Folate concentrations were determined by a previously
described microbiological assay using Lb. rhamnosus NCIMB 10463
as the indicator organism (Laino et al., 2012 ~ ). Briefly, diluted sam-
ples or different concentrations of HPLC-grade folic acid (Fluka
BioChemica, SigmaeAldrich, Switzerland) were placed with the
indicator strain and incubated statically during 48 h at 37 C in 96-
well sterile microplates containing the folate-free medium (Difco,
USA). The optical density was read at 580 nm (OD580) using a
microplate reader (VERSAmax tuneable microplate reader, Molec-
ular Devices, USA). The folate concentration of the samples was
determined by comparing the OD with those obtained with the
standard curve prepared using commercial folic acid.