Konstantinidis et al_folate secretion

Published: 2 August 2021| Version 2 | DOI: 10.17632/53yyysbsf9.2
Contributor:
Dimitrios Konstantinidis

Description

This data-set captures folate secretion by the parental L. plantarum and 4 evolved isolates (evolved in co-culture). The samples originate from bacteria monocultures in CDM44 (supplemented amino acids, omitted folate and PABA).

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Folate was extracted from 2 mL of culture supernatant by addition of an equal amount of 20% AcN: H2O. The samples were left at 4 oC for 10 min and then were transferred to dark glass vials. Next, they were concentrated using a vacuum concentrator, at 30 oC for 2 hours and subsequently reconstituted in 200 μL of 20% AcN:H2O. The reconstituted samples were filtered through a 0.22 μm syringe filter. The liquid chromatography method for the detection of folate was run on a Waters Acquity UPLC H-Class instrument with a PDA detector and a quaternary solvent system. The established method was 8 minutes long, with a flow rate of 0.35 mL/min and run on a BEH Hilic column (130 Å, 1.7 µm, 2.1 mm X 150 mm, Waters, part number 176001094). The column was heated to 35 oC, samples were kept at 6 oC and a total volume of 5 μL was injected. The method used 50% [v/v] acetonitrile (CAN; Biosolve, ULC grade) as washing buffer and 50% [v/v] methanol (Biosolve, ULC grade) as purging buffer. The organic mobile phase was acetonitrile, 10 mM ammonium acetate (Sigma-Aldrich, HPLC grade) was used as buffer and the pH was adjusted to 9. The two buffers used were 50% ACN + 10 mM Ammonium acetate pH 9 and 100% ACN + 10 mM Ammonium acetate pH 9. The full method is described on Table S4. The elution time of folate was estimated by using pure grade standards (≥97%, Sigma-Aldrich). All chromatograms were annotated with the vendor specific program Empower 3, and manually curated for peak identification. The readout for all chromatograms is the baseline corrected area under the curve of the vitamin peak.