SPSFAM ERP

The borohydride reduction is most effective if performed at 40 o C. Ultimately, 30 o C was chosen for incubation with the sucrase/ β -amylase/pullulanase mixture because the sucrase enzyme is less stable at 40 o C than 30 o C. Borohydride reduction is best performed at 40 o C, as is fructanase incubation. 7. Discussion The Single Lab Validation as outlined in this report shows that the Fructan Assay Kit (K-FRUC) method is fit for purpose and applicable for the measurement of fructan (inulin, FOS, levan and branched agave fructan) either as pure ingredients or as present in a wide range of food, feed and plant materials as required by the AOAC SMPR 2018.00.2. The K-FRUC test kit is user-friendly and 20 samples can be analyzed by a single operator within 4 h. This SLV included investigation into a variety of performance characteristics including Working range, Selectivity, Recovery, Limit of Detection (LOD), Limit of Quantification (LOQ), Trueness ( bias ), Precision (reproducibility and repeatability), Robustness and Stability. The K-FRUC assay was shown to be linear over a range of 0.1-54 µg per test. For animal feed and food samples treated as per the method outlined in this report the working range of 0.1-54 µg of fructan (as fructose plus glucose) per test equates to a concentration range of 0.1 to 100 g/100 g of the sample analyzed. The method as described in the Fructan Assay Kit is absolutely specific for fructan (inulin, FOS, levan and agave). Recovery of fructan (inulin), over fructan concentrations 0.2 to 100 g/100g and from a wide range of animal feeds and pet foods, is quantitative (97-103%) over fructan concentrations 0.1 to 100 g/100g. The Limit of Detection and Limit of Quantification were determined to be 0.036 g/100g and 0.119 g/100g respectively. Since there are no official certified reference materials for fructan, the reference material used for this analysis was Raftiline (native chicory inulin) which is a commercially available, almost pure, inulin. Accuracy of the Fructan Assay Kit method was assessed by comparison of the mean fructan content (x) obtained for Raftaline [a suitable

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