نبذة مختصرة : Optimisation of cultivation conditions in the industrial production of probiotics is crucial to reach a high-quality product with high probiotic functionality. The production process includes a fermentation step to produce biomass, accompanied by centrifugation to concentrate the cells. Subsequently, the cells are treated with stabilising solutions (lyoprotectants) before they are subjected to freezing. The frozen cell pellets can be subjected to freeze-drying to yield a dried final product. The probiotic product needs to withstand adverse environmental conditions both during production and after consumption (gastro-intestinal tract). The objective of this study was to elucidate the cellular response to various production process parameters and evaluate their influence on freeze-drying tolerance. In addition, the stability and probiotic activity of freeze-drying product was studied. Parameters such as temperature, pH, oxygen, media components during fermentation, and the pre-formulation hold time prior to freeze-drying were in focus. Furthermore, flow cytometry-based descriptors of bacterial morphology were evaluated for their potential correlation with process-relevant output parameters and physiological fitness during cultivation to avoid suboptimal growth. Additionally, a pipeline was developed for online flow cytometry combined with automated data processing using the kmeans clustering algorithm, which is a promising process analytical technology tool. The effects of temperature, initial pH, and oxygen levels on cell growth and cell size distributions of Limosilactobacillus reuteri DSM 17938 were investigated using multivariate flow cytometry. Morphological heterogeneities were observed under non-optimal growth conditions, with low temperature, high initial pH, and high oxygen levels triggering changes in morphology towards cell chain formation. High-growth pattern characterised by smaller cell sizes and decreased population heterogeneity was observed using the pulse width distribution parameter. This ...
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