Lipases in Wheat Breadmaking: Analysis and Functional Effects of Lipid Reaction Products

The baking activity of two different lipases was evaluated by a microbaking test on a 10 g flour basis, and the altered lipid composition of lipase-treated wheat lipids was quantitated. To identify and quantitate the various lipid classes, pure glycolipids and phospholipids were isolated from a wheat flour lipid extract by a silica gel batch procedure and silica gel column chromatography. These reference compounds were used to establish a high-performance liquid chromatographic method with evaporative light scattering detection, which was able to separate all of the wheat lipid classes and lipase reaction products. Wheat lipids, dough lipids, and dough lipids after lipase addition were quantitated using cholesterol as an internal standard. Especially digalactosyl diglycerides (−0.9 mmol/kg flour), monogalactosyl diglycerides (−0.4 mmol/kg), and <i>N</i>-acyl-phosphatidyl ethanolamine (−0.3 mmol/kg) were hydrolyzed, and a concomitant formation of digalactosyl monoglycerides (+0.6 mmol/kg), monogalactosyl monoglycerides (+0.6 mmol/kg), and <i>N</i>-acyl-lysophosphatidyl ethanolamine (+0.5 mmol/kg) was found. The lipase-induced changes of the lipid fraction caused increases in bread volume of 56–58%, depending on the type and concentration of the added lipase. The current results confirm the important relationship between the lipid fraction composition and the baking performance of flour

Quantitation of Specific Barley, Rye, and Oat Marker Peptides by Targeted Liquid Chromatography–Mass Spectrometry To Determine Gluten Concentrations

Celiac disease is triggered by the ingestion of gluten from wheat, barley, rye, and possibly oats. Gluten is quantitated by DNA-based methods or enzyme-linked immunosorbent assays (ELISAs). ELISAs mostly detect the prolamin fraction and potentially over- or underestimate gluten contents. Therefore, a new independent method is required to comprehensively detect gluten. A targeted liquid chromatography–tandem mass spectrometry method was developed to quantitate seven barley, seven rye, and three oat marker peptides derived from each gluten protein fraction (prolamin and glutelin) and type (barley, B-, C-, D-, and γ-hordeins; rye, γ-75k-, γ-40k-, ω-, and HMW-secalins). The quantitation of each marker peptide in the chymotryptic digest of a defined amount of the respective reference gluten protein type resulted in peptide-specific yields, which enabled the conversion of peptide into protein concentrations. This method was applied to quantitate gluten in samples from the brewing process, in raw materials for sourdough fermentation, and in dried sourdoughs

Targeted liquid chromatography tandem mass spectrometry to quantitate wheat gluten using well-defined reference proteins

<div><p>Celiac disease (CD) is an inflammatory disorder of the upper small intestine caused by the ingestion of storage proteins (prolamins and glutelins) from wheat, barley, rye, and, in rare cases, oats. CD patients need to follow a gluten-free diet by consuming gluten-free products with gluten contents of less than 20 mg/kg. Currently, the recommended method for the quantitative determination of gluten is an enzyme-linked immunosorbent assay (ELISA) based on the R5 monoclonal antibody. Because the R5 ELISA mostly detects the prolamin fraction of gluten, a new independent method is required to detect prolamins as well as glutelins. This paper presents the development of a method to quantitate 16 wheat marker peptides derived from all wheat gluten protein types by liquid chromatography tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring mode. The quantitation of each marker peptide in the chymotryptic digest of a defined amount of the respective reference wheat protein type resulted in peptide-specific yields. This enabled the conversion of peptide into protein type concentrations. Gluten contents were expressed as sum of all determined protein type concentrations. This new method was applied to quantitate gluten in wheat starches and compared to R5 ELISA and gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD), which resulted in a strong correlation between LC-MS/MS and the other two methods.</p></div

Selected wheat marker peptides.

<p>Amino acid sequences of the 16 peptides (P1-16), their specificity for wheat gluten protein types, and the detected peptide scores in the flour.</p

Schematic diagram showing the development of a method for the quantitation of gluten contents based on peptide yields.

<p>(A) Peptide identification and selection of 16 wheat marker peptides, (B) development of the liquid chromatography tandem mass spectrometry (LC-MS/MS) method with an isotopically labelled peptide as internal standard and optimization of the LC-MS/MS conditions, (C) quantitation of peptide yields in reference gluten protein types and conversion of peptide into protein type and gluten concentrations.</p

Linear Pearson correlations between gluten contents and concentrations of peptides from all wheat gluten protein types.

<p>(A) Peptide P4 from low-molecular-weight glutenin subunits (LMW-GS), (B) P7 from high-molecular-weight glutenin subunits (HMW-GS), (C) P8 from γ-gliadins, (D) P11 from α-gliadins, (E) P14 from ω5-gliadins, (F) P15 from ω1,2-gliadins. The presented peptides showed the highest correlation coefficients within the respective protein type (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192804#pone.0192804.t003" target="_blank">Table 3</a>).</p

Concentrations of the marker peptides (P1-16) in the respective protein type [μg/g] and the wheat flour mixture [μg/g].

<p>The concentrations of protein types in flour by LC-MS/MS [%] were calculated based on peptide concentrations in the specific protein types and compared to the contents [%] quantitated by RP-HPLC. The contents determined by RP-HPLC were taken as 100% to evaluate the recovery of LC-MS/MS. Protein type concentrations had to be multiplied by the individual correction factor to adjust to recoveries of 100%.</p

Optimized LC-MS/MS parameters for the 16 wheat marker peptides.

<p>Multiple reaction monitoring (MRM) parameters of P1-16 and the isotopically labelled peptide standard (*P11) as well as the corresponding response factors (<i>RF</i>), each referred to *P11.</p

Limits of detection (LOD) and quantitation (LOQ) for the marker peptides P1-16 in potato flour [μg/g].

<p>Correlation coefficients (r) were determined between peptide concentrations and gluten concentrations in the potato flour spiked to different gluten contents with the wheat flour mixture.</p

Gluten contents [μg/g] of wheat starches W4, W6, W8, W11, W13, W14 and W15.

<p>Results from different methods, LC-MS/MS, GP-HPLC-FLD and R5 ELISA, were compared.</p