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.

To identify the source of the 11-mer peptide, the analysis of all the experimental data lead us to hypothesize a secondary origin of the peptide, derived from the preliminary digestion of casein present in the commercial MRS growth medium used. A protease secreted by the probiotic strain B. longum subsp. infantis CECT 7210 may release 11-mer peptide by hydrolyzing the casein present in the culture. A mechanism by which functional molecules are released from casein was previously described by Janer et al. (2005) in B. animalis subsp. lactis species. These authors described a zinc metallopeptidase (PepO) able to hydrolyze α s1 -casein (f1-23), which was suggested to play a role in the increased growth of B. animalis subsp. lactis in milk. Accordingly, we performed the purification and identification of the protease responsible for 11-mer production. To obtain the protease, we applied a strategy based on fractionation by anionic chromatography. Following this protocol, a protease-positive anionic extract was obtained, and a SDS-gel band confirmed a unique band. MALDI-TOF peptide mass fingerprinting analysis identified the purified protease as "MalE-type ABC sugar transport system periplasmic component." This is a periplasmic binding protein, part of the Maltose ABC sugar transport system, which is involved in the transport of maltose and maltodextrins. Although preliminary studies with this component were performed in Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli (reviewed in Ehrmann et al., 1998) , it has been found in other bacteria, including bifidobacteria (Balac_0483 in assembly NC_012814.1 from B. animalis subsp. lactis Bl-04, BLD_1008 in assembly ACD98454.1 from B. longum DJO10A). With the data obtained by the whole genome sequencing of B. longum CECT 7210 strain (Chenoll et al., 2015 ; genome reference LN824140), the protein has been identified in its genome, with a molecular weight of 46.89 KDa, confirming that the protein is produced by this probiotic strain. Once identified, the MalE-type ABC sugar transport system periplasmic component was characterized in terms of its protease activity. The results obtained in inhibition studies with glucose, lactose, and maltose supernatants showed the highest activity when the probiotic was grown in maltose as the only carbon source. Moreover, maltose-grown purified fractions were around 28 times higher, with a maximum of 1000 mU mL −1 of activity. These results, together with the quantification of 11-mer peptide when these supernatants and fractions were incubated with β-casein, confirmed that protease activity is enhanced when maltose is the sole carbon source. This supports the identification of the MalE-type ABC sugar transport system periplasmic component as the molecule responsible for this protease activity. Although protease activity in MalE has not been described previously, our data point to a mechanism whereby the formation of the 11-mer peptide from β-casein is caused by the direct action of the purified protein, with an efficiency of 28 ± 1 µg/mL of reaction. Finally, optimum pH and temperature for purified protease activity have been established and the influence of zinc, potassium, and calcium as cofactors on 11-mer production discarded.