Rhodamine B-based lipase activity assay with natural substrates-development and application

For the various application fields of lipases in food, an accurate and reliable prediction of their performance is needed. This is not provided by the current artificial substrate-based assays, because natural substrates should be applied. The fluorescent dye Rhodamine B (RB) enables the tracking of released fatty acids in real time and therefore offers the possibility to assess lipase activity towards any substrate. A high-throughput assay including RB was established, validated and applied to a range of five fats/oils and two isolated lipids for a range of seven lipases. The results show that lipase reactions under different conditions are not comparable. Besides the substrate and the lipase, the milieu of the reaction has a decisive impact. For the prediction of lipase reactions in foods, further aspects such as the accessibility of substrates need to be taken into consideration. We developed a high-throughput Rhodamine B-based assay for the determination of lipase activities towards natural substrates. When applied to a range of five fats/oils and two isolated lipids in combination with seven lipases, we found individual patterns of lipase activities for each lipase and each substrate.image