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.

Purification was performed following Chenoll et al. (2011) . Briefly, the supernatant was added to sodium phosphate buffer (20 mM, pH 5.8) and then applied to a cationic exchange column (HiPrep 16/10 SP FF, GE Healthcare, Amersham Biosciences AB, Sweden) by means of a chromatography system (ÄKTA Explorer, Amersham Pharmacia Biotech). All those proteins of cationic nature adsorbed were then eluted with sodium phosphate buffer (20 mM, pH 5.8) and equilibrated with NaCl 1M without gradient. The proteins were eluted simultaneously in a few fractions in minimal volume (10 mL). Each collected fraction was subjected to an ultrafiltration process using 5,000 Da filters (Extreme Amicon, Millipore, Billerica, MA, USA) followed by another stage of reverse phase chromatography with a RESOURCE RPC 3 mL column (GE Healthcare). An aliquot of these fractions was taken to dryness to eliminate the dissolvent and re-suspended in a volume of MEM buffer (Sigma-Aldrich) just before performing in vitro inhibition assays. In total, 28 fractions were selected for rotavirus inhibition assays (strategy B) with SA11 virus in MA-104 cell line as explained above. The fractions that gave a positive inhibition result were analyzed by MALDI-TOF peptide mass fingerprinting at the "Centro Nacional de Investigaciones Cardiovasculares" (CNIC, Madrid, Spain), to determine the molecular weight(s) of the peptide(s) present in each fraction. Sequences obtained were compared against the database of proteins/peptides by means of the BLAST online tool.