Electrophoretic and immunochemical determination of bioindustrial enzymes in apple juice

The present study was undertaken to develop a specific and sensitive method which allows the detection of trace amounts of the potentially allergenic bioindustrial enzymes Pectinex Ultra-SPL (pectinase) and Gamylo 200L (amylase) in apple juice. This was achieved by an immunoblotting procedure based on sodium dodecyl sulfate (SDS) electrophoretic separation, followed by an immunochemical detection step using polyclonal rabbit antisera directed against these enzymes and, secondly, alkaline phosphatase labelled antibodies in combination with a highly sensitive enhanced chemiluminescent (ECL) detection method. In a juice spiked with enzyme at various concentrations between 10,000 and 1 ppm (1.0-0.0001%), the detection limit was 10 ppm (=0.001%) for Pectinex Ultra-SPL and 1 ppm (=0.0001%) for Gamylo 200L, respectively. Since the enzyme preparations contain less than 5% of enzymes, the detection limit for the pure enzymes is markedly lower, at least below 1 ppm. Whereas in the samples withdrawn during the initial stages of the industrial production process of apple juice, Pectinex Ultra-SPL and Gamylo 200L components were detectable in concentrations between 50 and 100 ppm, after clearing, ultrafiltration and pasteurization, neither pectinase nor amylase was detectable in the final consumer's product. Under the conditions employed, the bioindustrial enzymes Pectinex Ultra-SPL and Gamylo 200L were virtually removed during the course of production. The risk of sensitizing consumers by intake of apple juice seems to be marginal. This view is supported by the results of an animal model (anaphylaxis test), in which neither gastro-enteral reactions in guinea pigs and mice, nor IgE or IgG production by orally feeding these animals with apple juice concentrate, could be induced.