Prior to the enzymatic assays, the samples were prepared according to the following protocol. Firstly, 0.7 g of sample was suspended in 3.5 mL of 0.2 M phosphate buffer and vortexed thoroughly (Vortex RS-VA 10, Phoenix Instrument, Garbsen, Germany). Secondly, the samples underwent sonification (Time = 2 min, Amplitude = 60, Pulse = 6 s, Cole–Parmer Instrument Co., Vernon Hills, IL, USA). Afterwards, the samples were centrifuged (12,000 rpm, Time = 20 min, Centrifuge MPW-251, MPW, Warszawa, Poland) and the supernatant was transferred to the sterile Eppendorf tubes.
With the use of spectrophotometric methods, the activity of the fecal enzymes α-glucosidase, α-galactosidase, β-glucosidase, β-galactosidase, and β-glucuronidase was determined. The protocols used in the study were based on the reaction of α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase, and β-glucuronidase with 4-nitrophenyl α-D-glucopyranoside (TCI, Tokyo, Japan), 4-nitrophenyl β-D-glucopyranoside (TCI, Tokyo, Japan), 4-nitrophenyl α-D-galactopyranoside (TCI, Tokyo, Japan), 4-nitrophenyl β-D-galactopyranoside (TCI, Tokyo, Japan), and 4-nitrophenyl β-D-glucuronide (TCI, Tokyo, Japan), respectively.
The used substrates were specific to the respective enzymes present in the fecal sample. The reaction mixture contained 0.5 mL of phosphate buffer (pH = 7, 0.02 M), 0.05 mL of substrate solution (20 mM), and 0.25 mL of sample. Incubation was performed at 37 °C for 15 min (α-glucosidase, α-galactosidase and β-glucuronidase) or 60 min (β-glucosidase and β-galactosidase).
An observed hue shift in the sample to yellow proved the reaction had taken place. The intensity of the color was directly proportional to the amount of p-nitrophenol released. The reactions were inhibited using 0.25 M sodium carbonate after the given reaction time. The absorbance of the samples was measured using the spectrophotometer Rayleigh UV-2601 (BFRL, Beijing, China) at a wavelength of λ = 400 nm. The unit of enzyme activity refers to the amount of p-nitrophenol (expressed in µM) which was released during 1 h of reaction for 1 mg of protein in 1 mL of sample [µMh·mg−1].
Śliżewska K., Włodarczyk M., Sobczak M., Barczyńska R., Kapuśniak J., Socha P., Wierzbicka-Rucińska A, & Kotowska A. (2023). Comparison of the Activity of Fecal Enzymes and Concentration of SCFA in Healthy and Overweight Children. Nutrients, 15(4), 987.