Evaluating Food Additives on Gut Bacteria

The growth experiments were repeated at least twice and with three technical replicates for each treatment group. For initial growth experiments, a single colony of each F. prausnitzii strains was transferred to M2G broth and incubated at 37°C overnight (14 − 16 hours). Aliquots (100 μl) of these cultures were used to inoculate Hungate tubes containing anaerobically prepared M2G broth medium supplemented with either the food additives, or additional sterile water, to provide a final volume of 10 ml. Microbial growth was assessed hourly by measuring the culture optical density at 600 nm (OD₆₀₀). Based on the results, a second series of experiments with all three F. prausnitzii strains were performed. All three strains were cultured with M2G broth and growth monitored as described above. Once the cultures had reached mid-exponential phase of growth, either the sodium sulfite or polysorbate 80 stock solutions were added to provide a final concentration of 0.1% and bacterial growth monitored longitudinally for another 8 hours, as described above. As controls, separate cultures received a similar volume of sterile anaerobic water or were left untreated.
The growth studies with Proteobacteria strains were conducted within a 96-well microtiter plate format. Here, the inoculating cultures were prepared by using a single colony of each strain transferred to 5 ml of LB broth within a 50 ml centrifuge tube (Corning, US) and incubated for 5 hours aerobically with shaking at 200 rpm at 37°C. Aliquots (0.135 ml) of either sterile LB broth, or LB broth prepared to contain 0.1% final concentration one of the food additives described above (with the exceptions of aluminum silicate and aspartame) were dispensed into individual wells, which were then inoculated with 0.015 ml of a 1:100 dilution (with LB) of the inoculating cultures described above. As controls, wells containing 0.15 ml of uninoculated broth (with and without food additives) were also prepared and positioned around the edge of the microtiter plates. The plates were covered with Breathe-Easy sealing membranes and placed within a Multiskan GO microplate reader (ThermoFisher, US) for measurements of aerobic growth. The plate reader was programmed to shake at 300 rpm and OD₆₀₀ measurements were taken every 30 minutes for 17 hours. A parallel series of growth studies were also done with the 7 Proteobacteria strains using a 96-well microtiter plate format under “microaerobic” conditions. Here, the LB medium was prepared with an additional step of gassing the solution with a steady stream of nitrogen gas for 60 minutes, and the manipulations described above were performed within a Coy Anaerobic chamber filled with a mixture of CO₂:H₂:N₂ (15:5:80). The “microaerobic” conditions resulted from the periodic introduction of surrounding atmosphere during interchange operations, which resulted in transient readings of ~300 ppm oxygen within the chamber atmosphere, before returning to zero. Bacterial growth was monitored by OD₆₀₀ measurements using a FLUOStar Omega (BMG Labtech, Germany) plate reader permanently housed within the chamber using the same operational parameters described above.