Papain

Anti-Listeria active metabolites of the LAB isolates were examined for their sensitivity to proteinase K (AppliChem GmbH, Darmstadt, Germany), papain (Carl Roth, Karlsruhe, Germany), and trypsine (Carl Roth, Karlsruhe, Germany), using a well diffusion assay, as described above. Three 6.5 mm wells were cut in line, ensuring a 6 to 8 mm distance between each. In total, 100 µL of a proteolytic enzyme solution, which contained 100 µL of the corresponding enzyme (1 mg/mL of a solution of 5 µL of calcium chloride solution 1 M and 895 µL of dH₂O), was placed into the middle well, and the outer wells were filled with 100 µL of CFS. Plates were incubated for 24 h at 37 °C. The enzyme degradation of anti-Listeria active metabolites in the CFS was shown by a characteristic crescent-shaped zone of inhibition, opened toward the proteolytic enzyme. Uninoculated MSM+ broth was used as a negative control. The well diffusion assay was performed with three independent CFSs of each LAB.

Pea seeds (Pisum sativum L., cultivar Navarro) were provided by Norddeutsche Pflanzenzucht Hans-Georg-Lembke KG (Holtsee, Germany). Trypsin and Esperase^® 8.0 L were obtained from Sigma-Aldrich (Munich, Germany). Papain was from Carl Roth GmbH (Karlsruhe, Germany). L. plantarum (DSM 20174) was purchased from the German collection of microorganisms and cell cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany). Broad Range™ Unstained Protein Standard, 4–20% Criterion™ TGX stain-free™ precast polyacrylamide gels, Coomassie Brilliant Blue R-250 were from Bio-Rad Laboratories GmbH (Feldkirchen, Germany). Sodium dihydrogen phosphate, sodium dodecyl sulfate, sodium tetraborate decahydrate, o-phthaldialdehyde, and sodium monohydrogen phosphate were purchased from Sigma-Aldrich (Munich, Germany). All chemicals used in this study were of analytical grade unless otherwise indicated.