Abstract
Beer is a very harsh environment for bacterial growth due to various inhibitory factors. Hop
compounds are believed to be the key stress factor, with additional stress from alcohol, low pH and
lack of nutrients. However, a few specialist microorganisms, in particular Lactobacillus brevis,
possess the ability to withstand the harsh conditions, resulting in customer dissatisfaction and
economic loss. Therefore, it is important to understand the bacterial stress response and the
underlying tolerance mechanisms, and consequently perform a fast detection and an effective
cleaning process in order to prevent biological contaminations in breweries. The present PhD thesis
has explored the physiological response of L. brevis towards oxidizing disinfectants and beerassociated
stress (mainly hop stress) at the single cell level, as well as the genetic difference
between beer tolerant and sensitive strains.
The first study was devoted to developing a rapid colony-based method for investigating the
influence of oxidizing disinfectants on hop tolerant L. brevis strains. The method was based on an
automated microscope, combined with the membrane impermanent dye (propidium iodide, PI). It
provided comparable results to determination of colony forming units (CFU), only faster. The
results showed that there was no clear relationship between the tolerance to hop compounds and to
peracetic acid (PAA). A phenotypic heterogeneity was observed, not only between two strains, but
also within one strain. Moreover, the addition of PI to the agar showed that PAA does not destroy
the cell membrane. Furthermore, the dead cells appeared randomly within a micro-colony during
growth. This novel approach allowed the rapid analysis of bacterial viability after a disinfectant
treatment, and potentially the method could be applied to detect spoilage bacteria in breweries.
In the second study, for the first time, the physiological response of L. brevis to beer-associated
stress (mainly hop stress) was investigated at the single cell level. Based on the use of different
fluorescent probes, cell viability was assessed by fluorescence microscopy combined with flow
cytometry, and the intracellular pH (pHi) was determined by fluorescence ratio imaging microscopy
(FRIM). Different physiological subpopulations (viable, intermediate, damaged and debris) could
be observed when the cells were exposed to hop compounds. The viability results indicated that a
large proportion of cells were killed in all the tested strains, but a small subpopulation from the hop
tolerant strains eventually recovered, as revealed by pHi measurements. In addition, the study dealt
with the influence of manganese and ethanol on hop tolerance of L. brevis. It was found that Mn2+
caused a short-term protection against hop compounds in all strains, but it did not benefit the
sensitive strains during long-term incubation. On the other hand, ethanol resulted in an additional
short-term damage, but the subsequent growth pattern indicated a slight cross-resistance toward hop
compounds.
The third study focused on understanding the phenotypic difference in strains of L. brevis isolated
from beer, by investigating the genome of three tolerant strains and three sensitive strains. One of
the sensitive strains was derived from one tolerant strain by plasmid curing with novobiocin. The
genetic difference between the original strain and the plasmid-cured strain confirmed that the
plasmid-localized genes, such as horA and hitA, play an important role in beer spoilage ability.
However, the presence of horA and hitA in another sensitive strain and the absence of these two
genes in one of the tolerant strains, indicated that the beer-spoilage phenotype did not solely depend
on the previously described hop-tolerance genes. Furthermore, genes encoding a Clp protease, a
replication protein and a manganese transporter other than HitA were identified as novel beer
tolerance genes.
In conclusion, this PhD project provides new insight into how beer isolated bacteria respond to
brewing-associated stresses at the single cell level. Additionally, the micro-colony based method, as
well as the increased knowledge on beer-spoilage genes, could be further utilised for detection of
beer spoilage bacteria in breweries.
compounds are believed to be the key stress factor, with additional stress from alcohol, low pH and
lack of nutrients. However, a few specialist microorganisms, in particular Lactobacillus brevis,
possess the ability to withstand the harsh conditions, resulting in customer dissatisfaction and
economic loss. Therefore, it is important to understand the bacterial stress response and the
underlying tolerance mechanisms, and consequently perform a fast detection and an effective
cleaning process in order to prevent biological contaminations in breweries. The present PhD thesis
has explored the physiological response of L. brevis towards oxidizing disinfectants and beerassociated
stress (mainly hop stress) at the single cell level, as well as the genetic difference
between beer tolerant and sensitive strains.
The first study was devoted to developing a rapid colony-based method for investigating the
influence of oxidizing disinfectants on hop tolerant L. brevis strains. The method was based on an
automated microscope, combined with the membrane impermanent dye (propidium iodide, PI). It
provided comparable results to determination of colony forming units (CFU), only faster. The
results showed that there was no clear relationship between the tolerance to hop compounds and to
peracetic acid (PAA). A phenotypic heterogeneity was observed, not only between two strains, but
also within one strain. Moreover, the addition of PI to the agar showed that PAA does not destroy
the cell membrane. Furthermore, the dead cells appeared randomly within a micro-colony during
growth. This novel approach allowed the rapid analysis of bacterial viability after a disinfectant
treatment, and potentially the method could be applied to detect spoilage bacteria in breweries.
In the second study, for the first time, the physiological response of L. brevis to beer-associated
stress (mainly hop stress) was investigated at the single cell level. Based on the use of different
fluorescent probes, cell viability was assessed by fluorescence microscopy combined with flow
cytometry, and the intracellular pH (pHi) was determined by fluorescence ratio imaging microscopy
(FRIM). Different physiological subpopulations (viable, intermediate, damaged and debris) could
be observed when the cells were exposed to hop compounds. The viability results indicated that a
large proportion of cells were killed in all the tested strains, but a small subpopulation from the hop
tolerant strains eventually recovered, as revealed by pHi measurements. In addition, the study dealt
with the influence of manganese and ethanol on hop tolerance of L. brevis. It was found that Mn2+
caused a short-term protection against hop compounds in all strains, but it did not benefit the
sensitive strains during long-term incubation. On the other hand, ethanol resulted in an additional
short-term damage, but the subsequent growth pattern indicated a slight cross-resistance toward hop
compounds.
The third study focused on understanding the phenotypic difference in strains of L. brevis isolated
from beer, by investigating the genome of three tolerant strains and three sensitive strains. One of
the sensitive strains was derived from one tolerant strain by plasmid curing with novobiocin. The
genetic difference between the original strain and the plasmid-cured strain confirmed that the
plasmid-localized genes, such as horA and hitA, play an important role in beer spoilage ability.
However, the presence of horA and hitA in another sensitive strain and the absence of these two
genes in one of the tolerant strains, indicated that the beer-spoilage phenotype did not solely depend
on the previously described hop-tolerance genes. Furthermore, genes encoding a Clp protease, a
replication protein and a manganese transporter other than HitA were identified as novel beer
tolerance genes.
In conclusion, this PhD project provides new insight into how beer isolated bacteria respond to
brewing-associated stresses at the single cell level. Additionally, the micro-colony based method, as
well as the increased knowledge on beer-spoilage genes, could be further utilised for detection of
beer spoilage bacteria in breweries.
| Originalsprog | Engelsk |
|---|
| Forlag | Department of Food Science, Faculty of Science, University of Copenhagen |
|---|---|
| Status | Udgivet - 2017 |