Merson, R. L. (1990) Kinetic parameters for inactivation of Bacillus stearothermophilus at high temperatures. Journal of Food Science 55(2), 488—493. Davies, F. L. (1975) Heat resistance of Bacillus species. Journal of the Society of Dairy ¹echnology 28, 69—78. El-Bisi, M. H. and Ordal, Z. O. (1956a) The effect of certain sporulation conditions on the thermal death rate of Bacillus coagulans var. thermoacidurans. Journal of Bacteriology 71, 1—9. El-Bisi, M. H. and Ordal, Z. O. (1956b) The effect of certain sporulation conditions on the thermal death rate of Bacillus coagulans var. thermoacidurans. Journal of Bacteriology 71, 10—16. Esselen, W. B. and Pflug, I. J. (1956) Thermal resistance of putrefactive anaerobe PA3679 spores in vegetables in temperature range of 250—290°F. Food ¹echnology 11, 557. Etoa, F.-X. and Michielis, L. (1988) Heat-induced resistance of Bacillus stearothermophilus spores. ¸etters in Applied Microbiology 6, 43—45. Foschino, R., Galli, A. and Ottogalli, G. (1990) Research on the Microflora of UHT milk. Annali di Microbiologia ed Enzimologia 40, 47—59. Fox, K. and Pflug, I. J. (1968) Effect of temperature and gas velocity on dry heat destruction rate of bacterial spores. Applied Microbiology 16, 343—348. Franklin, J. C. (1970) Spores in milk: Problems associated with UHT processing. Journal of Applied Bacteriology 33, 180—191. Gaze, J. E. and Brown, K. L. (1988) The heat resistance of spores of Clostridium botulinum 213B over the temperature range 120 to 140°C. International Journal of Food Science and ¹echnology 23, 373. Hammer, P., Lembke, F., Suhren, G. and Heeschen, W. (1995) Characterization of a heat-resistant mesophilic Bacillus species affecting quality of UHT milk—a preliminary report. Kieler Milchwirtschaftliche Forschungsberichte 47, 297—305. Holdsworth, S. D. (1992) Kinetic factors for microbial destruction by wet heat. In Aseptic Processing and Packaging of Food Products, ed. S. D. Holdsworth. Elsevier Applied Science, London, New York, pp. 378—386. Jong, P. de (1996) Modelling and optimization of thermal processes in dairy industry. Thesis University Delft, Netherlands. Kessler, H. G., Pfeifer, J. and Schwoeppe, C. (1994) Untersuchungen ueber hitzeresistente mesophile Bacillus—Sporen in UHT Milch, Deutsche Milchwirtschaft 13, 588—592.
855
Klijn, N., Langeveld, L. P. M., and Weerkamp, A. H. (1994) Identification of a mesophilic Bacillus sp. producing spores with unusual heat-resistance. Proceedings of the 24th International Dairy Congress 1994, Australia, Ec17, posterabstract. Klijn, N., Herman, L., Langeveld, L. P. M., Wagendorp, A. A., Huemer, I. A. and Weerkamp, A. H. (1997) Genotypical and phenotypical characterization of Bacillus sporothermodurans strains, surviving UHT sterilisation. International Dairy Journal 7, 421—428. Lo´pez, M., Gonza´les I., Condo´n, S. and Bernardo, A. (1996) Effect of pH heating medium on the thermal resistance of Bacillus stearothermophilus spores. Journal of Food Microbiology 28, 405—410. Mayou, J. L. and Jezeski, J. J. (1977) Effect of using milk as a heating menstruum on the apparent heat resistance of Bacillus stearothermophilus spores. Journal of Food Protection 40, 228—231. Mazas, M., Gonzales, I., Lopez, M., Gonzales, J. and Sarmiento, R. M. (1995) Effects of sporulation media and strain on thermal resistance of Bacillus cereus spores. International Journal of Food Science and ¹echnology 30, 71—78. Navani, S. K., Scholefield, J. and Ribby, M. R. (1970) A digital computer program for the statistical analysis of heat resistance data applied to Bacillus stearothermophilus spores. Journal of Applied Bacteriology 33, 609—620. Peri, C., Pagliarini, E. and Pierucci, S. (1988) A study on optimizing heat treatment of milk. I. Pasteurization. Milchwissenschaft 43, 636—639. Petterson, B., Lembke, F., Hammer, P., Stackebrandt, E. and Priest, F. G. (1996) Bacillus sporothermodurans, a new species producing highly heat-resistant endospores. International Journal of Systematic Microbiology 46, 759—764. Sala, F. J., Ibarz, P., Palop, A., Raso, J. and Condo´n, S. (1994) Sporulation temperature and heat resistance of Bacillus subtilis at different pH values. Journal of Food Protection 58, 239—243. Segner, W. P., Frazier, W. C. and Calbert, H. E. (1963) Thermal inactivation of heat-resistant bacterial spores in milk concentrate at ultra high temperatures. Journal of Dairy Science 46, 891—896. Shehata, A. E., Khalafalla, S. M., Elmagdoub, M. N. I. and Hofi, A. A. (1977) Heat resistance parameters for spores of some Bacillus species in milk. Milchwissenschaft 32, 136—139. Stadhouders, J., Hup, G. and Langeveld, L. P. M. (1980) Some observations on the germination, heat resistance and outgrowth of fast-germinating and slow-germinating spores of B. cereus in pasteurized milk. Netherlands Milk and Dairy Journal 34, 215—228. Vogelsang, H. W. J. and de Jong, P. (1996) Reactorkundige beheersing van kaasmelkkwaliteit. NP¹ Procestechnologie 3(51), 15—20. Yokoya, K. and York, G. K. (1965) Effect of some environmental conditions on the thermal death rate of endospores of aerobic thermophilic bacteria. Applied Microbiology 13(6), 993—999.
Thermal Death Kinetics of Spores of Bacillus sporothermodurans Isolated from UHT Milk Ingrid A. Huemer*, Nicolette Klijn, Henri W. J. Vogelsang and Leo P. M. Langeveld Netherlands Institute for Dairy Research (NIZO), P.O. Box 20, 6710 BA Ede, The Netherlands (Received 7 August 1998; accepted 26 November 1998) ABSTRACT In recent years reports have been published on non-sterility problems in UHT milk caused by the survival of very heat-resistant spores, which have been identified as belonging to the species Bacillus sporothermodurans. In order to solve the problems in dairy practice more information is needed on the thermal death kinetics of these spores. The heat resistance of spores of three Bacillus sporothermodurans strains isolated from non-sterile UHT milk was determined in the temperature range of 110—145°C and was compared with the heat resistance of Bacillus stearothermophilus spores. For the low temperatures (110—125°C) the heating was carried out in tubes. For the higher temperatures (130—145°C) a direct UHT sterilizer was used. D values of 3.4—7.9 s (B. stearothermophilus D "0.9 s) indicate an exceptionally high heat resistance of spores of B. sporothermodurans under UHT time (TDT) curves show different slopes for B. sporothermodurans and B. stearothermophilus, with conditions. Thermal death z"13.1—14.2°C and 9.1°C, respectively. To our knowledge there are no observations of other spores in the literature for which such high D values and z values have been demonstrated. 1999 Published by Elsevier Science Ltd. All rights reserved Keywords: heat resistance; Bacillus sporothermodurans; spores TDT curve
INTRODUCTION
Michielis (1988), who found a ‘heat-induced resistance’ after sublethal heat treatment of spores. All these observations, as well as so-far unknown mechanisms contributing to the heat-resistance properties of bacterial spores, indicate the difficulties in obtaining comparable kinetic data. The objective of the present investigation was to obtain more knowledge on the heat resistance of B. sporothermodurans spores in milk in order to have reliable data for design of optimal thermal process conditions. A second aim was to obtain information about the time-temperature range fit for the specific selection of B. sporothermodurans spores from other heat-resistant spores in raw milk.
The occurrence of heat-resistant mesophilic sporeforming bacteria (HRS) causing non-sterility problems in UHT milk products has been reported during recent years by several authors (Foschino et al., 1990; Kessler et al., 1994; Hammer et al., 1995). Molecular investigations of the 16S rRNA of these sporeformers showed that a new Bacillus species is involved (Klijn et al., 1994), which has been recently described by Petterson et al. (1996) as Bacillus sporothermodurans. Data concerning the phenotypic characterization of B. sporothermodurans have been published (Petterson et al., 1996; Klijn et al., 1997), but besides the practical observations no information on the heat resistance of these spores has been published. This is essential information for dairy practice in order to solve the problems in relation to the survival of spores in the UHT process. Microbial heat resistance is affected by various environmental factors, such as sporulation temperature (ElBisi and Ordal, 1956; Cook and Gilbert, 1968; Beaman and Gerhardt, 1986), pH and composition of the sporulation medium (Amaha and Ordal, 1957; Yokoya and York, 1965; Busta, 1967; Davies, 1975; Mayou and Jezeski, 1977; Stadhouders et al., 1980; Mazas et al., 1995) and the heating menstruum (Cook and Gilbert, 1968; Shehata et al., 1977; Mayou and Jezeski, 1977; Cook and Gilbert, 1968; Condo´n and Sala, 1992; Sala et al., 1994; Lo´pez et al., 1996). Another phenomenon influencing the heat resistance of spores was investigated by Etoa and
MATERIALS AND METHODS Bacterial strains The following strains were used for heat-resistance studies: Bacillus sporothermodurans J16 (obtained from the Federal Dairy Research Center, Kiel, Germany; NIZO B1106), Bacillus sporothermodurans B93-20-12 (NIZO B1107, isolated from UHT milk), Bacillus sporothermodurans MB921 (NIZO B1109, isolated from UHT milk) and Bacillus stearothermophilus C953 (NIZO B469). For the isolation of strains B93-20-12 and MB921 from UHT milk, the milk samples were heated for 30 or 90 min at 100°C. All strains were grown on ONA>-medium consisting of 25 g L\ Nutrient Broth (Oxoid, Hampshire, UK), 15 g L\ Bacto Agar (Oxoid), 1 mg L\ Vitamin B12
*Corresponding author. 851
852
I. A. Huemer et al.
(Sigma, St. Louis, Mo.), 8.4 mg L\ MnSO ) H O and 1 g L\ CaCl ) 2H O, pH 6.8. Preparation of spore suspensions The strains (one colony) were spread on the surface of large ONA> plates (diameter 14 cm, 100 mL medium) and were incubated for seven days at 37°C (B. sporothermodurans) or 55°C (B. stearothermophilus). For obtaining the spore suspension B. sporothermodurans J16 the ONA> plates were spread from a colony grown from the original stock culture of B. sporothermodurans J16. For obtaining the spore suspension J16 the ONA> plates were spread from a colony obtained after 10 successive transfers and cultures of strain J16. Sporulation was checked microscopically. About 0.1%—1% of the B. sporothermodurans and B. stearothermophilus cells had sporulated. To harvest spores, the dense carpet of bacteria was removed with 5 mL of cold, sterile peptone physiological salt solution (1 g L\ Peptone, Oxoid; 8.5 g L\ NaCl) transferred to a sterile vessel and heated at 80°C for 13 min in order to kill vegetative cells. The spore content was then between 3;10 and 3;10 mL\. The spore suspensions were stored at 4°C until required. Stocks of the spore suspensions are kept at !135°C. Heat resistance studies Sterile skim milk was inoculated with the spores about 1 h before heat treatment and was stored at 5°C. For the determination of the heat resistance at low temperatures (110—125°C) the dilution rate was 1 : 100. Samples were heated in sterile glass tubes (inner diameter 7.0 mm, wall thickness 1 mm). The tubes were filled with 2 mL of sample, closed with a sterile rubber stopper and clasped in a rack, which was submerged in a silicone oil bath set at the desired temperature. The temperature equilibration time was measured in a separate tube provided with a thermocouple and heated in the same way. A time—temperature profile is shown in Fig. 1. To reach 120°C in the sample, 2.8 min were required. Next to the temperature equilibration time, the samples were heated at different temperatures for different time
periods. After heat treatment the rack with tubes was transferred to a water-ice bath for cooling. Heat resistance studies at UHT temperatures (130—145°C) were carried out by using a direct steam injection-vacuum sterilizer with a capacity of 60 L h\ (VTIS, Alfa-Laval, Lund, Sweden). Portions of 1 : 10,000 inoculated skim milk were heated for different time-temperature combinations. The heated milk was cooled in the equipment to about 15°C and was filled aseptically into sterile bottles which were kept at 5°C until further treatment. The concentration of spores in the milk samples was determined before and after heat treatment within about 20 h. For the tube method zero time (time with initial spore concentration) was considered the time at which the contents of the tubes reached the desired temperature. In case of the UHT treatment the initial number of spores was determined after heating the untreated sample for 13 min at 80°C to kill possible contaminants in the milk which could have been caused by the not fully aseptic handling of the sample before the sterilisation procedure. After appropriate dilution in peptone physiological salt solution, survivors were enumerated by plating on ONA> with incubation for 48 h at 37°C for the detection of Bacillus sporothermodurans and 55°C for Bacillus stearothermophilus. The arithmetic mean of the results from duplicate plates was taken as the number of survivors in each dilution. After each experiment the identity of the isolates were checked by RAPD-analyses (Klijn et al., 1997) in order to eliminate possible contaminations. Computer-assisted calculation of kinetic data Activation energy (E ) and pre-exponential factor (k ) were calculated with the computer program KINEST (Vogelsang and de Jong, 1996), using the end concentrations (N), the initial concentrations (N ), the measured heating time (h) and the measured temperature (¹) as given data, according to the following first-order reaction: ln
N "k e\# 02h. N
The decimal reduction times (D values) were calculated from the number of decimal reductions of spores (log(N /N)) in dependence on the heating time, whereby only the linear part of the measured survivor curves was included in the calculations. The best values of E and k were computed by minimizing the difference between experimental and calculated log(D) values. Next, z values were calculated using the activation energy (E ) estimated with KINEST and the average temperature (¹) of the investigated temperature range according to the equation of de Jong (de Jong, 1996): ln(10)R¹ z" . E !ln(10)R¹ Comparison between measured and theoretical kinetic data Fig. 1. Time—temperature profile in a tube with skimmilk heated in an oil bath at 120°C (䉭) and measured destruction curve of Bacillus sporothermodurans J16 spores (䉱).
Based on the kinetic data E and ln k calculated from measurements of the temperature range 110—120°C and
853
Heat resistance of Bacillus sporothermodurans spores
135—145°C for B. sporothermodurans J16 and the measured time-temperature profiles in the tubes, the theoretical heat inactivation of B. sporothermodurans J16 spores was calculated for the temperature range 120—135°C with a computer model based on the abovementioned equations. Then measurements were performed within this temperature range both with the tube method and with the direct UHT sterilizer. In this way the theoretical spore destruction was compared with the measured destruction in both heating methods, in order to estimate the reliability of the data obtained in the tube methods at high temperatures.
RESULTS AND DISCUSSION Effect of subcultivation on the heat resistance Table 1 shows the D values obtained for two spore suspensions of J16 that differed in the number of culture passages applied before preparation of the spore suspension. The D values obtained for the spore suspension of the more original culture of J16 were about twice as high as the D values of the spore suspension of a culture which was obtained after several culture steps (J16 ). In order to maintain the natural heat resistance properties of the spores under laboratory conditions it seems to be necessary to avoid continous subculturing of the original isolates. Heat resistance Thermal death time (TDT) curves, representing the logarithm of the decimal reduction time (D values) in seconds plotted against temperature, are shown for two of the investigated strains in Fig. 2. Linear relationships of TDT curves obtained above temperatures of 121°C have been questioned by numerous authors (Busta, 1967; Burton et al., 1977; David and Merson, 1990). In this work straight lines were obtained in all cases for the complete temperature interval studied, with linear correlation coefficients higher than 0.99. This finding is in agreement with data published by Esselen and Pflug (1956) and Gaze and Brown (1988), who reported no variation in the z value. In Table 2 the reaction kinetic data of the heat inactivation of spores of all investigated strains are collected. The activation energy for the heat inactivation of
Table 1. The Effect of Subcultivation on the Thermal Resistance of B. sporothermodurans Spores D values (s) 2
Spore suspensions of B. sporothermodurans J16
¹ (°C) 110 J16 J16
120
1084 79 1428 135
130
135
140
23.9 6.7 42.2 14.3
1.6 5.0
J16 : A Spore suspension prepared after 10 culture passages. J16 : B Spore suspension prepared from original stock culture.
Fig. 2. Thermal death time curves of B. stearothermophilus spores (䉬) and B. sporothermodurans spores J16 (䉭); best fit lines through experimental data.
Table 2. Reaction Kinetic Data for the Heat Inactivation of Bacillus Spores in Skimmilk Strains
z (°C)
E (kJ mol\)
ln k (s\)
B. B. B. B.
9.1 13.1 14.2 13.2
345.7 241.9 224.4 240.0
101.5 69.6 64.0 69.3
stearothermophilus C953 sporothermodurans J16 sporothermodurans B93-20-12 sporothermodurans MB921
B. stearothermophilus spores calculated in this work, E "345.7 kJ mol\, is in good agreement with that found by Peri et al. (1988), E "345.4 kJ mol\, for about the same temperature range. Reported decimal reduction times of D "246 s (Behringer and Kessler, 1992) and D "210 s (Mayou and Jezeski, 1977) for the heat inactivation of B. stearothermophilus spores in skim milk, and those found by other researchers as gathered by Holdsworth (1992), compare well with our findings, D "191 s. At low-temperatures (Fig. 2) spores of B. sporothermodurans strains were slightly less heat resistant than spores of B. stearothermophilus, and about equally resistant at about 120°C. From the review of Holdsworth (1992) it can be seen that the heat resistance of spores of B. sporothermodurans at about 120°C may be very close to that of spores of, e.g. Bacillus subtilis (up to 30 s). So, it is not possible to specifically select for B. sporothermodurans spores by laboratory heating experiments at that temperature or lower temperatures. All investigated B. sporothermodurans spores showed a considerably higher heat resistance at UHT temperatures, with D values ranging from 3.4 to 7.9 s, com pared with B. stearothermophilus with a D value of 0.9 s (see Fig. 2). This is caused by the high z value. The similar high z values (z"13.1—14.2°C) as well as the similar relatively low values of the activation energies calculated (E "224.4—241.9 kJ mol\) indicate a common exceptionally high heat resistance in the UHT temperature range of the B. sporothermodurans spores
854
I. A. Huemer et al.
Table 3. Comparison between Measured and Calculated D Values for B. sporothermodurans J16 2 D values (s) 2
¹ (°C)
120 125 130 135
Measured in tubes
Measured with UHT pilot
Calculated
150 69 48 30
n.d. 85 43 15
199 84 31 13
n.d.: not determined.
investigated in this work. This is uncommon for aerobic spores. In the survey by Holdsworth (1992) high z values for other aerobic spores are mentioned. However on checking the literature concerned, the promised article had not been published (Fox and Pflug, 1968), the results at high temperatures had been gathered by tube experiments (Navani et al., 1970) or the z value had been estimated only up to 128°C (Segner et al., 1963). Comparison between measurement and theoretical kinetic data The linear correlation coefficient for the best fit between the measured data in the temperature range 110—145°C and mathematical model for B. sporothermodurans J16 spores is 0.992. At temperatures above 125°C the measurements obtained with the tube method had a deviation compared to the calculated values based on the model, whereas in the measurements with the UHT sterilizer the expected values were obtained. It can therefore be concluded that the tube method results in reliable measurements at temperatures below 125°C. At higher temperatures the Dvalues obtained in the tube method are higher than those based on the caculated model and the UHT measurements (Table 3). This might be caused by the heating-up time, but real-time monitoring of the temperature did not support this hypothesis. Apparently, there is at a certain point a difference between batch heating in tubes and the UHT process. Therefore, it was concluded that measurement at temperatures higher than 125°C cannot be carried out with the tube method described, but should be performed with a UHT sterilizer.
is not possible to specifically select for B. sporothermodurans spores. Only the unique heat resistance in the temperature range of 130—140°C might be used as a selective tool for the isolation of B. sporothermodurans from raw milk. D values of 3.4—7.9 s indicate an exceptionally high heat resistance of spores of B. sporothermodurans under UHT conditions (for B. stearothermophilus, D "0.9 s). TDT curves show different slopes for spores of B. sporothermodurans and B. stearothermophilus, with z" 13.1—14.2°C and 9.1°C, respectively. To the best of our knowledge there are no observations regarding other aerobic spores in the literature for which such high D values and z values have been demonstrated. Based on the data presented, solutions to inactivate these spores in UHT milk processing can be designed.
NOMENCLATURE D 2
e E k N N R ¹ h z
decimal reduction time; time (s) required for the number of spores to be reduced by a factor of ten at a given temperature ¹; the index ¹ is expressed in °C base of natural logarithms activation energy (kJ mol\) pre-exponential factor (s\) initial concentration of spores (cfu mL\) end concentration of spores (cfu mL\) universal gas constant 8.314 (J mol\ K\) absolute temperature (K) heating time (s) temperature change (°C) required to cause a tenfold change in D 2
ACKNOWLEDGEMENTS The authors thank Arjen A. Wagendorp for the genetic characterization of the isolates and Klaas Leffring for the carefully carried out UHT experiments. Part of the research work presented here was supported by Tetra Pak Research, Stuttgart, Germany (Dr Fritz Lembke). We thank Stork CRO, Nieuw Vennep, The Netherlands, for kindly providing equipment for the tube heating method.
REFERENCES Conclusions The observations presented in this article show that non-sterility in UHT milk can be caused by the survival of spores of B. sporothermodurans present in the milk prior to the heat treatment. B. sporothermodurans spores have an exceptionally high heat resistance at UHT temperatures, which is definitely different from the kinetic pattern of B. stearothermophilus spores. However, due to the specific kinetic characteristics of heat inactivation in the temperature range 110—120°C, the spores of B. sporothermodurans have heat resistance similar to or even lower than those of B. stearothermophilus, and their heat resistance is even very close to that of spores of B. subtilis. This means that within the temperature range that can be applied by common heating equipment in a laboratory, it
Amaha, M. and Ordal, J. Z. (1957) Effect of divalent cations in the sporulation medium on the thermal death rate of Bacillus coagulans var. thermoacidurans. Journal of Bacteriology 74, 596—604. Beaman, T. C. and Gerhardt, P. (1986) Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization and thermal adaption. Applied and Environmental Microbiology 52, 1242—1246. Behringer, R. and Kessler, H. G. (1992) Thermal destruction kinetics of spores of selected Bacillus strains in skimmilk and skimmilk concentrate. International Dairy Journal 2, 233—242. Burton, H., Perkin, A. G., Davies, F. L. and Underwood, H. M. (1977) Thermal death kinetics of Bacillus stearothermophilus spores at ultra high temperatures. III. Relationship between data from capillary tube experiments and from UHT sterilizers. Journal of Food ¹echnology 12, 149—161.
Heat resistance of Bacillus sporothermodurans spores Busta, F. F. (1967) Thermal inactivation characteristics of bacterial spores at ultra high temperatures. Applied Microbiology 15, 640—645. Cook, A. M. and Gilbert, R. J. (1968) Factors affecting the heat resistance of Bacillus stearothermophilus spores. II. The effect of sporulating conditions and nature of the heating medium. Journal of Food ¹echnology 3, 295—302. Condo´n, S. and Sala, F. J. (1992) Heat resistance of Bacillus subtilis in buffer and foods of different pH. Journal of Food Protection 55, 605—608. David, J. R. D. and Merson, R. L. (1990) Kinetic parameters for inactivation of Bacillus stearothermophilus at high temperatures. Journal of Food Science 55(2), 488—493. Davies, F. L. (1975) Heat resistance of Bacillus species. Journal of the Society of Dairy ¹echnology 28, 69—78. El-Bisi, M. H. and Ordal, Z. O. (1956a) The effect of certain sporulation conditions on the thermal death rate of Bacillus coagulans var. thermoacidurans. Journal of Bacteriology 71, 1—9. El-Bisi, M. H. and Ordal, Z. O. (1956b) The effect of certain sporulation conditions on the thermal death rate of Bacillus coagulans var. thermoacidurans. Journal of Bacteriology 71, 10—16. Esselen, W. B. and Pflug, I. J. (1956) Thermal resistance of putrefactive anaerobe PA3679 spores in vegetables in temperature range of 250—290°F. Food ¹echnology 11, 557. Etoa, F.-X. and Michielis, L. (1988) Heat-induced resistance of Bacillus stearothermophilus spores. ¸etters in Applied Microbiology 6, 43—45. Foschino, R., Galli, A. and Ottogalli, G. (1990) Research on the Microflora of UHT milk. Annali di Microbiologia ed Enzimologia 40, 47—59. Fox, K. and Pflug, I. J. (1968) Effect of temperature and gas velocity on dry heat destruction rate of bacterial spores. Applied Microbiology 16, 343—348. Franklin, J. C. (1970) Spores in milk: Problems associated with UHT processing. Journal of Applied Bacteriology 33, 180—191. Gaze, J. E. and Brown, K. L. (1988) The heat resistance of spores of Clostridium botulinum 213B over the temperature range 120 to 140°C. International Journal of Food Science and ¹echnology 23, 373. Hammer, P., Lembke, F., Suhren, G. and Heeschen, W. (1995) Characterization of a heat-resistant mesophilic Bacillus species affecting quality of UHT milk—a preliminary report. Kieler Milchwirtschaftliche Forschungsberichte 47, 297—305. Holdsworth, S. D. (1992) Kinetic factors for microbial destruction by wet heat. In Aseptic Processing and Packaging of Food Products, ed. S. D. Holdsworth. Elsevier Applied Science, London, New York, pp. 378—386. Jong, P. de (1996) Modelling and optimization of thermal processes in dairy industry. Thesis University Delft, Netherlands. Kessler, H. G., Pfeifer, J. and Schwoeppe, C. (1994) Untersuchungen ueber hitzeresistente mesophile Bacillus—Sporen in UHT Milch, Deutsche Milchwirtschaft 13, 588—592.
855
Klijn, N., Langeveld, L. P. M., and Weerkamp, A. H. (1994) Identification of a mesophilic Bacillus sp. producing spores with unusual heat-resistance. Proceedings of the 24th International Dairy Congress 1994, Australia, Ec17, posterabstract. Klijn, N., Herman, L., Langeveld, L. P. M., Wagendorp, A. A., Huemer, I. A. and Weerkamp, A. H. (1997) Genotypical and phenotypical characterization of Bacillus sporothermodurans strains, surviving UHT sterilisation. International Dairy Journal 7, 421—428. Lo´pez, M., Gonza´les I., Condo´n, S. and Bernardo, A. (1996) Effect of pH heating medium on the thermal resistance of Bacillus stearothermophilus spores. Journal of Food Microbiology 28, 405—410. Mayou, J. L. and Jezeski, J. J. (1977) Effect of using milk as a heating menstruum on the apparent heat resistance of Bacillus stearothermophilus spores. Journal of Food Protection 40, 228—231. Mazas, M., Gonzales, I., Lopez, M., Gonzales, J. and Sarmiento, R. M. (1995) Effects of sporulation media and strain on thermal resistance of Bacillus cereus spores. International Journal of Food Science and ¹echnology 30, 71—78. Navani, S. K., Scholefield, J. and Ribby, M. R. (1970) A digital computer program for the statistical analysis of heat resistance data applied to Bacillus stearothermophilus spores. Journal of Applied Bacteriology 33, 609—620. Peri, C., Pagliarini, E. and Pierucci, S. (1988) A study on optimizing heat treatment of milk. I. Pasteurization. Milchwissenschaft 43, 636—639. Petterson, B., Lembke, F., Hammer, P., Stackebrandt, E. and Priest, F. G. (1996) Bacillus sporothermodurans, a new species producing highly heat-resistant endospores. International Journal of Systematic Microbiology 46, 759—764. Sala, F. J., Ibarz, P., Palop, A., Raso, J. and Condo´n, S. (1994) Sporulation temperature and heat resistance of Bacillus subtilis at different pH values. Journal of Food Protection 58, 239—243. Segner, W. P., Frazier, W. C. and Calbert, H. E. (1963) Thermal inactivation of heat-resistant bacterial spores in milk concentrate at ultra high temperatures. Journal of Dairy Science 46, 891—896. Shehata, A. E., Khalafalla, S. M., Elmagdoub, M. N. I. and Hofi, A. A. (1977) Heat resistance parameters for spores of some Bacillus species in milk. Milchwissenschaft 32, 136—139. Stadhouders, J., Hup, G. and Langeveld, L. P. M. (1980) Some observations on the germination, heat resistance and outgrowth of fast-germinating and slow-germinating spores of B. cereus in pasteurized milk. Netherlands Milk and Dairy Journal 34, 215—228. Vogelsang, H. W. J. and de Jong, P. (1996) Reactorkundige beheersing van kaasmelkkwaliteit. NP¹ Procestechnologie 3(51), 15—20. Yokoya, K. and York, G. K. (1965) Effect of some environmental conditions on the thermal death rate of endospores of aerobic thermophilic bacteria. Applied Microbiology 13(6), 993—999.