Impact of Cultivation Parameters on Cell Physiology of Limosilactobacillus reuteri

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 parameter can be used to distinguish larger cells from smaller cells and to separate singlets from doublets (i.e., single cells from aggregated cells). Although, oxygen is known to inhibit growth in L. reuteri, controlled oxygen supply resulted in noticeable effect on the cell metabolism, in a higher degree of unsaturated fatty acids in the cell, and improved freeze-drying stress tolerance. Another important component that was examined was the addition of exogeneous fatty acid source in the form of Tween 80. A chemically defined minimal medium was developed, with 14 amino acids identified as essential for growth. The addition of Tween 80 to the medium improved biomass yield, growth rate, and shortened cultivation time. L. reuteri DSM 17938 may not be able to efficiently synthesise unsaturated fatty acid without an exogenous fatty acid source, but this requires further investigation. Lastly, the pre-formulation hold time during the manufacture of probiotics was found to significantly affect long-term stability, with direct freeze samples showing better freeze-drying stability compared to those subjected to rest for 3 h incubation at room temperature. These findings suggest that an optimised production process and formulation of agents can lead to the successful production of high-quality probiotics with excellent stability

Non-inhibitory levels of oxygen during cultivation increase freeze-drying stress tolerance in Limosilactobacillus reuteri DSM 17938

The physiological effects of oxygen on Limosilactobacillus reuteri DSM 17938 during cultivation and the ensuing properties of the freeze-dried probiotic product was investigated. On-line flow cytometry and k-means clustering gating was used to follow growth and viability in real time during cultivation. The bacterium tolerated aeration at 500mL/min, with a growth rate of 0.74 +/- 0.13h(-1) which demonstrated that low levels of oxygen did not influence the growth kinetics of the bacterium. Modulation of the redox metabolism was, however, seen already at non-inhibitory oxygen levels by 1.5-fold higher production of acetate and 1.5-fold lower ethanol production. A significantly higher survival rate in the freeze-dried product was observed for cells cultivated in presence of oxygen compared to absence of oxygen (61.8%+/- 2.4% vs. 11.5%+/- 4.3%), coinciding with a higher degree of unsaturated fatty acids (UFA:SFA ratio of 10 for air sparged vs. 3.59 for N-2 sparged conditions.). Oxygen also resulted in improved bile tolerance and boosted 5 ' nucleotidase activity (370U/L vs. 240U/L in N-2 sparged conditions) but lower tolerance to acidic conditions compared bacteria grown under complete anaerobic conditions which survived up to 90min of exposure at pH 2. Overall, our results indicate the controlled supply of oxygen during production may be used as means for probiotic activity optimization of L. reuteri DSM 17938

Non-inhibitory levels of oxygen during cultivation increase freeze-drying stress tolerance in Limosilactobacillus reuteri DSM 17938

The physiological effects of oxygen on Limosilactobacillus reuteri DSM 17938 during cultivation and the ensuing properties of the freeze-dried probiotic product was investigated. On-line flow cytometry and k-means clustering gating was used to follow growth and viability in real time during cultivation. The bacterium tolerated aeration at 500 ml/min, with a growth rate of 0.74 ± 0.13 h-1 which demonstrated that low levels of oxygen did not influence the growth kinetics of the bacterium. Modulation of the redox metabolism was, however, seen already at non-inhibitory oxygen levels by 1.5-fold higher production of acetate and 1.5-fold lower ethanol production. A significantly higher survival rate in the freeze-dried product was observed for cells cultivated in presence of oxygen compared to absence of oxygen (61.8 ± 2.4 % vs 11.5 ± 4.3 %), coinciding with a higher degree of unsaturated fatty acids (UFA:SFA ratio of 10 for air sparged vs 3.59 for N2 sparged conditions.). Oxygen also resulted in improved bile tolerance and boosted 5’nucleotidase activity (370 U/L vs 240 U/L in N2 sparged conditions) but lower tolerance to acidic conditions compared bacteria grown under complete anaerobic conditions which survived up to 90 min of exposure at pH 2. Overall, our results indicate the controlled supply of oxygen during production may be used as means for probiotic activity optimisation of L. reuteri DSM 17938

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

Optimisation of cultivation conditions in the industrial production of probiotics is crucial to reach a high-quality product with retained probiotic functionality. Flow cytometry-based descriptors of bacterial morphology may be used as markers to estimate physiological ftness during cultivation, and can be applied for online monitoring to avoid suboptimal growth. In the current study, the efects of temperature, initial pH and oxygen levels on cell growth and cell size distributions of Limosilactobacillus reuteri DSM 17938 were measured using multivariate fow cytometry. A pleomorphic behaviour was evident from the measurements 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 signifcant morphological heterogeneities within the bacterial cell population under non-optimal growth conditions, and pointed towards low temperature, high initial pH, and high oxygen levels all being triggers for changes in morphology towards cell chain formation. However, cell size did not correlate to survivability after freeze-thaw or freeze-drying stress, indicating that it is not a key determinant for physical stress tolerance. The fact that L. reuteri morphology varies depending on cultivation conditions suggests that it can be used as marker for estimating physiological ftness and responses to its environment

Chitosan flocculation associated with biofilms of C. saccharolyticus and C. owensensis enhances biomass retention in a CSTR

Cell immobilization and co-culture techniques have gained attention due to its potential to obtain high volumetric hydrogen productivities (QH2). Chitosan retained biomass in the fermentation of co-cultures of Caldicellulosiruptor saccharolyticus and C. owensensis efficiently, up to a maximum dilution rate (D) of 0.9 h−1. Without chitosan, wash out of the co-culture occurred earlier, accompanied with approximately 50% drop in QH2 (D > 0.4 h−1). However, butyl rubber did not show as much potential as carrier material; it did neither improve QH2 nor biomass retention in continuous culture. The population dynamics revealed that C. owensensis was the dominant species (95%) in the presence of chitosan, whereas C. saccharolyticus was the predominant (99%) during cultivation without chitosan. In contrast, the co-culture with rubber as carrier maintained the relative population ratios around 1:1. This study highlighted chitosan as an effective potential carrier for immobilization, thereby paving the way for cost – effective hydrogen production