AOAC OMB Final Action Recommendation (December 2019)-2016.14

2016.14 (October 2019) – FOS-03 MANUSCRIPT

FOR ERP USE ONLY DO NOT DISTRIBUTE

Appendix A: Sucrase/β-amylase/pullulanase/maltase enzyme mixture check test 1 To check performance and suitability of sucrase enzyme mixture, equal aliquots (100 µL) of 2 sucrose solution (0.75 mg/mL) and fructooligosaccharide (FOS) ingredient solution (0.75 mg/mL) 3 are mixed and treated with the sucrase enzyme mixture (200 µL) to be tested. Water is added 4 (100 µl) and the mixture is incubated at 40 °C for 90 min (or the relevant conditions to be tested). 5 After centrifugation (5 min at 10 000 x g ), an aliquot of the supernatant (100 µL) is transferred into 6 a vial suitable for the autosampler instrument, diluted with water (400 µL) and mixed. This solution 7 is injected onto an HPAEC-PAD instrument (conditions described below) and the chromatographic 8 profile obtained is compared with the one obtained from the same solution (sucrose/FOS) not 9 treated with the enzyme mixture (200 µL of water instead of the enzyme mixture). 10 Note: It is highly recommended to perform this test with a FOS ingredient of low DP (average 3- 11 5) which has been produced by partial hydrolysis of inulin, as these seem to be the most sensitive 12 to sucrase side activities or impurities. 13 Chromatographic conditions 14 Mobile phase A : 300 mM NaOH 15 Mobile phase B : Purified water with resistivity ≥18 MΩ 16 Mobile phase C : 500 mM NaOAc + 150 mM NaOH 17 Flow rate : 1.0 mL/min 18 Column : CarboPac PA1, 250 x 4 mm, 10 µm (ThermoFisher Scientific, or 19 equivalent) 20 Oven temperature : 30 °C 21 Injection volume : 25 µL 22 Detector parameters : Quadruple waveform described in Table 2016.14D , gold working 23 electrode and Ag/AgCl reference electrode (or equivalent).

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