Spontaneous Lactobacillus delbrueckii phage-resistant mutants with acquired bile tolerance

Abstract

Three commercial phage-sensitive strains of Lactobacillus delbrueckii (strains Ab1, YSD V and Ib3) and four spontaneous phage-resistant mutants (strains A7, A17, V2 and I39) isolated from them, all with a probiotic potential previously demonstrated were studied for their tolerance of bile salts (ox gall). Minimal Inhibitory Concentrations (MICs) ranged from 0.30% to 0.35% (w/v) of ox gall. These strains were exposed to gradually increasing concentrations of ox gall with the aim of isolating bile resistant derivatives. Stable derivatives able to tolerate up to 0.9% of ox gall were obtained from L. delbrueckii Ab1, as well as from its spontaneous phage-resistant mutants A7 and A17. Random Amplified Polymorphic DNA (RAPD-PCR) analysis revealed a strong genetic homology between the ox gall-tolerant derivatives and their respective non-adapted original strains. These derivatives maintained, in general, the phage resistance phenotype of the non-adapted strains, with only one exception (phage-resistant mutant A7). After progressive ox gall adaptation, the phage-resistant mutant A7 also exhibited progressive reversion of the phage resistance phenotype. The derivative with the highest ox gall-acquired tolerance (A0.97) became sensitive to the phage, but derivatives with low (A0.37) and intermediate (A0.67) ox gall-acquired tolerance retained phage resistance. The technological properties of ox gall derivatives were comparable to those of their respective parent strains. However, the cells of the former were smaller than those of the original strains. Finally, the tolerant derivatives grew faster in the presence of ox gall than the parent strains. Our results demonstrated that it was possible to obtain, by a natural selection strategy, probiotic strains with acquired ox gall-tolerance from three (L. delbrueckii Ab1 and their phage-resistant mutants A7 and A17) of seven tested strains. Since such derivatives keep both phage resistance and other useful technological properties, they could be used for production of functional foods