D fructose d glucose 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.

d-Allose, d-glucose, α-Glc1P, d-gluconic acid, lactose, d-mannose, and d-xylose were purchased from Fujifilm Wako Pure Chemical (Osaka, Japan); N-acetyl-d-glucosamine, d-galactose, and sucrose were purchased from Nacalai Tesque (Kyoto, Japan); d-glucosamine was purchased from Tokyo Chemical Industry (Tokyo, Japan); cellobiose, d-galacturonic acid, and d-glucuronic acid were purchased from Sigma (St. Louis, MO, USA). β-(1 → 4)-Mannobiose was prepared as previously described^{29 (link)}. Lactoless L3 (β-galactosidase) was provided by Daiwa Kasei (Shiga, Japan). SP from Bifidobacterium longum was prepared according to the method described by Nishimoto and Kitaoka^{5 (link)}. One unit of SP was defined as the amount of enzyme required to phosphorolyze 1 μmol of sucrose in 1 min. SP activity was measured as follows: a reaction mixture (50 μL) containing appropriate concentration of enzyme, 20 mM sucrose, 40 mM sodium phosphate, 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH buffer (pH 7.0), and 0.2 mg/mL bovine serum albumin (BSA, Nacalai Tesque) was incubated at 37 °C for 10 min. The enzymatic reaction was terminated by incubating the sample at 80 °C for 3 min, and the liberated d-fructose was measured using a d-Fructose/d-Glucose Assay Kit (Megazyme, Brey, Ireland).

Lactobacillus plantarum 299 V® was used as a positive control for inulin fermentation and identified by PCR using the pair of primers Plantarum (Supplementary Table S1). Bacteria were sub-cultured at 1/100 dilution in standard minimal medium (M9) supplemented with 1% inulin for 24 h in anaerobic conditions using an anaerobic sachet (BD BBL™ GasPak™ anaerobic indicator, BD, Mississauga, ON, Canada) and the optical density at 600 nm (OD600) was determined in a Spark® multimode microplate reader (Tecan Group Ltd., Männedrof, Switzerland) every hour for a day. In parallel, the release of free fructose and glucose in the supernatants during inulin fermentation were determined after 3 h of culture using a D-fructose/D-glucose assay kit (Megazyme International Ltd., Wicklow, Ireland).

Phytic acid concentration was measured using K-PHYT 05/07 kit assay (Megazyme Intl., Wicklow, Ireland), following the manufacturer’s instructions.
The in vitro protein digestibility (IVPD) of muffins was determined according to Akeson and Stahmann [28 (link)] with some modifications [29 (link)]. IVPD was expressed as the percentage of the total protein solubilized after a sequential enzymatic treatment mimicking the in vivo digestion enzymatic hydrolysis. The protein concentration was determined following the Bradford method [30 (link)].
The starch hydrolysis (HI) degree was determined according to the method (mimicked the in vivo digestion) proposed by De Angelis et al. [31 (link)]. The glucose released after the enzymatic process was measured with D-Fructose/D-Glucose Assay Kit (Megazyme, Wicklow, Ireland). HI was expressed as the percentage of potentially available starch hydrolyzed after 180 min. Wheat flour bread (WB) leavened with baker’s yeast was used as the control to estimate the hydrolysis index (HI = 100). The predicted GI (pGI) was calculated using the equation—pGI = 0.549 × HI + 39.71 [32 (link)].

In vitro starch hydrolysis was determined as previously described (24 (link)). The procedure mimicked the in vivo digestion of starch. Aliquots of doughs, containing 1 g of starch (determined in dough), were subjected to enzymatic process (pancreatic amylase and pepsin-HCl), and the released glucose content was measured with D-Fructose/D-Glucose Assay Kit (Megazyme, Wicklow, Ireland). Simulated digests were dialyzed (cut-off of the membrane: 12,400 Da) for 180 min. Aliquots of dialysate, containing free glucose and partially hydrolysed starch, were sampled every 30 min and further treated with amyloglucosidase. Then, free glucose was determined using the above-mentioned enzyme-based kit and finally converted into hydrolysed (digested) starch in the dough. Control white wheat bread was used as the control to estimate the hydrolysis index (HI = 100). The pGI was calculated using the equation: pGI = 0.549 × HI + 39.71 (25 (link)). Each sample was analyzed in triplicate.