Rotilabo mini tip syringe filter

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.

To determine FODMAP conversion in the carbohydrate model system, 4 measuring intervals were defined based on the results of the previous growth experiments—6 h, 24 h, 48 h, and 72 h of incubation. The respective cell mass was transferred from the Honeycomb format plates into 2 mL centrifugation tubes. Proteins were removed by Carrez-precipitation. Hence, 15 µL of Carrez I solution—potassium hexacyanoferrate(II)trihydrate (Merck, Darmstadt, Germany) in 1000 mL H₂O, and 15 µL of Carrez II Solution—300 g zinc acetate dehydrate (VWR International GmbH, Darmstadt, Germany) in 1000 mL H₂O was added to precipitate the proteins. After centrifugation at 16,000× g for 30 min at 4 °C, the supernatant was transferred to a volumetric flask and filled up to 1 mL. Next, the solution was filtered through a 0.2-µm filter (Rotilabo Mini-Tip syringe filter; Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and diluted to receive carbohydrate concentrations between 5–200 ppm and transferred into vials (1.5 mL clear glass; VWR International GmbH, Darmstadt, Germany) for further analysis.