Prominence system

Lignin-derived aromatic monomers were quantified via HPLC analysis as described previously [11 (link)]. Prior to HPLC analysis, samples were centrifuged at 20,000 × g for 10 min, and supernatants were frozen at −20 °C. The analysis was performed on a Shimadzu Prominence system equipped with a RFQ Fast Acid column (50 × 7.8 mm, Phenomenex, Aschaffenburg, Germany) as described previously [30 (link)]. Detection was carried out via a UV detector at 220 nm and crosschecked at 270 nm. As external standards, SA, CA, HBA, PCA, FA, VA, and vanillin were used in concentrations of 1, 5, and 10 mM.

FA and VA were quantified with HPLC analysis as described previously (Margesin et al. 2021 (link)) in reducing intervals (28–8 h intervals, Additional file 1: Table S1). In short: after centrifugation (10 min, 20 000 × g) to remove larger particles the supernatants were frozen (− 20 °C) and at least 0.7 mL of the supernatant was filtered (0.2 µm RC filter) for HPLC measurement. The measurement was performed at 70 °C using a Shimadzu Prominence system equipped with a RFQ Fast Acid column (50 × 7.8 mm, Phenomenex, Torrance, CA, Germany) and a mobile phase of 5 mM sulfuric acid as described previously (Wagner et al. 2017 (link)). A UV detection at 220 nm combined with a crosscheck at 270 nm was applied. The calibration was performed via injection of 1, 5 and 10 mM FA and VA external standards.
The rate constant of decline (k), DT50 was calculated using Computer Assisted Kinetic Evaluation (CAKE) software (available online with public free access, https://cake-kinetics.org/, accessed: 21.04.2022) using a convergence tolerance of 1 × 10^–5, 100 max. iterations and iteratively reweighted least squares (max. reweightings: 200, error variance tolerance: 1 × 10¹⁰) and a simple first order fit.

Pediocin PA-1 carrying the Met31Leu substitution [pediocin PA-1(M31L)], was selected for the study, as the antimicrobial activity of this linear analog is similar to the wild-type bacteriocin naturally produced by Pediococcus acidilactici, while its stability is improved by the lack of Met31which is sensitive to oxidation. Briefly, pediocin PA-1(M31L) was synthesized as described previously (Bédard et al., 2018 (link)) by standard solid phase peptide synthesis (SPPS) on prelude peptide synthesizer from Gyros Protein Technologies (Tucson, AZ, United States) using HMBP-ChemMatrix^® resin. The peptide purification was carried out by RP-HPLC with a Shimadzu Prominence system on a Phenomenex Kinetex^® EVO C18 column (250 mm × 21.2 mm, 300 Å, 5 μm) and UV detection at 220 and 254 nm using 0.1% AcOH/H₂O (A) and 0.1% AcOH/CH₃CN (B), at 14 mL/min flow rate.