Abstract
Bacillus cereus spores are a concern to the food industry, especially to the producers of heat sensitive food products like egg white that cannot be retorted. The research presented in this thesis investigated the impact of environmental conditions (temperatures of 4 - 25 ˚C, pH 4 - 9, the addition of egg yolk or the germinant L-alanine) on the survival or germination of B. cereus spores; the up or down regulation of genes; and the resistance of the spores to inactivation by thermal processing or pulse electric field (PEF) processing.
The B. cereus spores used in this thesis were isolated from commercial egg white during a shelf life study. Incubation at 4 °C and pH 4 prevented germination of the spores. Addition of the germinant L-alanine only induced germination if the spores were also heat treated (70 °C for a minimum of 10 min). An interesting finding was that the spores were not inert during cold storage in phosphate buffer (50mM, pH7.4) for 6 days (was not tested further), as evidenced by a reduction in their D-values at 88 and 92 °C by 13.9 and 8.2 min (p < 0.05) respectively. This loss of thermal resistance was accompanied by the appearance of deformations/cracks on the spore surface (scanning electron microscopy).
Exposure to either 0.05 or 0.5 M sodium phosphate buffer or sodium chloride solutions for 6 days at 4 ˚C resulted in the accumulation of sodium ions and the reduction of potassium ions (as monitored by inductively coupled plasma spectrometry) and a reduction in D88-values, in the absence of detectable germination. The D88-values of spores suspended in either sodium phosphate buffer (0.5 and 0.05 M) or sodium chloride (0.05 and 0.5 M) solutions reduced significantly by 10 min on day 6, while spores suspended in 0.5M calcium chloride and 0.5 M potassium phosphate buffer solutions showed a slight and significant (p<0.05) increase by approximately 2 min. Exposure of spores to Na+ in sodium phosphate buffer (pH7.0, 0.05 and 0.5 M) and sodium chloride (0.05 and 0.5 M) resulted in the cells accumulating Na+ (66.0±2.9, 193.1±4.6, 136.2±9.9 and 70.5±2.7 μg/g) at the significant expense of K+ (10.8±0.5, 7.5±0.2, 8.1±0.4 and 3.6±0.4 μg/g respectively) as compared to spores in deionized water (control) that showed lower Na+ (8.6±0.6μg/g) and higher K+ (31.7±2.4 μg/g) content. It was postulated that the reduction in the thermal resistance on exposure to Na+ may be due to the activation of the Na+ K+ antiporter leading to K+ expulsion in turn associated with hydration. Though the mechanism needed further proof, it was evident that spores were responsive during cold storage and indicating a possibility to reduce the D-values without inducing any germinants in the medium. The accumulation of sodium ions and the reduction of potassium ions were associated with a loss of thermal resistance B. cereus spores regardless of any onset of metabolic activity (resazurin assay) or germination (plating methods).
To understand the molecular mechanism behind the loss of resistance during cold storage, differential gene expression was monitored using mRNA sequencing. Genes coding for uracil permease (BC_3890), Mg2+ P-type ATPase-like protein (BC_1581), ABC transporter ATP-binding protein (BC_0815) and 2-keto-3-deoxygluconate permease (BC_4841) were found to be upregulated indicating a possible transport of ions that may have disrupted the integrity of spores during cold storage (for 6 days) confirming that spores were not completely inert.
Since spores were not found to be inert during cold storage, their response to Pulsed electric field treatment was investigated. The resistance of Bacillus spores towards the electroporation during PEF treatment is postulated mainly due to the presence of cortex and a densely crosslinked peptidoglycan structure of the spore coat. The effect of PEF treatment with mild heat (< 80 ˚C) on B. cereus spores was investigated by monitoring inactivation, germination and D-values. PEF treatment of B. cereus spores in phosphate buffer, when combined with heat (80 ˚C) at 9.4, 8.8, 8.1 and 7.3 kV/cm resulted in the inactivation 3.4±0.01, 1.9±0.01, 1.8±0.02 and 1.8±0.01 log CFU spores/mL. At 75 ˚C the spore numbers decreased by log 1.6±0.02, 1.5±0.01, 1.4±0.01 and 1.3±0.01 CFU spores/mL at 9.4, 8.8, 8.1 and 7.3 kV/cm, respectively. PEF treatment (9.4 kV/cm) at 80 and 75 ˚C, led to a loss of thermal resistance (D88 values) by 11.5 and 13.2 min, respectively. This effect of PEF was also investigated by mRNA sequencing, which revealed upregulation of chitooligosaccharide deacetylase gene (BC1768) that encodes for an enzyme targeting GlcNAc residue of N-acetyl-D-glucosamine-(β-1,4)-N-acetylmuramyl-L-alanine-D-isoglutamine. GlcNAc is one of the most important cell wall component and the deacetylases are usually involved in the hydrolyses indicating disruption to the spore covering, which could be the inner, outer membrane or cortex as all three contain peptidoglycan layers of different thickness. The loss of structural integrity might have led to the subsequent loss of thermal resistance. Also, gene (BC2729) encoding for penicillin-binding protein, required for both the initiation of division and continued septal ingrowth in sporulation during septation was found to be downregulated (-3.5 log2-transformed fold change value) suggesting a possible arrest in the usual process of sporulation or maintaining the resistance against harsh conditions. PEF treatment on B. cereus spores was found to be a stress inducer that was interfering with the permeability barriers (inner or outer membrane) in the dormant spores and reducing the spore’s thermal resistance. Thus, PEF treatment could be a suitable hurdle step to reduce the degree of heating required to inactivate B. cereus spores.
The thesis revealed the selective permeability of B. cereus spores during cold storage to be their “Achilles heel”, which reduces their thermal resistance. This is also the first evidence of the impact of PEF treatment and cold storage on differential gene expression in B. cereus spores and is believed to provide new strategies for inactivation regimes.