Effects of heat and high pressure on Bacillus cereus spores: strain variability, kinetics and combination with the antimicrobial carvacrol

Bacillus cereus is an important food-borne pathogen. It is primarily of concern in mildly processed refrigerated foods with an extended shelf-life because it can form heat-resistant spores and because some spores can germinate and grow at temperatures as low as 4°C. B. cereus can cause two different types of food-borne disease, designated as the emetic and diarrheal syndrome, respectively. The taxonomy of these bacteria is complex and has evolved considerably over the last decade. B. cereus is genetically much related to six other Bacillus species, and together, these are now referred to as B. cereus sensu lato. Based on molecular-genetic criteria, B. cereus sensu lato is subdivided into seven phylogenetic groups, but these do not exactly coincide with the original species delineation, and food-borne pathogens of B. cereus sensu stricto are distributed over at least four phylogenetic groups. Adding further to the complexity, food-borne pathogens are also increasingly found among some of the other species of B. cereus sensu lato. The advanced taxonomic insights together with the trend towards milder processing in the food industry call for more detailed studies on properties of spores from the different phylogenetic groups of B. cereus, and in particular on their resistance to traditional and novel food preservation methods. In the first part, the thermal inactivationkinetics of spores from 39 B. cereus sensu lato strains representing six phylogenetic groups (group II to VII) were studied at three or more different temperatures for five different holding times in potassium phosphate buffer. Survival curves and thermal death time curves were established and the kinetic parameters DT (decimal reduction time at temperature T) and z (temperature dependence of DT) were derived by linear regression. Most strains (38/39) had survival curves without a pronounced shoulder or tail, as reflected by linear regression coefficients R2 generally higher than 0.95. Generally, there was a considerable strain-to-strain variation in most phylogenetic groups. However, spores from psychrotolerant strains (group VI) showed the lowest heat resistance, while spores from thermotolerant strains (groups III and VII) were generally most heat resistant. Further analysis revealed a positive correlation betweenspore heat resistance and both minimal and maximal growth temperature of the strains. In contrast, the z value was negatively correlated with the minimal and maximal growth temperature. The data of phylogenetic group-specific heat resistance provided here will contribute to a refinementof the existing risk assessment models of B. cereus, taking into account group-specific D and z values, as well as novel insights in the pathogenicity of strains in each phylogenetic group. In the second part, since the susceptibility of B. cereus spores of differentgenetic groups to high pressure (HP) at moderate temperature has not been investigated, we studied the effect of HP treatment at 600800 MPa, 2060°C, for 15 minholding time on spores from the same collection of B. cereus strains (except two strains) suspended in potassium phosphate buffer. The results revealed that HP treatment induced spore germination as well as spore inactivation. Both outcomes exhibited a considerablestrain to strain variation, but no significant differences betweendifferent phylogenetic groups. Spore inactivation increased only marginally with pressure (from 600 to 800 MPa), and more strongly with temperature (from 20 to 60°C). However, even under the most severe treatments, a 6-D reduction the commonly used standard for pasteurization of low acid foods with a long refrigerated shelf life could not be reached.  High pressure treatment at elevated temperature (>60°C) (HPHT) has been shown to have potential for food appertization because it can effectively inactivate bacterial spores. However, little information is available about inactivation of B. cereus spores by this process. An interesting question, both from a basic scientific point of view and withan outlook on industrial applications, is whether and to what extent the HP component of HPHT processes contributes to spore inactivation. In the literature, both synergistic and antagonistic interactions between pressure and heat have been observed. Therefore, in the third part, the inactivation kinetics of spores from the heat resistant B. cereus strain F4430/73 by high pressure treatment at elevated temperature (HPHT; 600 MPa, 60100°C) and by conventional thermal treatment (HT: 0.1 MPa, 60100°C) were compared in MES buffer system. HT inactivation could be described by a first-order kinetic model, while HPHT inactivation showed a fast followed by a slow phase. Significant spore inactivation occurred during pressure come-up and equilibration time (2.0 to 4.1 log10, from 70100°C). HPHT inactivation proceeded considerably faster than HT inactivation, even in the slow phase which also followed first-order kinetics. The D values in the slow phase at 0.1 and 600 MPa also showed a distincttemperature dependency, with z values significantly lower in the case of HT compared to HPHT. These results indicate a strong synergistic effect of HP and HT on inactivation of B. cereus spores, similar to whatmost other studies have observed for spores from other bacteria. Finally, in a fourth part, the effect of HPHT treatment at different temperatures (600 MPa, 50100°C with 5°C increments, 5 min holding time) on the release of dipicolinic acid from the spores and on the loss of sporerefractility was investigated, both in the absence and in the presence of the natural antimicrobial carvacrol. The objective here was to provide mechanistic insights in the impact of HPHT on B. cereus spores, but also to explore the potential of this natural antimicrobial to improve the efficacy of HPHT treatment in a hurdle-type approach. Spore inactivation by HPHT was less than about 1 log10 at 50 to 70°C, but gradually increased at highertemperatures up to about 5 log10 at 100°C. DPA release and loss of spore refractility in the spore population were higher at moderate (≤ 65°C) than at high (≥ 70°C) treatment temperatures, and we propose that moderate conditions induced the normal physiological pathway of spore germination resulting in fully hydrated spores, while at higher temperatures this pathway was suppressed and replaced by another mechanism of pressure-induced dipicolinic acid (DPA) release thatresults only in partial spore rehydration, probably because spore cortex hydrolysis is inhibited. Remarkably, carvacrol strongly suppressed DPArelease and spore rehydration during HPHT treatment at ≥ 65°C and also partly inhibited DPA release at ≥ 65°C. Concomitantly, HPHT spore inactivation was reduced by carvacrol at 6590°C but unaffected at 95100°C.In summary, this work provides novel information on the inactivation of B. cereus spores that will be useful to optimize processing of foods by heat and high pressure treatment, Firstly,we described the variation in resistance of different B. cereus spores belonging to different phylogenetic groups towards heat and high pressure at moderate temperature. Secondly, we provide detailed kinetic analysis of the HPHT inactivation of the spores of a heat-resistant B. cereus strain, showing a strongly synergistic interaction of heatand pressure. The addition of carvacrol does not increase the efficacy of spore inactivation by HPHT treatment and, at 6590°C, even decreases the efficacy. Thirdly, based on analysis of spore refractilityand DPA release, we propose that HP induces the normal physiological pathway of spore germination up to about 65°C, while at higher temperatures this pathway is suppressed and replaced by another mechanism of pressure-induced DPA release. 

Keywords:High temperature, High pressure

Disciplines:Food sciences and (bio)technology, Microbiology, Systems biology, Laboratory medicine, Other chemical sciences, Nutrition and dietetics, Agricultural animal production, Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering

Project type:PhD project