@article{UhrWielandHomannetal.2016,
  author    = {Uhr, Linda and Wieland, Phillis and Homann, Thomas and Huschek, Gerd and Rawel, Harshadrai Manilal},
  title     = {Identification and LC-MS/MS-based analyses of technical enzymes in wheat flour and baked products},
  series = {European food research and technology : official organ of the EuCheMS, Division of Food Chemistry},
  volume    = {242},
  journal   = {European food research and technology : official organ of the EuCheMS, Division of Food Chemistry},
  publisher = {Springer},
  address   = {New York},
  issn      = {1438-2377},
  doi       = {10.1007/s00217-015-2536-5},
  pages     = {247 -- 257},
  year      = {2016},
  abstract  = {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.},
  language  = {en}
}
@article{UhrBuchholzHomannetal.2014,
  author    = {Uhr, Linda and Buchholz, Tina and Homann, Thomas and Huschek, Gerd and Rawel, Harshadrai Manilal},
  title     = {Targeted proteomics-based analysis of technical enzymes from fungal origin in baked products},
  series = {Journal of cereal science},
  volume    = {60},
  journal   = {Journal of cereal science},
  number    = {2},
  publisher = {Elsevier},
  address   = {London},
  issn      = {0733-5210},
  doi       = {10.1016/j.jcs.2014.04.007},
  pages     = {440 -- 447},
  year      = {2014},
  abstract  = {The application of technical enzymes is a potential tool in modulating the dough and baking quality of cereal products. No endogenous amylases (alpha- and beta-forms) are present in mature wheat grains; they may be synthesized or activated during germination. Hence, microbial alpha-amylases are added to the dough, being resistant to the endogenous alpha-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 alpha-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. (C) 2014 Elsevier Ltd. All rights reserved.},
  language  = {en}
}