4.7. Urease Activity Determination

A modification of the Berthelot reaction was used to monitor the ammonium ions derived from hydrolysis of urea by urease activity [47,48]. The H. pylori pellets from the LAB supernatant exposures were resuspended in 200 µL sodium phopshate buffer (10 mM, pH 7). The samples were diluted 1/10 in the same buffer and 10 µL of this suspension were added in duplicate to 90 µL of urease reaction buffer (2.5% sodium citrate and 0.5 mM urea in 5 mM sodium phosphate buffer, pH 7) in a 96-well plate. The plate was incubated at 25 °C for 30 min. Afterwards, the modified Berthelot method was performed to measure the ammonium ions formed based on the colorimetric reaction of the 100 µL sample above with 50 µL of 2-phenylphenol-nitroprusside reagent (3.22% 2-phenylphenol sodium salt tetrahydrate and 0.015% sodium nitroprusside), followed by 25 µL buffered sodium hypochlorite (1% sodium phosphate, 0.5% sodium hypochlorite, pH 13) and 100 µL water. The plate was incubated under gentle agitation in the dark at 25 °C for 30 min and the color was read at 670 nm. Standard curves were prepared using ammonium sulfate (range: 50–3500 µM). The urease activity is represented as the fold change in the measured concentration of NH₄⁺ ions relative to the non-treated H. pylori control (BHI, 0 mM urea). A minimum of three biological replicates were performed with three technical replicates per H. pylori exposure and 2 technical replicates for the Berthelot reaction.