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.

Recent clinical trials have demonstrated that some probiotic strains are able to ameliorate acute rotaviral diarrhea (Grandy et al., 2010; Lee et al., 2014) . It is assumed that gut microbiota balance, the enhancement of mucosal barrier and the modulation of the immune response afford protection against rotavirus diarrhea (Kotzampassi and Giamarellos-Bourboulis, 2012) . However, few studies have investigated the mechanisms underlying the protective effect of probiotics against rotavirus. A previous work demonstrated the direct effect of strain B. longum subsp. infantis CECT 7210 against rotavirus replication (Muñoz et al., 2011) . Likewise, Lee et al. (2014) found that B. longum (IBG) and L. acidophilus (LA) strongly inhibited rotavirus infection using Vero cell line, but did not identify the mechanism further. In another study, Maragkoudakis et al. (2010) investigated the induction of NO-and H 2 O 2 reactive oxygen species (ROS) released by cell lines co-incubated with lactic acid bacteria, and results were strain and cell line specific. Other data report that probiotics can block viral attachment by competitive inhibition if they are able to bind viral receptors at the surface of intestinal cells (Colbère-Garapin et al., 2007) . In our study, a systematic approach was taken to identify the molecules directly involved in rotavirus inhibition. First of all, to determine the nature of the substance of interest, supernatants with and without enzymatic treatment were analyzed for functionality. In these assays, the high percentage of inhibition of the non-treated cell-free supernatants suggested that part of the activity of CECT 7210 strain against rotavirus was not directly exerted by cells, and that the molecule responsible for direct rotavirus inhibition was released to the supernatant, as previously hypothesized for other probiotic strains (Botić et al., 2007) . In assays performed to identify the nature of the functional compounds, treatment with different proteases eliminated inhibition capacity, confirming the proteinaceous nature of the substance responsible for inhibiting rotavirus infection. Similar assays were applied with lipases but inhibition capacity was not reduced (data not shown), refuting the potential lipidic nature of the substance of interest.