Mcilvaine buffer

Trivalent IPV, containing the inactivated Mahoney strain for type 1, MEF for type 2 and Saukett for type 3, was obtained from the process development department of Intravacc (Bilthoven, The Netherlands). The ten times concentrated trivalent bulk used in this study was determined at a nominal concentration of 400-80-320 DU/ml by ELISA as described (24 (link)).
The excipients sucrose, D-sorbitol, D-trehalose dihydrate, mannitol, L-glutamic monosodium salt monohydrate (MSG), glycine, myo-inositol, magnesiumchloride hexahydrate, lithium chloride and ovalbumin were all purchased from Sigma (St. Louis, MO). Peptone (vegetable) and dextran (6 kDa, from Leuconostoc ssp.) were from Fluka (Buchs, Switzerland) and sodium chloride was from Merck (Darmstadt, Germany). To prepare 10 mM McIlvaine buffer, 10 mM citric acid (Sigma-Aldrich, St. Louis, MO) was added to 10 mM disodium hydrogen phosphate dehydrate (Na₂HPO₄) (Fluka, Buchs, Switzerland) in a ratio of 1:6 resulting in a pH-value of 7.0. All excipients used were of reagent quality or higher grade.

In-vitro gastrointestinal digestion tests were performed in 130-mL reaction mixtures containing NaCl (4.39 g), KCl (0.22 g), CaCl₂ (0.04 g), McIlvaine buffer (pH 5.0), meat analogs (25 g, finely cut), and 0.0065% pepsin from porcine gastric mucosa (Sigma-Aldrich, St. Louis, MO, USA). The digestive reaction mixtures were incubated at 37 °C and 60 rpm for 90 min, and 1 N HCl was added to the reaction mixtures every 5 min, resulting in pH 3.0 after 40 min. The solution was clarified using a Nanosep® Centrifugal Device (Pall Corporation, Port Washington, NY, USA) as described in the instruction manual. The free amino nitrogen was measured using the ninhydrin method^{66 (link)}.

The assay for enzyme activity consisted of a 50 µL aliquot of diluted eluate, 50 µL of 20 mM 4-nitrophenyl-α-D-galactopyranoside (PNPG) (Sigma-Aldrich, St. Louis, MO, USA) and 150 µL of McIlvaine buffer (pH 7.0). The reaction was incubated for 10 min at 37°C, stopped by addition of 1 mL 0.5 M Na₂CO₃, and the amount of product formed determined spectrophotometrically using a plate reader (Tecan Infinite^® 200 PRO) at 410 nm. The pH optimum of SbAGA1 and SbAGA2 enzymes was determined using McIlvaine buffer (pH from 5.0 to 9.0; in 0.5 pH increments).