Pectin beads containing beta-lactamases were designed for the hydrolysis of colonic residual antibiotics responsible for the emergence of resistance. Beads were prepared by ionotropic gelation in CaCl2 and stabilized by coating with polyethylenimine (PEI) to resist disintegration in the upper GI tract. Particle characterization showed that dried beads had a diameter around 1 mm independently of the presence of PEI. Seven to ten percent (w/w) of PEI was located on bead surface forming a coating layer as observed by scanning electron microscopy. PEI improved considerably bead stability in simulated intestinal medium while affecting slightly the encapsulation efficiency of active beta-lactamases. Coated beads were able to preserve beta-lactamases from premature leakage in the upper GIT whereas, in simulated colonic medium, pectinases induced matrix degradation and reduction of beta-lactamase content especially in beads coated in a 0.8% PEI solution. Finally, the pharmacokinetics of amoxicillin in rat after oral administration was not modified by the co-administration of beads containing beta-lactamases. In conclusion, PEI-coated beads are stable in the upper GIT but remain sensitive to the action of pectinolytic enzymes allowing release of beta-lactamases in a colonic medium without modification of the absorption of a beta-lactam antibiotic when co-administered with loaded beads.