Immunoassay for Natamycin Trace Screening: Bread, Wine and Other Edibles Analysis

The antifungal drug natamycin (NAT) is widely used in medicine and in the food industry as preservative E235 for a wide variety of foods. The risk of the development of resistance to NAT and its spread in relation to other polyene antibiotics is fraught with the emergence of incurable infections. This work is devoted to the development of an immunoassay to investigate the prevalence of NAT use for food preservation. Two immunogen designs based on tetanus toxoid, conjugated to NAT through different sites of hapten molecules, were compared in antibody generation. Assay formats using heterologous coating antigens were superior for both antibodies. The ELISA variant demonstrated the highest sensitivity (IC₅₀ = 0.12 ng/mL), and a limit of detection of 0.02 ng/mL was selected for NAT determination. The optimized extraction procedure provided a recovery rate of 72-106% for various food matrixes with variations below 12%. Cyclodextrins, as well as NAT-cyclodextrin complex formulations, showed no interference with the quantification of NAT. One hundred and six food product brands, including baked goods, wines, beers, drinks, sauces, and yogurts, were tested to assess the prevalence of the undeclared use of NAT as a preservative. The screening examination revealed three positive yogurts with an undeclared NAT incorporation of 1.1-9.3 mg/kg.