The penetration of an exogenous Aspergillus niger pectinmethylesterase added by soaking or by vacuum-impregnation in apple cubes was examined by three different methods. The enzyme activity was first quantitatively assayed in different concentric zones of 1.5 cm apple cubes. Exogenous enzyme activity was distributed homogeneously after vacuum-impregnation, while the penetration of the enzyme was limited to the superficial zone (< 2 mm depth) after soaking for 1 h. The second and third methods consisted in blotting the inner surface of cubes on a nitrocellulose membrane subsequently revealed with antibodies directed against the exogenous enzyme and in observing by fluorescence microscopy apple cubes soaked or vacuum-impregnated with fluorescein-labelled enzyme, respectively. The quantitative results were confirmed by these qualitative visual methods at a macroscopic and at a microscopic scale, respectively. Vacuum-impregnation can thus be used to add this exogenous enzyme to fruit pieces more rapidly and more homogeneously than soaking in order to improve the firmness of thermally treated fruits. (c) 2005 Elsevier Inc. All rights reserved.