Alpha amylase

To determine changes of DP of graminan-type fructans after fermentation, a fingerprint analysis was performed after extraction of the wheat flour-water suspension. Following fermentation for 8 h and 15 min, the suspensions in the Einhorn locks were subjected to an ethanol extraction. The matrix was transferred into a centrifugation tube, 30 mL of 96% ethanol (VWR International GmbH, Darmstadt, Germany) was added and the mixture was heated to 80 °C for 20 min. After centrifugation at 9000× g for 6 min, the supernatant was filled up to the defined volume of 50 mL. A 3 mL aliquot of the extract was evaporated at 50 °C under a nitrogen stream to dryness and resuspended in 0.96 mL of water. Twenty µL each of amyloglucosidase (AMG) and alpha-amylase (Novozymes, Ireland) were added, and incubation was carried out at 50 °C for 30 min. Afterwards, protein precipitation, as described in Section 2.4, was conducted. The solution was sterile-filtered through a 0.2-µm filter (Rotilabo Mini-Tip syringe filter; Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and used further for HPAEC-PAD (high pressure anion-exchange chromatography-pulsed amperometric detection) analysis.