New method for selection of hydrogen peroxide adapted bifidobacteria cells using continuous culture and immobilized cell technology

BACKGROUND: Oxidative stress can severely compromise viability of bifidobacteria. Exposure of Bifidobacterium cells to oxygen causes accumulation of reactive oxygen species, mainly hydrogen peroxide, leading to cell death. In this study, we tested the suitability of continuous culture under increasing selective pressure combined with immobilized cell technology for the selection of hydrogen peroxide adapted Bifidobacterium cells. Cells of B. longum NCC2705 were immobilized in gellan-xanthan gum gel beads and used to continuously ferment MRS medium containing increasing concentration of H(2)O(2 )from 0 to 130 ppm. RESULTS: At the beginning of the culture, high cell density of 10(13 )CFU per litre of reactor was tested. The continuous culture gradually adapted to increasing H(2)O(2 )concentrations. However, after increasing the H(2)O(2 )concentration to 130 ppm the OD of the culture decreased to 0. Full wash out was prevented by the immobilization of the cells in gel matrix. Hence after stopping the stress, it was possible to re-grow the cells that survived the highest lethal dose of H(2)O(2 )and to select two adapted colonies (HPR1 and HPR2) after plating of the culture effluent. In contrast to HPR1, HPR2 showed stable characteristics over at least 70 generations and exhibited also higher tolerance to O(2 )than non adapted wild type cells. Preliminary characterization of HPR2 was carried out by global genome expression profile analysis. Two genes coding for a protein with unknown function and possessing trans-membrane domains and an ABC-type transporter protein were overexpressed in HPR2 cells compared to wild type cells. CONCLUSIONS: Our study showed that continuous culture with cell immobilization is a valid approach for selecting cells adapted to hydrogen peroxide. Elucidation of H(2)O(2 )adaptation mechanisms in HPR2 could be helpful to develop oxygen resistant bifidobacteria.