can be lost over time, this technology is worth investigation and could be used to produce cells with different physiologies and to apply stresses under well-controlled conditions. An example of a two-stage continuous fermentation process for the production of stress-adapted probiotics is illustrated inFigure 2a. This type of continuous system could provide a new tool for the more efficient screening of sublethal stresses than conventional batch cultures, because the system stabilizes rapidly to changing conditions in the second reactor (after 5 to 7 residence times, corresponding to a few hours). Currently, no data are available on the potential and limitations of continuous fermentations for probiotic production with controlled physiology.Membrane bioreactorsIn a membrane system with continuous feeding of fresh medium, cells are retained in the bioreactor by an ultrafiltration or microfiltration membrane whereas small molecules diffuse through the pores of the membrane according to their size. Therefore, inhibitory metabolic products are eliminated in the permeate and cells are concentrated on the retentate side. The concentrated cell fraction can be harvested batch-wise or continuously with no, or minimal, additional downstream treatment for cell concentration before freezing or freeze-drying. Few data have been reported on probiotic cell production in membrane reactors, although these systems could result in high cell yields and volumetric productivities. Taniguchi et al. reported final B. longumconcentrations in a membrane bioreactor that were seven times higher than those obtained during free-cell batch fermentations. Similarly, Corre et al. measured high cell yields and a 15-fold improvement of volumetric productivity compared with free-cell batch cultures forBifidobacterium bifidum. However, cell physiology and functionality were not studied. Indeed, cells are subjected to many stresses in membrane bioreactors, such as low nutrient concentration, oxygen, osmotic and mechanical stresses that could affect sensitive bacteria, but might also lead to cross-protection effects for other stresses. Finally, close cell-to-cell contacts owing to high cell density in the retentate section might induce quorum-sensing responses affecting cell physiology. Research is needed on these factors and their effects on physiology, fitness and functionality of probiotics to explore the potential of these systems for the production of stress-adapted cells.Two-stage continuous fermentation processes for the production of stress-adapted probiotics, with a first reactor (R1) operated with free or immobilized cells and a second
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