Flow cytometric analysis reveals culture-condition dependent variations in phenotypic heterogeneity of Limosilactobacillus reuteri

Industrial production of probiotic bacteria must be adapted to reach a stable product with resistance to unfavourable environmental conditions during storage and that display probiotic functionality. Flow cytometry-based descriptors, light scatter and pulse width of bacterial morphology were used as markers to estimate physiological fitness. The effects of temperature, pH and oxygen levels on cell growth and cell morphology of Limosilactobacillus reuteri DSM 17938 were measured. A change in morphology was observed from the measurement of light scatter and pulse width distributions. A pattern of high growth yielding smaller cells and less heterogeneous populations could be observed. Analysis of pulse width distributions revealed significant morphological heterogeneities within the bacterial cell population under non-optimal growth conditions. The fact that L. reuteri morphology varies depending on cultivation conditions suggests that it can be used as marker for estimating physiological fitness and responses to its environment. These subtle differences amongst cultures at the subpopulation level has large potential to be use as PAT tool in process control of morphology during fermentation. In this study, cell size and distribution did not correlate to survivability after freeze-thaw or freeze-drying stress but augured well for growth.