Bromocresol purple indicator

A glutaminase assay was conducted to determine the amount of glutaminase enzyme secreted by the cells undergoing treatment in order to determine whether they exhibited properties expressed in neurons. Intracellular activities of glutaminase were measured by quantifying enzymatic interconversion of L-glutamine to L-glutamate using a colorimetric assay. NH₃ levels are proportional to glutaminase, therefore, by quantifying NH₃ levels, glutaminase levels can be determined. The cell medium supernatant was collected at each treatment change during cell culture and kept frozen at -80°C for glutaminase quantification.
$\begin{array}{c}\text{Glutamine}+{\text{H}}_2\text{O\hspace{0.17em}}\stackrel{\text{Glutaminase}}{\to }\text{Glutamate}+\text{N}{\text{H}}_3\end{array}$
The assay was conducted by adding 50 μL of the sample, 10 μL of 0.1% w/v bromocresol purple indicator (Sigma-Aldrich, MO, USA), and 150 μL of 100 mM L-glutamine glutamine (pH 8.5) (Sigma-Aldrich, MO, USA) to a 96-well plate. The absorbance was then read at 340 nm at different time points during 24 h incubation at 37°C in the TECAN infinity 200 plate reader.
No blank was utilised for the colorimetric assay as the fresh base medium (DMEM/F12+Glutamax+10% penicillin+streptomycin) is basic and the medium supernatant becomes acidic after being in contact with the cells.

The mucinolytic activity of the B. boum strains was tested using a medium where porcine mucin (Sigma-Aldrich, St. Luis, MO, USA) was used as a selective factor [54 (link)]. Bifidobacterial cultures were grown in Wilkins–Chalgren broth (Oxoid) supplemented with soya peptone (5 g/L; Oxoid), were twice centrifuged (5000× g for 10 min), twice flushed with sterile saline, and resuspended in the PBS buffer. The cultures were inoculated (0.1%) into the broth with mucin and incubated at 37 °C for 48 h. Mucin utilization was accompanied by a change of bromocresol purple indicator (Sigma-Aldrich) from violet to yellow color as a consequence of reduced pH value through final products (mainly acetic and lactic acid) of specific fermentative metabolism of bifidobacteria. Cultivation was done under anaerobic conditions by displacing atmospheric oxygen with purified carbon dioxide.