AOAC OMB Final Action Recommendation (December 2019)-2016.14

2016.14 (Jan. 2019) FOS-03 MLT Report FOR ERP USE ONLY DO NOT DISTRIBUTE

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

Elution gradient :

500 mM NaOAc + 150 mM NaOH, % C

Water, % B

Flow rate, mL/min

300 mM NaOH, % A

Time, min

0.0

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

30.0 30.0 16.6

60.0 41.0 22.7

10.0 29.0 60.7

15.0 40.0 40.1 45.0 45.1 50.0 50.1 60.0

0.0 0.0

0.0 0.0 0.0 0.0 0.0

100.0 100.0

100.0 100.0

0.0 0.0

30.0 30.0

10.0 10.0

60.0