Targeted proteomics-based analysis of technical enzymes from fungal origin in baked products

•The paper addresses the detection issues of technical enzymes in bakery.•Possibilities of targeted proteomics-based analysis of food proteins.•An alternative to antibody recognition for specific peptides is put forward.•Method can be adapted for other protein based supplementary ingredients.

The application of technical enzymes is a potential tool in modulating the dough and baking quality of cereal products. No endogenous amylases (α- and β-forms) are present in mature wheat grains; they may be synthesized or activated during germination. Hence, microbial α-amylases are added to the dough, being resistant to the endogenous α-amylase/trypsin inhibitors. Here, we report on the initial identification of two technical enzymes from a commercial sample based on an in-gel tryptic digestion coupled with MALDI-MS analysis. The primary component of the protein fraction with 51.3 kDa was α-amylase from Aspergillus species. A second major protein with 24.8 kDa was identified as endo-1,4-xylanase from Thermomyces lanuginosus. In the following experimental work up, a targeted proteomics approach utilizing the combination of specific proteolytic digestion of the added amylase and xylanase in wheat flour, dough or baked products, solid phase extraction of released peptides and their detection using LC-MS/MS was optimized. The targeted (MRM) MS/MS peptide signals showed that the peptide “ALSSALHER” (MW = 983) originating from amylase and “GWNPGLNAR” (MW = 983) from xylanase can be used to identify the corresponding technical enzymes added. Consequently, locally available baked products were tested and found to contain these enzymes as supplementary ingredients.