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Non-thermal inactivation of vegetative bacteria : kinetics methods and mechanisms


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Zhang, Dong Lai 2008 , 'Non-thermal inactivation of vegetative bacteria : kinetics methods and mechanisms', PhD thesis, University of Tasmania.

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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 aw 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 aw 0.90) at 25°C, 35°C and 45°C. 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 108 to less than the
detection level (1.3 x 10 1 cells ml1), 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°C), 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 aw or high temperature retain viability after being rendered
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
aw (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
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.

Item Type: Thesis - PhD
Authors/Creators:Zhang, Dong Lai
Keywords: Listeria monocytogenes, Food, Foodborne diseases
Copyright Holders: The Author
Copyright Information:

Copyright 2008 the Author - The University is continuing to endeavour to trace the copyright owner(s) and in the meantime this item has been reproduced here in good faith. We would be pleased to hear from the copyright owner(s).

Additional Information:

Available for library use only and copying in accordance with the Copyright Act 1968, as amended. Thesis (PhD)--University of Tasmania, 2008. Includes bibliographical references. Ch. 1. Literature review -- Ch. 2. An investigation of the role of temperature in the inactivation rate of vegetative bacteria -- Ch. 3. Testing intracelullar ATP level as a rapid method for assessing microbial inactivation -- Ch. 4. Elongation factor EF-TU as an indicator of cell viability -- Ch. 5. Mechanisms of non-thermal inactivation - a time course study -- General summary and conclusion

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