Bovine blood hemoglobin

Overnight culture (L. rhamnosus OSU-PECh-69) was centrifuged at 4122× g for 15 min at 4 °C (Sorvall Legend XF; Thermo Scientific, Waltham, MA, USA) to separate cells from the supernatant and then adjusted to 3.6 × 10⁶ CFU/mL. Proteolytic activity was determined as described by Anson [24 (link)] with some modifications. Briefly, an acidic buffer containing acetic acid, boric acid, and phosphoric acid at 0.025 M of final concentration at pH 5 was mixed with 0.5% of bovine blood hemoglobin (Sigma-Aldrich, St. Louis, MO, USA). The solution was sterilized using a 0.10 μm filter (Millipore, Burlington, MA, USA) and stored at −20 °C until use. Each reaction contained 300 μL of the hemoglobin solution and 100 μL of sample. The mix was incubated at 37 °C for 1 h. The reaction was stopped by adding 100 μL of trichloroacetic acid (50% w/v) and cooled at 4 °C for 10 min. The mix was centrifuged at 16,000× g for 10 min at room temperature, and 200 μL of supernatant was transferred in a 96-well plate (Flat bottom; Corning, Corning, NY, USA). Finally, the samples were read at 280 nm in a microplate reader spectrophotometer (Multiskan GO; Thermo Scientific, Walthman, MA, USA). One unit of proteolytic activity was defined as a change of 0.01 absorbances per min (U/min). The specific activity was correlated with the protein concentration (U/min × mg protein).