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Ó Springer-Verlag 1998
O R I G I N A L PA P E R
Â Paula Marõa Periago ? Pablo Salvador Fernandez Â Â Marõa Jose Ocio ? Antonio Martõnez Â Â
Apparent thermal resistance of Bacillus stearothermophilus spores recovered under anaerobic conditions
Received: 1 July 1997
AbstractmThe effect of recovering Bacillus stearothermophilus spores under anaerobic conditions on their apparent thermal resistance was studied. Spores were suspended in bidistilled water as a reference medium, heated at 115, 117, 119, 121, 123 and 125 °C and recovered under aerobic and anaerobic conditions. D values (decimal reduction time) obtained following recovery under anaerobic conditions were lower than those obtained under aerobic conditions. Reductions of between 31 and 48% were found for all the temperatures studied. When spores were suspended in mushroom extract and recovered under anaerobic conditions the apparent heat resistance was much lower than that obtained under aerobic conditions (D121 °C was 4.3 min and 1.7 min, under aerobic and anaerobic conditions, respectively). Heating the spores in mushroom extract and recovering the spores under anaerobic conditions produced an additive effect, decreasing the apparent heat resistance of the B. stearothermophilus spores. Key wordsmAnaerobiosis ? Injury ? Bacillus stearothermophilus ? Process optimization
Bacillus stearothermophilus spores have been used as a test of the efficiency of thermal processes (e. g. sterilization by steam under pressure) and as an indicator of spoilage of food by thermophilic species. They are heat resistant, and are relatively easy to work with, as they germinate and
Â P. M. Periago ? P. S. Fernandez ( ) ? M. Jose Ocio ? A. Martõnez1 Â Â Centro de Edafologõa y Biologõa Aplicada del Segura, Â Â Avenida de La Fama s/n, Apartado de Correos 4195, E-30080 Murcia, Spain Present address: 1 Instituto de Agroquõmica y Tecnologõa de Alimentos, Â Â Apartado de Correos 73, Paterna, Spain
grow under aerobic conditions at 60 °C and results can be obtained rapidly (48 h). Data obtained by suspending them in reference media have traditionally been used to establish the F0 value (sterilization value for a reference temperature of 121.1 °C and a Z value of 10 °C) required for the thermal processing of foods. If the food is highly contaminated with thermophiles, the heat treatment required to achieve commercially acceptable sterilization of the canned food can be very severe. Depending on the warehousing and distribution temperatures, a 2D to 6D sterilization process should be achieved for preservation against spoilage by thermophilic spore-forming organisms, which could require an F121.1 °C of up to 24 min . This is the case for canned mushrooms, where the heat treatment required, due to their high level of contamination, results in a considerable loss of yield, an increase in quality degradation, and very high costs of production (energy and labour) . Acidification is currently used during the canning of mushrooms to improve the product quality and to save energy . It has been shown that heat can damage spores and a manifestation of this injury is that heated spores become sensitive to the presence of different environmental factors. A fraction of heat-treated B. stearothermophilus spores was unable to grow when the recovery medium was acidified . The recovery of Clostridium sporogenes, C. botulinum and B. stearothermophilus heat-treated spores is also affected by the presence of NaCl in the recovery medium [5 ± 7]. Although spore injury may have beneficial effects in food processing, and there is information concerning the effect of different environmental factors on the heat resistance of B. stearothermophilus spores, very little is known about the effect of anaerobic conditions on the ability of this facultative anaerobe to grow from both unheated and heat-treated spores. It has been shown that the increase of the redox potential of the heating medium can either increase or reduce the heat resistance of different nonsporulated organisms . A significant effect of redox potential and oxygen concentration has been identified for different foodborne pathogens (Listeria monocytogenes, Salmonella enteritidis and Escherichia coli O157 : H7),
appeared in most cases both after aerobic and anaerobic incubation. . Germany) using glass beads to remove spore clumps. The proportion of NaCl in YDTAS (0.5 mm outer diameter. stearothermophilus spores incubated under anaerobic conditions in reference medium. Figures 1 and 2 show two typical examples of survival curves of spores heated in bidistilled water and mushroom extract at 121 °C incubated under both aerobic and anaerobic conditions. Materials and methods Spore production. 1. Spores were stored in bidistilled water at 4 °C until use. previously described to occur under aerobic conditions [12. Despite the heat shock activation.  was used. Aqueous suspensions of spores (0. B. and incubating for 48 h at 55 °C in aerobic and anaerobic conditions. Four replicate experiments were performed. the tubes were cooled in ice/water. The contents were flushed into a sterile tube containing 10 ml sterile distilled water using a syringe. For sporulation the proceÂ dure described by Fernandez et al.1-ml samples were dispensed in tubes containing 9. The average percentage of unheated spores suspended in bidistilled water that were inhibited by the anaerobic incubation was 19%.9 ml of distilled water and serial dilutions were performed. Effect of anaerobic conditions on apparent heat resistance Survival curves were obtained by plotting the logarithm of the number of colony-forming units (CFU) of spores per millilitre against heating time. Heat-resistance studies. Danaerobiosis being the value obtained following recovery in an anaerobic atmosphere and Daerobiosis the D value obtained under aerobic conditions. After the heating period. Activated spores were plated in aerobic and anaerobic conditions. Preparation of the mushroom extract. 75 mm length).7 mm inner diameter. UK). Two capillary tubes were used for each time ± temperature combination and another two were used as controls. For each temperature. cut. There is some evidence to suggest that. the same NaCl concentration was added to the recovery medium. When spores were heated in mushroom extract containing 2% NaCl. an initial shoulder.1 °C. which induced a decrease in the DT values obtained (time at a specified temperature to reduce the microbial population by 90%). Adams  pointed out that the redox potential of the recovery medium may be critical for heated clostridial spores. the second one contained 2% NaCl (w/v). 15]. as there has been no systematic study of the effect of anaerobic conditions on spore heat resistance.01 ml) were centrally injected using a micropipetting aid into ring-marked microhaematocrit capillary tubes (0. four replicate experiments were performed. stearothermophilus spores heated over the commercial sterilization temperature range is unknown. Samples containing Results Effect of anaerobic conditions on unheated spores An initial number of 100. Suspensions of spores in bidistilled water were activated by being heated at 100 °C for 15 min. Five different heating times. Fresh. a part of the spores could be injured. D values for individual experiments were obtained as the inverse negative straight portion of the survival curve (Table 1). although this kind of injury is poorly understood. evenly distributed. Inhibition of unheated spores due to the anaerobic conditions was calculated as the percentage of unheated spores that did not grow in anaerobic conditions. and were heated in a stirred oil bath. the aim of this work was to obtain heat-resistance data for between 115 and 125 °C for B. The extracts were placed in tubes and again sterilized at 121 °C for 20 min and stored at 4 °C until use. thermophilic spores have to germinate and grow in strong anaerobic conditions to produce spoilage.64 where anaerobic conditions during heating and recovery increased their apparent heat resistance . after heating. to evaluate the effect of anaerobiosis on unheated spores. Therefore. The final pH was 6. Percentages of the reduction in D values obtained after incubation in anaerobic conditions were expressed as (1±Danaerobiosis/Daerobiosis) ´ 100. Regression lines were established for each replicate at all the temperatures and conditions of this study. The number of unheated spores was based on duplicate counts from appropriate dilutions in YDTAS .5 ml) were mixed for 5 min at room temperature with a mixer (Heidolph Electro. Spores heated in natural mushroom extract were recovered in standard YDTAS. Feeherry et al. the effect of anaerobic conditions on B. compared to the counts obtained in aerobic conditions. Calculations. this injury revealing sensitivity to anaerobic conditions. The jars were then sealed and thermally processed at 121 °C for 20 min. Spores were activated as previously described. However. and ranged from 10 to 29%. Two batches were prepared: one batch was prepared with mushrooms without added NaCl. Recovery of heat-treated spores. activated spores (0.6 for both batches. In hermetically sealed foods. The processed product was then homogenized using a Polytron tissue homogenizer (Brinkman Polytron) and filtered through cheesecloth.000 spores per capillary tube was obtained in aerobic conditions.01 ml) and bidistilled water (0. sliced and placed in 454-g jars.02%) was taken into account to calculate the appropriate amount of this compound that had to be added. then 0. washed in soap and immersed in 70% ethanol. were used for each temperature. stearothermophilus ATCC 12980 was obtained from the Spanish Type Culture Collection. 118 and 121 °C in mushroom extract. Anaerobic conditions were obtained using BBL anaerobic jars (BBL GAS Pack Anaerobic System. Correlation coefficients of survival curves were always above 0.000 ± 300. and to compare these results with data produced by heating the spores in a vegetable substrate (mushroom extract) and then recovering them under anaerobic conditions. The tubes were then sealed by pulling off both ends in a gas oxygen flame.  reported an increased sensitivity of heatinjured spores to anaerobic conditions during incubation after heating at 121. before heat-resistance studies.98 and they all crossed at least one log cycle. Spores were heated to between 115 and 125 °C in bidistilled water and at 116. and the pH values were measured. The thermal resistance of spores in bidistilled water and mushroom extract was determined. Viable counts were based on duplicate counts from appropriate dilutions in YDTAS  and incubated for 48 h at 55 °C in aerobic conditions and for 120 h at 55 °C in BBL anaerobic jars. The heat-resistance test was carried out using a manual adaptation of the micromethod of Stern and Proctor . raw mushrooms (250 g) were washed. Effect of anaerobic conditions on heat-activated spores.
The correlation coefficient of the regression lines obtained by plotting the heating temperature versus D values was 0. activated (heatshocked at 100 °C for 15 min) spores suspended in bidistilled water.3) Danaerobiosisb (min) 24.70 (0.4) 10.9 31. There were significant differences (P #0. between 10 and 29%. Calculated as (1±Danaerobiosis/Daerobiosis) ´ 100 Decimal reduction time Temperature evolution inside the capillary tubes was monitored using type k thermocouples connected to a datalogger (Digitron Instruments.15 (1. In all cases.60 (0.82 (0.0) 13. e.70 (4. .4) 0.1) Reduction (%)a 33.5) 2.39 (0. The D values obtained after heating the spores in bidistilled water and recovering in aerobic and anaerobic conditions were statistically analysed using an ANOVA table (Statgraphics.0) 2.38 (5. The heating rate was fast (the time taken to reach the desired temperature was 6 s).3) 5.3) 10.7) 4. stearothermophilus spores heated at 121 °C in natural mushroom extract and recovered under aerobic and anaerobic conditions Table 2mMean D values for B.1 34.6) 1.12 (2.72 (0.4 35. Numbers in parentheses are standard deviations of D values Temperature ( °C) 115 117 119 121 123 125 a b Fig.1) Daerobiosisb (min) 37. Significant reductions in D values were also observed when heattreated spores were recovered anaerobically. i. For spores heated in mushroom extract.55 (0.5 47.90 (1. UK). 3) for spores heated in bidistilled water.3) 4.58 (0.05) between the D values obtained in aerobic and anaerobic conditions for all the heating temperatures studied.65 Fig.26 (0.2) Danaerobiosis (min) 2. Number in parentheses are standard deviations of D values Temperature (°C) 116 118 121 Substrate Mushroom + 2% NaCl Mushroom (no NaCl added) Mushroom (no NaCl added) Daerobiosis (min) 8. USA) (Table 1).1 47. The z value obtained in aerobic conditions was 6. This result does not agree with observations of Feeherry et al.17 (0.2) 1.76 (0.67 (Fig. stearothermophilus spores heated in mushroom extract and recovered under aerobic and anaerobic conditions.3)a 19. D values obtained after heating the spores in mushroom extract are shown in Table 2. indicates that anaerobic conditions have some effect on the ability of the spores to germinate and grow. 1mSurvival curves for B. who reported that .32 (0.07 (0.9 Effect on z values The z values (degrees of temperature for a ten-fold change in the D values) were obtained by plotting the logarithm of the D values against the heating temperature and were calculated as the negative inverse of the slope of the regression line obtained. the percentage reduction was higher than that observed following heating in bidistilled water. Discussion The percentage inhibition of unheated.27 (0.74 and in anaerobic conditions it was 6. stearothermophilus spores heated at 121 °C in bidistilled water and recovered under aerobic and anaerobic conditions Table 1mMean D values for Bacillus stearothermophilus spores heated in bidistilled water and recovered under aerobic or anaerobic conditions.999 in both cases.2) 1.1) 3. straight lines were produced for the temperature interval studied. The percentage reduction of D values caused by recovery in anaerobic conditions ranged from 31 to 48% (Table 1). 1985 ± 1992. 2mSurvival curves for B.
According to the D121 °C value calculated in optimal conditions. This appears to indicate an additive effect of the heating substrate. Okereke A. to achieve a 2D to 6D process when there is risk of spoilage by thermophilic bacteria (considering an initial contamination with 102 spores).2 min would be necessary. if the remaining conditions were considered to be optimal. as described previously .8 min in aerobic conditions). stearothermophilus spores. where high heat treatments are necessary to achieve microbiological stability. In these conditions. Therefore. particularly against thermophiles. the apparent heat resistance is lower than that obtained when NaCl is not present in mushroom extract (Table 2). The presence or level in the food of other environmental factors such as pH. Kilara A.2 min in aerobic conditions). pH.6 min. 4. Rodrigo M. NaCl and anaerobic conditions on the apparent heat resistance of termophilic spores. NaCl. Spores heated in mushroom substrate have a lower heat resistance than that observed when using a reference medium. 3mThermal death time curves for B. Sanchez T. Beelman RB. independently of the sterilization temperature.6 min). the lethality achieved by a heat treatment. In this study.7 min and D121 °C = 1. Ocio MJ. Interaction between different environmental factors affects B. recovery in anaerobic conditions significantly reduced their apparent heat resistance at all the temperatures studied.1 °C. it was observed that when spores are heated in mushroom substrate containing 2% NaCl (w/v). has been reported after anaerobic incubation . Fernandez PS. Thus. injury should play an important role in the delay or reduction of spoilage of hermetically sealed foods by thermophilic bacteria . 54%. References 1. The combined effects of heating substrate and pH .9 min. further studies should be carried out to establish the combined effect of food substrate. A low initial level of contamination with spores of thermophilic bacteria must be maintained in foods. higher than that obtained in the absence of NaCl (63%). if the quality of the food itself is to be retained. These results are particularly relevant to the canned mushrooms industry. This heat treatment would cause a severe loss in the quality of the final product. D121 °C = 4. 16] than that described by other authors.2 min in anaerobic and D116 °C = 8. When heat-treated spores were suspended in the reference medium. i.66 Fig. stearothermophilus in bidistilled water recovered under aerobic conditions was similar  or slightly higher [12. which would mean a significant reduction of the heat treatment.6 ± 39. and the effect of anaerobic conditions on heat-injured spores was independent of the heating temperature or length of heat treatment. Kuhn GD (1984) J Food Proc Preserv 8: 1 ± 14 Â 4. increasing the number of heated spores that can be recovered in anaerobic conditions. To define an adequate combination of all of these factors could be the key to determining good-quality products whilst at the same time maintaining their microbial stability against spoilage by thermophilic bacteria. There is also a reduction if spores are incubated in anaerobic conditions (D116 °C = 2. Gomez FJ. or of anaerobic conditions and recovery in acidified medium  have been shown to reduce the spores' thermal resistance. stearothermophilus spore heat resistance. AcknowledgementmThis project was funded by CYCIT (Project ALI94-0959-CO2-02). The thermal resistance of B. Therefore. etc. the reduction in D values obtained in anaerobic conditions was 75%. an F121 °C (sterilization value at 121 °C) of 19. this effect should be taken into account in processing and storage of these foods. McCord J. at the same time.4 min. indicating that the heat resistance of the spores used was high. A similar reduction in D121.4 ± 20. and the highest one found in this study. Â Â Martõnez A (1994) Lett Appl Microbiol 19: 146 ± 148 Â . The percentage of reduction achieved (60 ± 65%) is higher than that observed in the reference medium (31 ± 48%). If the effect of anaerobic conditions is taken into account (D121 °C = 2. Interactions between different factors are of the greatest importance in establishing an appropriate sterilization process for protection against spoilage by thermophilic bacteria and. i. since if spore levels were too high it would be impossible to ensure that all the spores were inactivated or injured during the thermal process.7 min in anaerobic and D118 °C = 10. stearothermophilus spores heated in bidistilled water obtained after aerobic and anaerobic incubation (heating temperature versus D value) anaerobic conditions did not affect the recovery of unheated spores. and recovered with the same NaCl content (2% NaCl). e. These results show the importance of anaerobic conditions for the apparent heat resistance of B. Witowski ME. Incubation in anaerobic conditions (Table 2) decreases the spores' D value considerably related to that observed under aerobic conditions (D118 °C = 3. The percentage reduction of D values ranges from 31 to 48%. Beelman RB (1990) J Food Sci 55: 1327 ± 1330 3. will influence the apparent heat resistance as well. e. as suggested by Pflug . the calculated F121 °C would be 10. Pflug IJ (1987) J Food Prot 50: 608 ± 615 2.
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