E. Baril* 1,2, L. Coroller 2, I. Leguerinel 2, P. Mafart 2
1 ADRIA Developpement, Z.A. de Creac’h Gwen, 29 196 Quimper, France
2 Université Européenne de Bretagne, France - Université de Brest, EA3882 Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, IFR148 ScInBioS, UMT 08.3 PHYSI'Opt, 6 rue de l'Université, 29334 Quimper, France.
(Eugenie.baril@univ-brest.fr)
Introduction
Microbiological risk assessment is modelled tacking to account many environmental steps from raw ingredient to food consumption. However predictive microbiology lack of data about the bacterial sporulation behaviour (Nauta et al., 2003).
The effects of the sporulation temperature on heat resistance of Bacillus cereus spores have been widely investigated. Sporulation temperature has been shown among the major factors that impact on spore heat resistance (Palop et al., 1999). D100°C values were 10-fold higher for Bacillus cereus spores when the sporulation temperature increased from 20°C to 45°C (Gonzalez et al., 1999). Then, a linear relationship has been established between sporulation temperature and heat resistance of spores (Leguerinel et al., 2007) :
Where: T*spo is the reference sporulation temperature,
ZTspo is the distance of Tspo from T*spo which leads to a ten fold reduction in decimal reduction time.
Sporulation protocols used to investigate sporulation temperature effects were biased because of allowing growth before sporulation on rich nutrient media. These last ones allowed cellular growth until nutriment depletion (occurring at stationary phase) then initiation of sporulation. Incubation temperature during growth and sporulation were kept constant and identical. Consequently in those conditions, spore properties depend on adaptation during growth as well on sporulation environment.
In contrast, synchronous sporulation consisted in transferring vegetative cells from rich nutrient medium to a poor one(Jenkinson et al., 1980; Mandelstam & Higgs, 1974). It allows separating events due to adaptation during growth or sporulation processes.
In order to estimate a possible effect of stress adaptations during growth on spore heat resistance, our study compared the spore production and the spore heat resistance as a function of growth and sporulation temperatures.
Materials and methods
The studied bacterial strain was the Bacillus cereus psychrotrophic strain KBAB4, isolated from soil in France. Minimal, optimal and maximal growth temperatures of KBAB4 strain were estimated respectively at 7°C, 30°C and 43°C (Auger et al., 2008).
Growth was carried out in nutrient broth until the beginning of stationary phase, and then cells were transferred in the sporulation media. The sporulation media were a phosphate buffer to prevent growth and ensure sporulation.
On one hand, growth (in nutrient broth) and sporulation (in phosphate buffer) were performed at the same temperature (12°C, 20°C, 30°C or 35°C). On an others hand, growth were performed at optimal temperature (30°C) in nutrient broth then sporulation occurred at stress temperature (12°C, 20°C or 35°C) in phosphate buffer. Media were incubated until more than 99% of cells were spores.
Sporulation kinetics were estimated by counting viable cells on nutrient agar. Whole population and cells resistant to heat treatment of 70°C 5 minutes (spores) were enumerated. Harvested spores were
stored one month at 4°c before use. Spore heat resistance was determined by following thermal death kinetics at 85°C and 90°C.
Results and discussion
Times to obtain more than 99% of spores were rather independent of the growth temperatures (Table 1). Whatever growth and sporulation conditions, spore concentrations were close to 108spores/ml.
Table 1: Needed incubation times to standardize cellular physiological state.
Growth temperature Incubation time of growth
Sporulation temperature
Incubation time of sporulation
12°C 60 hours 12°C 7 days
30°C 6 hours 12°C 7 days
20°C 30 hours 20°C 6 days
30°C 6 hours 20°C 6 days
30°C 6 hours 30°C 3 days
30°C 6 hours 30°C 3 days
35°C 7 hours 35°C 3 days
30°C 6 hours 35°C 3 days
D85°C and D90°C values of spores formed at 12°C after growth at 12°C were not significantly different from those formed at 12°C after growth at 30°C. Growth temperature does not seem to have any significant effect on spore heat resistance (Figure 1). This suggests that the spore heat resistance properties are not influenced by adaptation of the vegetative cells during growth phase.
-5 -4 -3 -2 -1 0
0.00 5.00 10.00 15.00 20.00
time of heat treatment (minutes)
Survivors log(N/N0)
g12°C s12°C g30°C s12°C g20°C s20°C g30°C s20°C g30°C s30°C g30°C s30°C g35°C s35°C g30°C s35°C
Figure 1: Log (N/N0) versus heating time at 90°C for different incubation temperatures of growth and sporulation.
Spores of B. cereus KBAB4 produced at 12°C, 20°C and 35°C were less resistant than spores produced at 30°C. This suggests the existence of an optimal sporulation temperature, allowing formation of highly heat resistant spores. However, this optimal temperature concept is not consistent with the linear relationship between sporulation temperature and heat resistance of spores. This linear relationship could be improved. Few studies dealt with incubation temperature higher than the optimal one. Therefore lower heat-resistance possibly induced by sporulation at temperatures close to the upper limit of growth is neglected.
This work is supported by the Agence Nationale de la Recherche (ANR) (France) as part of an ANR-07-PNRA-027-07 MEMOSPORE contract, by the industrial association Bretagne Biotechnologies Alimentaires (BBA) and the French National Association of the Technical Research (ANRT).
References
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Jenkinson, H. F., Kay, D. & Mandelstam, J. (1980). Temporal dissociation of late events in Bacillus subtilis sporulation from expression of genes that determine them. Journal of Bacteriology 141, 793-805.
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