Abstract
Aiming at a superior performance, survival and stability of dairy starter cultures requires deeper
insights into physiological dynamics and relationships. This PhD thesis contributes to a more
comprehensive physiological understanding of Lactococcus lactis under conditions encountered
during industrial production by employing flow cytometry for viability assessment, cell size
comparison, intracellular pH (pHi) determination and cell sorting. The physiological studies of
L. lactis were complemented by examining the growth behavior, glucose consumption, lactate
production, culturability on solid medium and (specific) acidification activity in milk in response to
the extracellular pH (pHex) during batch fermentations and in response to stress during downstream
processing and storage as frozen, freeze- and spray-dried cells.
In this PhD thesis, in situ flow cytometric viability assessment was found to facilitate the
differentiation and accurate quantification of L. lactis cells in different physiological states, which
agreed with the reproductive viability of reference samples and of exponential cells. The high
viability of one particular L. lactis strain demonstrated its robustness during fermentation,
downstream processing and storage in the absence of a protectant. However, storing freeze-dried
L. lactis cells at 30 °C negatively affected the culturability and acidification activity.
The reactivation of freeze-dried cells in fermentation medium prior to flow cytometric viability
assessment, cell size comparison and pHi determination reflected the increasing physiological
impairment during this accelerated stability test, while a preincubation in buffer led to inconsistent
flow cytometric results. The comparison of reproductive and growth-independent viability
suggested the presence of cells showing metabolic activity and membrane integrity but being
non-culturable during storage.
Comparing the size of L. lactis cells during pH-controlled fermentations facilitated the
identification of relationships to growth behavior, glucose metabolism and specific acidification
activity. For instance, a higher specific acidification activity was maintained during fermentation
at pHex 5.5 which was linked to higher cell sizes and ongoing glucose metabolism.
In this PhD thesis, flow cytometric pHi determination was employed to examine the response
of L. lactis cells towards different pHex under non-growing conditions in buffer and showed the
relevance of the glucose availability and of the growth phase. Kinetics of pHi and pH gradients
(pHi – pHex) were recorded during L. lactis fermentations and provided valuable insights into the
relationships between pHi perturbations and recovery with regard to growth, growth inhibition and
survival. On the one hand, cells were able to tolerate and grow despite small negative or even
absent pH gradients at a high pHex of 7.5 although lag phase was prolonged and growth was
delayed. On the other hand, challenging cells by shifting the pHex upwards or downwards during
fermentations caused an immediate adaptation of the pHi and the growth behavior. The impact
of pHex on the physiology of L. lactis during fermentations was further shown by the earlier
inhibition of growth at low or decreasing pHex and by the continuation of growth until glucose
limitation at higher pHex. In this context, the pHi heterogeneity was found to reflect the
pH-dependent stress level causing these differences in growth which were presumably related to
the differences in non-dissociated lactic acid concentrations.
Because a negative impact of fluorescent labeling and cell sorting on L. lactis physiology was
detected in this PhD thesis, it was not possible to take advantage of applying flow cytometric
cell sorting for the subsequent characterization and recultivation of sorted cells.
In conclusion, this PhD thesis demonstrates both the complexity and the potential of flow cytometry
in physiological studies of L. lactis. An improved understanding of L. lactis physiology at the
single-cell level as well as of cell population heterogeneity will provide the basis for optimizing
industrial production processes in terms of biomass and activity.
insights into physiological dynamics and relationships. This PhD thesis contributes to a more
comprehensive physiological understanding of Lactococcus lactis under conditions encountered
during industrial production by employing flow cytometry for viability assessment, cell size
comparison, intracellular pH (pHi) determination and cell sorting. The physiological studies of
L. lactis were complemented by examining the growth behavior, glucose consumption, lactate
production, culturability on solid medium and (specific) acidification activity in milk in response to
the extracellular pH (pHex) during batch fermentations and in response to stress during downstream
processing and storage as frozen, freeze- and spray-dried cells.
In this PhD thesis, in situ flow cytometric viability assessment was found to facilitate the
differentiation and accurate quantification of L. lactis cells in different physiological states, which
agreed with the reproductive viability of reference samples and of exponential cells. The high
viability of one particular L. lactis strain demonstrated its robustness during fermentation,
downstream processing and storage in the absence of a protectant. However, storing freeze-dried
L. lactis cells at 30 °C negatively affected the culturability and acidification activity.
The reactivation of freeze-dried cells in fermentation medium prior to flow cytometric viability
assessment, cell size comparison and pHi determination reflected the increasing physiological
impairment during this accelerated stability test, while a preincubation in buffer led to inconsistent
flow cytometric results. The comparison of reproductive and growth-independent viability
suggested the presence of cells showing metabolic activity and membrane integrity but being
non-culturable during storage.
Comparing the size of L. lactis cells during pH-controlled fermentations facilitated the
identification of relationships to growth behavior, glucose metabolism and specific acidification
activity. For instance, a higher specific acidification activity was maintained during fermentation
at pHex 5.5 which was linked to higher cell sizes and ongoing glucose metabolism.
In this PhD thesis, flow cytometric pHi determination was employed to examine the response
of L. lactis cells towards different pHex under non-growing conditions in buffer and showed the
relevance of the glucose availability and of the growth phase. Kinetics of pHi and pH gradients
(pHi – pHex) were recorded during L. lactis fermentations and provided valuable insights into the
relationships between pHi perturbations and recovery with regard to growth, growth inhibition and
survival. On the one hand, cells were able to tolerate and grow despite small negative or even
absent pH gradients at a high pHex of 7.5 although lag phase was prolonged and growth was
delayed. On the other hand, challenging cells by shifting the pHex upwards or downwards during
fermentations caused an immediate adaptation of the pHi and the growth behavior. The impact
of pHex on the physiology of L. lactis during fermentations was further shown by the earlier
inhibition of growth at low or decreasing pHex and by the continuation of growth until glucose
limitation at higher pHex. In this context, the pHi heterogeneity was found to reflect the
pH-dependent stress level causing these differences in growth which were presumably related to
the differences in non-dissociated lactic acid concentrations.
Because a negative impact of fluorescent labeling and cell sorting on L. lactis physiology was
detected in this PhD thesis, it was not possible to take advantage of applying flow cytometric
cell sorting for the subsequent characterization and recultivation of sorted cells.
In conclusion, this PhD thesis demonstrates both the complexity and the potential of flow cytometry
in physiological studies of L. lactis. An improved understanding of L. lactis physiology at the
single-cell level as well as of cell population heterogeneity will provide the basis for optimizing
industrial production processes in terms of biomass and activity.
| Originalsprog | Engelsk |
|---|
| Forlag | Department of Food Science, Faculty of Science, University of Copenhagen |
|---|---|
| Antal sider | 160 |
| Status | Udgivet - 2016 |