Identification of a Peptide Produced by Bifidobacterium longum CECT 7210 with Antirotaviral Activity

Chenoll, Empar; Casinos, Beatriz; Bataller, Esther; Buesa, Javier; Ramón, Daniel; Genovés, Salvador; Fábrega, Joan; Rivero Urgell, Montserrat; Moreno Muñoz, José A.

A volume of 1 L culture of strain B. longum subsp. infantis CECT 7210 in MRSC medium was obtained and centrifuged at 12,000 × g for 15 min under refrigerated conditions. Proteins were precipitated by the addition of ammonium sulfate (80% saturation) and collected by centrifugation (30 min, 12,000 × g). Pellet was resuspended in Tris-HCl buffer (Tris-HCl 20 mM, pH 8.5), dialyzed with a 10 KDa membrane at 4 • C and applied to an anionic exchange column (HiPrep 16/10 Q XL, GE Healthcare) by means of a chromatographic system (ÄKTA Explorer). Anionic proteins were eluted with buffer Tris-HCl (20mM, pH 8.5, CaCl 2 5 mM) using a gradient of NaCl from 0 to 1 M. As an indicator of the purification process, samples were monitored at 280 nm throughout elution and protease activity measured with BSA as a substrate following Bradford (1976) . Protease-positive fractions were then concentrated by 10 KDa ultrafiltration (Millipore), diluted with distilled water and applied to gel filtration chromatography (1% column volume) with a HiLoad Superdex 75 prep grade column (GE Healthcare Europe GmbH, Barcelona, Spain) with fractionation in a range of 3,000-70,000 Da. Sample was monitored with absorbance at 280 nm, and fractions with proteins were collected, desalted by PD10 columns (GE Healthcare Life Sciences) and concentrated by 10 KDa ultrafiltration. A volume of 30 µL of protease-positive fraction was electrophoresed on a 12.5% polyacrylamide SDS-PAGE gel. Gel was stained with silver nitrate and a 209-7.1 KDa commercial molecular weight marker (SDS-PAGE Standards, Broad Range) was used. The single band was separated and analyzed by MALDI-TOF peptide mass fingerprinting at CNIC, to determine the molecular weight and sequence of the protein. Sequence obtained was compared against the database of proteins/peptides by means of the BLAST online tool.