Rapid assessment of Oenococcus oeni activity by measuring intracellular pH and membrane potential by flow cytometry, and its application to the more effective control of malolactic fermentation.

The aim of this study is to highlight the changes in the physiological cellular state of Oenococcus oeni during malolactic fermentation (MLF), and to use its cellular parameters to improve existing knowledge of O. oeni behaviour and to more effectively control the performance of the bacteria during MLF in wine. To do this, measurements of intracellular pH, transmembrane potential and vitality were performed using flow cytometry with different fluorescent probes: CFDA-SE and CDCF, DiBAC and CFDA, respectively. The kinetics of the cellular changes in these parameters were determined during MLF in FT80 synthetic medium and in white wine, as were the kinetics of malic acid consumption. pHin measurement throughout the entire growth shows that the pH was equal to the pH of the culture medium during the early stage, increased to pH6 in the exponential phase, and then decreased to equilibrate with the pH of the medium in the late stationary phase. Membrane potential increased in early MLF and then decreased. The decrease in pHin and membrane potential occurred when all of the malic acid was consumed. Finally, we showed that the higher the ΔpH (pHin-pHex) in O. oeni cells was, the shorter the lag phase of the MLF was. To better manage the initiation of MLF in wines, the physiological state of O. oeni cells must be taken into account. These results allow us to understand the sometimes random initiation of MLF in wines inoculated with O. oeni and to suggest ways to improve this control.

Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp. lactis TOMSC161.

This work aimed at characterizing the biochemical and biophysical properties of the membrane of Lactococcus lactis TOMSC161 cells during fermentation at different temperatures, in relation to their freeze-drying and storage resistance. Cells were cultivated at two different temperatures (22 and 30 °C) and were harvested at different growth phases (from the middle exponential phase to the late stationary phase). Bacterial membranes were characterized by determining the fatty acid composition, the lipid phase transition, and the membrane fluidity. Cultivability and acidification activity losses of L. lactis were quantified after freezing, drying, and 3 months of storage. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to lipid composition, and it was established that the cyclopropanation of unsaturated fatty acids with concomitant membrane rigidification during growth led to an increase in the freeze-drying and storage resistance of L. lactis. As expected, cultivating cells at a lower fermentation temperature than the optimum growth temperature induced a homeoviscous adaptation that was demonstrated by a lowered lipid phase transition temperature but that was not related to any improvement in freeze-drying resistance. L. lactis TOMSC161 was therefore able to develop a combined biochemical and biophysical response at the membrane level during fermentation. The ratio of cyclic fatty acids to unsaturated fatty acids (CFA/UFA) appeared to be the most relevant parameter associated with membrane rigidification and cell resistance to freeze-drying and storage. This study increased our knowledge about the physiological mechanisms that explain the resistance of lactic acid bacteria (LAB) to freeze-drying and storage stresses and demonstrated the relevance of complementary methods of membrane characterization.

Rapid enumeration of Oenococcus oeni during malolactic fermentation by flow cytometry.

AIMS: The aim of this study was to provide a method to rapidly enumerate Oenococcus oeni cells during malolactic fermentation (MLF). To keep MLF under control, it is important to monitor the growth of the bacteria O. oeni. However, the enumeration of O. oeni using the plate count technique requires a very long incubation time of about 10 days or more, which is not adapted to monitoring MLF in real time. METHODS AND RESULTS: Flow cytometry (FCM), in combination with several fluorescent probes, is a rapid method for counting large numbers of bacterial cells. However, probes based on fluorescein [FDA, carboxyfluorescein diacetate (cFDA)] did not give good results for O. oeni. For the first time, we propose using the BacLight™ kit for enumeration of O. oeni, and we compare the results with three methods: plate count and FCM, in combination with either fluorescein probes or the BacLight™kit. The last method provides a perfect correlation with the plate count method. CONCLUSIONS: FCM coupled with the Baclight™ kit makes it possible to count O. oeni cells during MLF with a perfect correlation with the plate count method. SIGNIFICANCE AND IMPACT OF THE STUDY: The result is obtained in 20 min vs 10 days with the reference method which will be very useful for wine microbiologists. Moreover, it should be emphasized that FDA/cFDA staining is not recommended because it can lead to an erroneous count during the latency period or at the end of growth due to the variation of intracellular pH in the O. oeni cells during growth.

Assessment of the dynamics of the physiological states of Lactococcus lactis ssp. cremoris SK11 during growth by flow cytometry.

AIMS: The aim of this study was to improve knowledge about the dynamics of the physiological states of Lactococcus lactis ssp. cremoris SK11, a chain-forming bacterium, during growth, and to evaluate whether flow cytometry (FCM) combined with fluorescent probes can assess these different physiological states. METHODS AND RESULTS: Cellular viability was assessed using double labelling with carboxyfluorescein diacetate and propidium iodide. FCM makes it possible to discriminate between three cell populations: viable cells, dead cells and cells in an intermediate physiological state. During exponential and stationary phases, the cells in the intermediate physiological state were culturable, whereas this population was no longer culturable at the end of the stationary phase. CONCLUSIONS, AND IMPACT OF THE STUDY: We introduced a new parameter, the ratio of the means of the fluorescence cytometric index to discriminate between viable culturable and viable nonculturable cells. Finally, this work confirms the relevance of FCM combined with two fluorescent stains to evaluate the physiological states of L. lactis SK11 cells during their growth and to distinguish viable cells from viable but not culturable cells.