Next-generation multiparameter flow cytometry assay improves the assessment of oxidative stress in probiotics

peer-reviewedStability of probiotic products’ potency throughout shelf life is essential to ensure systematic delivery of the dosages required to provide clinically-proven health benefits. Due to the oxygen sensitivity of gut-derived microorganisms, methods for the rapid and accurate monitoring of oxidative stress in probiotics are greatly needed as they can be instrumental to both bioprocess optimization and quality control. This study introduces a next-generation flow cytometry method multiplexing the CellROX® Green and Propidium Iodide probes for the simultaneous measurement of free total reactive oxygen species (ROS) and membrane integrity, respectively. The multiparameter method was compared to the single-parameter assays, measuring either ROS or membrane integrity, for the ability to evaluate the fitness of Lactobacillus rhamnosus GG (LGG) after freeze drying, spray drying and H2O2-mediated oxidative stress. Each stand-alone assay detected only three cell populations, showing either differential membrane integrity (Syto 24+/PI-, Syto 24+/PI+, Syto 24-/PI+) or ROS levels (ROS-, low-ROS, high-ROS), and no correlation could be drawn between these groups. Conversely, the multiparameter method detected up to five physiologically distinct cell populations and allowed the integrated assessment of their membrane integrity and oxidative stress. It also revealed a much larger fitness heterogeneity in LGG as each group of low-ROS and high-ROS cells was found to be formed by a healthier population with an intact membrane (L-ROS/PI-, H-ROS/PI-) and a population with damaged membrane (L-ROS/PI+, H-ROS/PI+). As the CRG probe only detects free unreacted ROS, these populations are suggested to reflect the dynamic lifecycle of ROS formation, accumulation and reactive depletion leading to oxidative damage of macromolecules and consequent cell death. With the stand-alone CRG assay being unable to detect ROS lifecycle, the multiparameter method here presented delivers a superior profiling of the heterogeneity generated by oxidative stress in bacteria and enables a more correct interpretation of CRG fluorescence data. We provide recent examples from literature where the use of a single-parameter fluorescence approach may have led to misinterpret oxidative stress data and eventually draw erroneous conclusions

Encapsulation of Lactobacillus reuteri in W1/O/W2 double emulsions : Formulation, storage and in vitro gastro-intestinal digestion stability

Encapsulation of Lactobacillus reuteri in the internal aqueous phase of W1/O/W2 emulsions was investigated. Microstructure of the emulsions was evaluated using a particle size analyzer and light microscopy. The encapsulation properties were evaluated in terms of encapsulation efficiency (EE) for freshly prepared samples and encapsulation stability (ES) during storage and in vitro digestion and the results were compared to control. Particle size analysis showed that encapsulation of the bacteria increased the droplet sizes of W1/O emulsions significantly (P 0.05) the droplet sizes for double emulsions (14.9 ± 0.3 μm) compared to control (15.5 ± 0.4 μm) which remained stable through storage period. The EE was 7.23 ± 0.07 Log CFU/mL. During cold storage, the ES decreased with a higher pace for control (from 6.18 ± 0.03 to <1 Log CFU/mL) compared to formulations with encapsulated bacteria (from 7.23 ± 0.07 to 2.82 ± 0.10 Log CFU/mL). Finally, during in vitro digestion, the ES decreased with a higher rate for control (from 6.25 ± 0.36 to 2.69 ± 0.06 Log CFU/mL) compared to encapsulated samples (from 6.69 ± 0.07 to 4.64 ± 0.06 Log CFU/mL). The results showed that encapsulation of Lactobacillus reuteri using double emulsions can protect the probiotics during storage and in vitro gastro-intestinal digestion