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Identification and LC-MS/MS-based analyses of technical enzymes in wheat flour and baked products
(2016)
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.
Soya glycinin was derivatized with different phenolic substances (caffeic-, chlorogenic-, gallic acid and quercetin). The protein derivatives formed have been characterized in terms of their properties where they showed changes in the content of free epsilon-amino groups, tryptophan and thiol groups. The derivatives have also been characterized in terms of their solubility at different pH-values to document the influence on the functional properties. Another objective of this paper was to demonstrate the influence on the digestibility of the proteins with one of the main enzymes of the gastro-intestinal tract (pancreatin) on the basis of in vitro experiments after derivatization with phenolic substances. The enzymatic digestion of the derivatized proteins was promoted.
alpha-Chymotrypsin was modified by covalent attachment of selected phenolic and related compounds (caffeic acid, chlorogenic acid, ferulic acid, gallic acid, quinic acid, m-/o-/p-dihydroxybenzene and p-benzoquinone) at pH 9. The derivatives formed were characterised in terms of their activity and selected physicochemical properties. In vitro experiments showed that the proteolytic digestion of food proteins with alpha-chymotrypsin derivatives was adversely affected. This decrease depended on the reactivity of the phenolic and related substances tested as well as on the kind of substrate applied. The derivatisation was accompanied by a reduction in the amount of free lysine and tryptophan residues. Moreover, the solubility of the derivatives decreased over a broad pH range, with a parallel increase in the hydrophobicity. The isoelectric point was shifted to a lower pH value, and formation of high-molecular-weight fractions was documented by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).