Purification and biochemical characterization of Brazil nut (Bertholletia excelsa L.) seed storage proteins.

Brazil nut storage proteins, 2S albumin, 7S vicilin, and an 11S legumin, were purified using column chromatography. Analytical ultracentrifugation of the purified albumin, vicilin, and legumin proteins, respectively, registered sedimentation coefficients of 1.8, 7.1, and 11.8 S. Under reducing conditions, the major polypeptide bands in 2S albumin were observed at 6.4, 10-11, and 15.2 kDa. The 7S globulin was composed of one 12.6 kDa, two approximately 38-42 kDa, and two approximately 54-57 kDa polypeptides, whereas the 11S globulin contained two major classes of polypeptides: approximately 30-32 and approximately 20-21 kDa. The 7S globulin stained positive when reacted with Schiff reagent, indicating that it is a glycoprotein. The estimated molecular mass and Stokes radius for 2S albumin and 7S and 11S globulins were 19.2 kDa and 20.1 A, 114.8 kDa and 41.1 A, and 289.4 kDa and 56.6 A, respectively. Circular dichroism spectroscopic analysis indicated the secondary structure of the three proteins to be mainly beta-sheets and turns. Emission fluorescence spectra of the native proteins registered a lambda(max) at 337, 345, and 328 nm for 2S albumin and 7S and 11S globulins, respectively. When probed with anti-Brazil nut seed protein rabbit polyclonal antibodies, 7S globulin exhibited higher immunoreactivity than 2S albumin and 11S globulin.

A sensitive and robust competitive enzyme-linked immunosorbent assay for Brazil nut ( Bertholletia excelsa L.) detection.

Undeclared Brazil nut residue in food products is of great concern because it can trigger life-threatening allergic reactions in sensitive patients. A rabbit polyclonal antibody-based competitive ELISA (IC(50) = 23.2 +/- 9 ng/mL, n = 76) with good sensitivity, detection range of 10-90 ng/mL, was developed. The ELISA could detect Brazil nut seed proteins over a pH range of 5-12. The optimal pH range for the detection assay was 7-10. Among the 66 tested foods/ingredients, only cinnamon exhibited statistically significant interference (1.36%, p = 0.05). Exposing Brazil nut seeds to processing did not adversely affect the nut seed protein detection using the assay. Brazil nut seed protein recovery from 100 mg of foods spiked with 10 and 1 microg of soluble Brazil nut proteins or 100 and 10 microg of defatted Brazil nut flour exhibited a wide recovery range, 63-315%, indicating protein-food matrix interaction.