OMB First to Final Action Recommendation Checklist 11-12-18

ERP - GLUTEN ASSAYS: AOAC 2015.05 FAR

Partially hydrolyzed secalin in sourdough As for the intact secalin, the recovery is also above 100% for the hydrolyzed secalin, as to be  expected from an R5 ELISA. However, the tendency of decreasing recovery with decreasing  concentrations of hydrolyzed secalin is not as pronounced as for intact secalin. Thus the hydrolyzed  spike is much more comparable to the calibrator of the system, due to more similar kinetic behavior  as explained above.   CVs for all concentration within the measurement range were excellent, only at the LoQ level, a  higher CV was observed. For detailed results see annex 4.  Table 4. Mean concentrations, SD and recovery of six independent   extractions of partially‐hydrolyzed secalin‐spiked sourdough samples  Hydrolyzed secalin (rye) in sourdough  spiked  mean*  SD  CV  recovery  mg/kg  mg/kg  mg/kg  %  %  15  34.9  2.0  5.6  229  10  23.2  1.1  4.8  226  5  10.9  0.9  7.8  207  2.5  5.10**  0.87  17.0  180  Blank  0.59  *   blank corrected  ** concentrations below standard 2 (5 mg/kg) were extrapolated using 2 nd  order polynomial function  3.3 Fermentation of secalin and hydrolyzed secalin in sourdough over 24 hours  To show the applicability of the competitive R5 ELISA for secalin measurement during sourdough  fermentation, sourdough was spiked with intact and hydrolyzed secalin. Rice sourdough was spiked  with 14 mg/kg secalin or hydrolyzed secalin, was mixed thoroughly and incubated for 24 h at room  temperature. Samples were taken at 0; 1; 2; 4; 6; 8 and 24 hours (4 replicates per time point). Samples  were extracted directly after sampling with 60% ethanol according to instructions for use of  RIDASCREEN® Gliadin competitive and application note for RIDASCREEN® Gliadin R7001 (sandwich  format).   As shown in figure 1, intact secalin was constantly hydrolyzed by sourdough cultures within 24 h by  about 40%. This “loss” is not due to a loss of protein mass but a loss of two or more connected epitopes  which are after cleavage no longer detectable by a sandwich system. Therefore –as stated in the test  kit insert‐ the R5 sandwich format is not suitable for measurement of fermented/hydrolyzed samples.  As expected, secalin showed an overestimation of 240% at the beginning of the experiment.    

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