Ultraflex workstation

The absolute masses of oligogalacturonides were obtained by MALDI-TOF MS, using an Ultraflex workstation (Bruker Daltonics, Billerica, MA, USA), operating in reflectron mode and positive polarity. Sample preparation was performed according to the protocol described by Gómez et al. [50 (link)] using 2,5-dihydroxybenzoic acid (DHB) as the matrix. Data were acquired and processed by means of the Flex Control and Flex Analysis software (Bruker Daltonics, Billerica, MA, USA), respectively.

The colony MALDI-TOF MS analysis of L. salivarius P1ACE3 was performed as previously described, with slight modifications [22 (link)]. Briefly, single colonies of this strain, grown at 37 °C for 48 h on TSB agar plates, were picked and mixed with 50 μL 100% isopropanol with 0.1% (v/v) trifluoroacetic acid (TFA). The mix was vortexed three times and centrifuged at 13,000 rpm for 30 s, after which 1 μL of supernatants was mixed with 1 μL of a sinapic acid matrix (Sigma-Aldrich, St. Louis, MO, USA) in 30% acetonitrile and 0.3% TFA, and then applied directly to the MS target plate and dried under a stream of warm air. The MALDI-TOF MS analysis of samples was done on an Ultraflex workstation (Bruker Daltonics, Billerica, MA, USA) equipped with a 337 nm nitrogen laser, at the Unidad de Espectrometría de Masas (CAI Técnicas Químicas, UCM, Madrid, Spain). The mass spectrometer was calibrated with protein calibration standard I (4000–20,000 m/z) from Bruker Daltonics. The FlexControl Software v.2.4. (Bruker Daltonics) was used for sample analysis and control of method parameters. The L. lactis subsp. lactis BB24 (nisin A producer) strain, grown at 32 °C for 48 h on MRS agar plates was used as a positive control in the colony MALDI-TOF MS analysis of L. salivarius P1ACE3.

Colony MALDI-TOF MS analysis of both Lp. plantarum BF12 and Lp. plantarum WT12 was performed as previously described by Lawrence et al. [30 (link)], with slight modifications. Briefly, single colonies of the strains grown on MRS agar plates (1.5%, w/v) at 30 °C for 48 h were picked and resuspended in 50 μL 100% (v/v) isopropanol with 0.1% (v/v) trifluoroacetic acid (TFA). The mixtures were vortexed and centrifuged at 11,000× g for 30 s. Subsequently, 1 μL of the corresponding supernatant was mixed with 1 μL of a sinapic acid matrix (Sigma-Aldrich, St. Louis, MO, USA) in 30% (v/v) acetonitrile and 0.3% (v/v) TFA. Then, the mixtures were transferred onto the MS target plate and dried. The MALDI-TOF MS analysis of samples was conducted on an Ultraflex workstation (Bruker Daltonics, Billerica, MA, USA), equipped with a 337 nm nitrogen laser, at the Unidad de Espectrometría de Masas (CAI Técnicas Químicas, Universidad Complutense de Madrid, Madrid, Spain). The mass spectrometer was calibrated with protein calibration standard I (4000–20,000 m/z) according to the manufacturer instructions. The FlexControl Software v.2.4. (Bruker Daltonics) was used for sample analysis and control of method parameters.