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.

Once we had confirmed the proteinaceous nature of the substance acting against rotavirus, we went on to identify the active compound. We performed a supernatant purification strategy based on cationic exchange chromatography. This strategy had been carried out successfully to purify a probiotic antibacterial compound . Following this strategy, 28 fractions with cationic nature were obtained. Based on the inhibition of rotavirus infection, only six fractions were positive and thus selected for MALDI-TOF further analysis. Their spectra pointed to one functional compound, the 11amino acid peptide , common in all the positive fractions. This peptide is part of cow milk β-casein and has a molecular mass of 1.28263 KDa. In order to verify the functionality of the 11-mer peptide, inhibition assays were run with the synthesized peptide in Wa, Ito, and VA70 rotavirus using both HT-29 and MA-104 cell lines. The results were similar to those previously published with the probiotic B. longum CECT 7210 (Muñoz et al., 2011) , reaching a maximum of 56% of infectious foci inhibition, and clearly confirming peptide 11-mer as being responsible for rotavirus inhibition. Reports on the direct activity of peptides against rotavirus are very scarce. Ijaz et al. (1998) found that a synthetic peptide from VP4 directly inhibited rotavirus. Their study hypothesized that VP4-peptide blocks the receptor sites on the host cells. In other studies, Kvistgaard et al. (2004) and Bojsen et al. (2007) found that some bovine macromolecular whey proteins may exert in vitro and in vivo inhibitory activity against rotavirus, the major component being bovine IgG. Regarding lactoferrin, few peptides have been reported. Superti et al. (1997) used different peptides coming from artificially hydrolyzed lactoferrin to demonstrate that some peptides are able to interfere with different stages in virus replication, and the importance of specific recognition events. Regarding casein as the source of antiviral activity, previous work in bovine milk demonstrated the antiviral activity of κ-casein against human rotavirus and concluded that the inhibitory mechanism of bovine κ-casein against human rotavirus involves direct binding to viral particles via glycan residues (Inagaki et al., 2014) . In our case, the 11-mer peptide does not contain glycan residues, and furthermore, its antiviral activity is conserved with synthesized peptide (with no glycan residues). Therefore, another mechanism must be involved in its activity against rotavirus. The facts that the peptide was effective in assays carried out with both strategies (A and B), and that we found different sensitivities depending on the virus and cell line, strongly suggest a two-way effect, in which the interaction of the peptide with both the mammalian cell surface and directly with the virus, must play a role in the inhibition mechanism, as hypothesized previously (Muñoz et al., 2011) . Moreover, positive results were obtained even at low peptide concentrations (1 µM), showing the peptide exerts a strong activity.