Non-thermal inactivation of vegetative bacteria : kinetics methods and mechanisms
Creators: name: Zhang, DL
Publisher: name: University Of Tasmania
Year: 2023

description: The inactivation of microorganisms with treatments of temperature, pH and water activity is of vital importance to the food industry. In a previous study in fermented meat products (Ross et al., 2004), it was observed that temperature is the dominant factor governing the rate of inactivation of E. coli. This study investigates whether this observation is true for other microorganisms and the mechanisms behind the phenomena. To better characterize and understand non-thermal inactivation, this dissertation involves three main phases: kinetics investigation between two species, E. coli and L. monocytogenes, methods development (luminometry) and studies on mechanisms of non-thermal inactivation of L. monocytogenes, including quantitative real time PCR (QPCR) and microarrays analysis. To study non-thermal inactivation kinetics, Listeria monocytogenes, a foodborne bacterial pathogen with different characteristics to E. coli, was selected in this dissertation. Specifically, sixty-three inactivation rates were determined for both species at a non-growth-permissive pH and water activity (pH 3.50 and a\\(_w\\) 0.90 respectively) at nine growth permissive temperatures. The results showed that inactivation rates of both species were very similar, and the inactivation rate responses of both were comparable to those previously and independently reported for a variety of E. coli strains under a wide range of growth-preventing pH and water activity conditions (McQuestin, 2006). Thus, it appears that the influence of non-lethal temperature on the rate of inactivation of vegetative bacteria in inimical environments is not species-dependent. For methodology, luminometry, in which intracellular ATP level is measured by its ability to generate light using the luciferin/luciferase enzyme system has been investigated as means of quantifying microbial loads. To assess luminometry as a more rapid method of enumeration of bacteria in inimical environments, exponential phase L. monocytogenes ScottA and Fw 03/0035 were inactivated under inimical conditions (pH 3.50 and a\\(_w\\) 0.90) at 25¬¨‚àûC, 35¬¨‚àûC and 45¬¨‚àûC. Samples were periodically withdrawn for parallel viable count and luminometric analysis. The results showed that inactivation rates and kinetics determined by the ATP method were not comparable to those from viable counts. However, when both methods were applied to conditions permitting cell growth, there was a good correlation. Thus, the A TP method is not sensitive enough to quantify microbial inactivation, particularly when cells are inactivated in sub-lethal conditions; but it is well correlated with microbial growth. To better understand the physiology of bacterial cells in inimical environments, particularly whether they are metabolically active, tuf gene expression was studied using QPCR methods. L. monocytogenes strains ScottA and Fw 03/0035 were inactivated with the same conditions as that described for the luminometry experiments. Although viable cells numbers decreased from 10\\(^8\\) to less than the detection level (1.3 x 10\\(^1\\) cells ml\\(^{-1}\\)), the tuf gene mRNA level remained unchanged. To determine whether this relatively high level of mRNA was due to unexpected stability of the mRNA or due to de novo synthesis, additional experiments were undertaken. Cells were inactivated under either mildly lethal temperature (55¬¨‚àûC), in the presence of rifampin (which inhibits DNA-dependent RNA polymerase) or a combination of both. The results show that when the antibiotic was present tuf gene expression was reduced much more completely, with a three log reduction compared with mildly lethal temperature with higher tuf gene levels of only a half log reduction. This raises the possibility that L. monocytogenes under mildly lethal conditions of pH and a\\(_w\\) or high temperature retain viability after being rendered non-culturable. To explore the genetic responses to the inactivation phenomena observed, genomic microarray analysis was performed to determine the effects oflow pH (3.5) and low a\\(_w\\) (0.90) on exponential phase L. monocytogenes ScottA, in a time-course experiment (5 min, 24 h, 48 h, 72 h).The results suggest that a large number of genes relevant to amino acid biosynthesis and metabolism are up-regulated, indicating a possible switch to alternative carbon sources as an energy supply and aid to maintenance of cell integrity. Genes belonging to the categories of structure and function of cell wall, cell movements, and carbohydrate metabolism were down regulated indicating lowered mobility. The regulatory network might play an important role in regulating cellular physiological status and may dictate the rate of inactivation. In this dissertation, the hypothesis that temperature is the main factor governing the rate of inactivation of vegetative bacteria was firstly investigated and suggested to be non-species dependent and this will be very useful to understand microbiological safety of non-thermal processed food. For physiology of non-thermal inactivation, the thesis is fully addressed that when cells encounter environmental stresses, the regulatory network might play an important role in up-regulating and down-regulating house keeping genes to cope with sublethal conditions and that when they reach the point of completely losing their culturability, they may still remain viable, thus entering the state of viable, but non-culturable, cells.