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The association of carboxymethyl starch (CMS) and alginate is proposed as a novel matrix for the entrapment of bioactive agents in microspheres affording their protection against gastrointestinal degradation. In this study, the enzyme diamine oxidase (DAO) from white pea (Lathyrus sativus) was immobilized by inclusion in microspheres formed by ionotropic gelation of CMS/alginate by complexation with Ca(2+) . The association of CMS to alginate generated a more compact structure presenting a lesser porosity, thus decreasing the access of gastric fluid inside the microspheres and preventing the loss of entrapped enzyme. Moreover, the immobilized enzyme remained active and was able to oxidize the polyamine substrates even in the presence of degrading proteases of pancreatin. The inclusion yield in terms of entrapped protein was of about 82%-95%. The DAO entrapped in calcium CMS/alginate beads retained up to 70% of its initial activity in simulated gastric fluid (pH 2.0). In simulated intestinal fluid (pH 7.2) with pancreatin, an overall retention of 65% of activity for the immobilized DAO was observed over 24 H, whereas in similar conditions the free enzyme was totally inactivated. Our project proposes the vegetal DAO as an antihistaminic agent orally administered to treat food histaminosis and colon inflammation., (© 2015 The Authors. Biotechnology and Applied Biochemistry published by Wiley Periodicals, Inc. on behalf of the International Union of Biochemistry and Molecular Biology, Inc.)