AOAC ISPAM Stakeholder Panel Meeting Book (August 26, 2018)

( 3 ) Similarly, we have tried extraction of non-gluten proteins with 0.4 M NaCl in PBS (pH = 7.5) followed by Dumas nitrogen analysis of the resulting centrifuged pellet. ( 4 ) Potentially, an approximation could be made on a sample just by a Dumas nitrogen and use a factor for each grain (e.g., for wheat, multiply protein by 0.8, for rye and barley, multiply protein by 0.55) based on Katharina Scherf’s empirical data. We assume the accuracy of such method should not be too critical, since our end criteria for ELISA recoveries will be quite wide. Inaccuracy of this method will be dwarfed by the size of the criteria. We could increase the width of the ELISA criteria to account for the uncertainty in the “reference” method’s accuracy. Example Samples of contaminant wheat, rye, and barley were obtained from Katharina Scherf’s laboratory, with gluten concentration as determined by the Wieser method. These samples were used to spike the very low oat flour. For this series, we used oat groats that were made by mechanically separating oats and then mechanically cleaning the groats again after dehulling. The groats were ground in a Retsch mill to pass a 1.0 mm screen. The sample of oat flour was homogenized in a food processor for 5 min prior to spiking. A large quantity (ca 5 kg) was homogenized by splitting in two portions and stirring by Robot-Coupe for 5 min. After initial mix, one half of each half was mixed together in the Robot-Coupe (ca 2.5 kg). The final flour was mixed in a large plastic bag. The oat flour was partitioned into equal portions with a large riffle splitter to approximate target weight for spiking. The exact weights of oat flour and spiked ground grains were recorded to calculate spiked concentration. Spiked amounts of wheat, barley, and rye were weighed in a metal measuring cup prior to addition to the oat flour. Each portion of oat flour was put into a separate Cuisinart DLC-X food processor bowl. The preweighed spiked ground grain was sprinkled evenly over the top of the flour. The sample was then mixed in the Cuisinart for 5 min, after which the motor was stopped and the bowl scraped with a tongue depressor and then the sample was mixed for an additional 5 min. Spiked data including masses are given in Table 1. In addition to the gluten concentration estimates we obtained from Katharina Scherf, we also analyzed the samples by liquid phase extraction of non-gluten proteins and Dumas nitrogen analysis of the centrifuged pellet. We did this two ways: the first by extraction in water (2x), followed by extraction in 0.5 M NaCl (2x). The second method we tried was by 0.4 M NaCl in PBS (pH 7.5) (3x). The second method was suggested by Katharina Scherf as a more optimal extraction, which will not solubilize certain omega gliadins which can be lost in pure water extracts. (Personal correspondence with K. Scherf) After spiking and homogenization, the sample set was analyzed with significant replication by the R5 method (R-Biopharm kit No. R7001 with cocktail extraction). The results are displayed in Figure 1.

Figure 1. Graphical representation of kit response.

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