Identification and LC-MS/MS-based analyses of technical enzymes in wheat flour and baked products

The use of technical enzymes in bakery industry is necessary for a consistent and good quality of baked products. Since the cultivation of cereals leads to low amounts of endogenous enzymes being present, a need of their commercial alternatives is becoming a routine process in order to meet the consumer quality demands. Targeted quantification proteomics-based methods are necessary for their detection to meet the regulatory criteria. Here, we initially report on the identification of Lipase FE-01, a lipase from fungus Thermomyces lanuginosus, as analyzed by SDS-PAGE, in-Gel digestion, and MALDI-TOF-MS. In further experiments, the focus of the study was directed toward an extensive use and optimization of in-solution enzymatic digestion in combination with LC-MS/MS techniques in identification of specific peptide markers and finally in utilization of the latter in delivering reproducible quantification data for several different technical enzymes (alpha-amylases, xylanase, and lipases from microbial origin) in complex matrices such as baked bread and wheat flour. Two digestion protocols (a fast option using thermocycler program and the well-established overnight method) were tested, and both of these can be successfully applied. The application of isotopically labeled analogs of the MRM targeted peptides as internal standards and the addition of an internal protein standard during the extraction/digestion experiment were compared to determine the optimal quantification algorithm of the recovered enzyme concentrations. Thus, a standardized sensitive LC-MS/MS method could be developed to determine technical enzymes as forthcoming ingredients in the prefabricated food formulations in concentrations lower than 10 ppm.