D mannitol assay kit

The content of fructans, fructose, glucose, and mannitol before and after bran bioprocessing was analysed enzymatically according to AOAC method 999.03 (with Fructan Assay Kit, Megazyme, Ireland), AOAC method 985.09 (with D-Fructose/D-Glucose Assay Kit, Megazyme, Ireland), and D-Mannitol Assay Kit (Megazyme, Ireland), respectively. Samples for the determination of fructose, glucose, and mannitol were freshly prepared on the day of analysis. Each sample suspension was heated at 80 °C for 10 min in a water bath. After cooling to room temperature and centrifugation at 4000 rpm for 10 min, 1.5 mL of the supernatant was centrifuged again at 14,700 rpm for 3 min [21 (link)]. The results for fructans represent the combined content of fructans and galactooligosaccharides, as the samples were not treated with α-galactosidase before analysis. A proximate total FODMAP content in 30 g of snack was calculated based on fructans, fructose, and mannitol content determined in the bran (3 g), oat flour (15 g), and rice protein (5 g) used for the preparation of the dough (Section 2.4.), considering the loss of water during baking (i.e., the baking loss). Baking loss was determined using the following formula [24 (link)]:
where m_pd represents the weight of dough before baking and m_bs the weight of baked snack.

For isolation of compatible solutes, cells were grown in mineral medium with 300 mM NaCl until they reached an OD₆₀₀ of 0.9. Additionally, solutes were determined directly after osmotic upshift. Therefore, cells were grown in mineral medium to the exponential growth phase (OD₆₀₀ of 0.4 to 0.5), and hyperosmotic stress was applied by the addition of 300 mM NaCl. Cells were incubated (37°C, 130 rpm) and partly harvested over a time period of 3 h. Solute extraction was performed with chloroform and methanol as described before (36 (link)). Mannitol, trehalose, and glutamate were determined enzymatically (trehalose assay kit K‐TREH, l‐glutamic acid assay kit K‐GLUT, and d-mannitol assay kit from Megazyme, Bray, Ireland) (36 (link)).