Modifications in the exposure to the solvent of hydrophobic residues, changes in their organization into surface hydrophobic patches, and alterations in the dimerization equilibrium of beta-lactoglobulin upon thermal treatment at neutral pH were studied. Exposure of tryptophan residues was temperature dependent and was essentially completed on the time scale of seconds. Reorganization of generic hydrophobic protein patches on the protein surface was monitored through binding of 1,8-anilinonaphthalenesulfonate, and was much slower than changes in tryptophan exposure. Different phases in surface hydrophobicity changes were related to the swelling and the subsequent collapse of the protein, which formed a metastable swollen intermediate. Heat treatment of beta-lactoglobulin also resulted in the formation of soluble oligomeric aggregates. The aggregation process was studied as a function of temperature, demonstrating that (i) dimer dissociation was a necessary step in a sequential polymerization mechanism and (ii) cohesion of hydrophobic patches was the major driving force for aggregation.

A study on the concentration dependence of the modifications ensuing from thermal treatment of bovine /3-lactoglobulin was carried out by using a combination of techniques. Heat-induced changes in tertiary structure were monitored by intrinsic tryptophan fluorescence, while modifications in protein surface hydrophobicity were studied both during their occurrence and at equilibrium by using the fluorescent hydrophobic probe 1,8-anilinonaphthalenesulfonate. The association equilibria in the heated and cooled protein and the stabilization of aggregates by intermolecular disulfides were studied by gel permeation chromatography and nonreducing, denaturing electrophoresis.Results indicate that irreversible modification of the tertiary structure is not concentration dependent, while the temperature required for the occurrence of protein swelling, the initial step in the formation of associated forms of the protein, increases with the protein concentration. Stabilization of aggregates by intermolecular disulfides was dependent on concentration only at temperatures below 75 °C.

An antibody-immobilized ELISA test for the detection of the triazole tetraconazole is described. The minimum detection limit was 2 ng/mL (ppb), and the linearity of the assay extended from 1.9 to 1000 ng/mL (ppb), with a variation of absorbance (ΔOD) of ∼0.83 ± 0.04. Good reproducibility and reliability of the ELISA assay was found for fruit commodities spiked with different amounts of tetraconazole. The effect of fruit matrices on the sensibility of the ELISA was investigated, and a matrix interference index (I m) is proposed for quantitation of tetraconazole recovery in the analyzed fruit homogenates. Analysis of fruit juices was optimized to minimize matrix interference. This study could represent an approach for validation of ELISA tests for analysis of tetraconazole in fruit commodities and it should be adaptable to other food matrices. Keywords: Immunoassay; ELISA; fungicide; tetraconazole; fruit analysis; matrix interference