Gopod assay kit

The same extracted sample used for lignin analysis (30 mg) was pretreated with 450 μL 1% w/v sulphuric acid in an autoclave (Astell, UK) at 121 °C for 1 h in 2 mL tubes or saccharified without pretreatment. Solids were washed three times with 1.5 mL 25 mm sodium acetate pH 4.5. Saccharifying enzyme mixture (Celluclast and Novozyme 188 (Sigma)) was prepared as described in Gomez et al. (2010). The FPU (filter paper unit) activity (65 FPU/mL) of the purified mixture was measured (Adney and Baker, 1996) along with β‐glucosidase activity (95.7 CBU/mL) (Ghose, 1987). Saccharification was performed with an enzyme loading of 0.6 FPU per 30 mg of sample in 25 mm sodium acetate pH 4.5 with 0.02% w/v NaN₃ in a total volume of 1.5 mL for 72 h at 50 °C with shaking. Triplicate reactions were performed per plant. Glucose released was quantified using the GOPOD assay kit (K‐GLUC) (Megazyme, Ireland) scaled for a 96‐well plate and expressed as a proportion of the 30 mg extracted sample.

The glucose adsorption capacity (GAC) of the berry PP was determined according to the method described by Bhutkar et al. [25 (link)], with certain modifications. The berry PP (1 g) was mixed with 25 mL of glucose solution of varying concentrations (0, 5, 10, 50, and 100 mmol/L) and incubated for 6 h at 37 °C, with periodical mixing at 120 rpm (GFL 1092, Thermolab, Germany). Next, the glucose concentration was determined using a glucose oxidase/peroxidase (GOPOD) assay kit (Megazyme, Ireland). The GAC was calculated as follows: $$\mathrm{GAC}\left(\mathrm{mmol}/\mathrm{L}\right)=\frac{{\mathrm{C}}_{1-}\left({\mathrm{C}}_2-{ \mathrm{C}}_{3 }\right)}{\mathrm{m}}\times \mathrm{V}$$
where C₁ is the concentration of the original glucose solution; C₂ is the concentration of the glucose in the samples with the berry PP and various concentrations of glucose solution (5, 10, 50, and 100 mmol/L) after 6 h of incubation; C₃ is the concentration of glucose in the samples with the berry PP aqueous solution after 6 h of incubation; m is the weight of the berry pomace (g); and V is the volume of the sample (mL).

The glucose diffusion was determined according to the method described by Bhutkar et al. [25 (link)], with certain modifications. In total, 1 g of berry PP was mixed with 25 mL of 20 mmol/L glucose solution or 25 mL of water. The mixtures were dialyzed against 200 mL of distilled water in dialysis bags at 37 °C, with periodical mixing at 120 rpm (GFL 1092, Thermolab, Germany). Samples from the dialysate were collected at various time intervals (30, 60, 120, and 180 min) and the glucose concentration was determined using a GOPOD assay kit (Megazyme, Ireland). A control sample was prepared that did not contain berry PP. The glucose dialysis retardation index (GDRI) was calculated as follows: $$\mathrm{GDRI}\left(\%\right)=100-\frac{{\mathrm{C}}_2-{ \mathrm{C}}_3}{{\mathrm{C}}_{1 }}\times 100$$
where C₁ is the glucose content of the control, C₂ is the glucose content of the samples with the berry pomace in the glucose solution, and C₃ is the glucose content of the samples with the berry pomace in the aqueous solution.

The β-galactosidase (β-gal) was produced from Escherichia coli BL21 containing gene from L. reuteri L103 following procedure explained by Iqbal et al. [47 (link)]. The enzyme activity was measured for chromogenic substrate oNPG and natural substrate lactose following methods by Splechtna et al. [48 (link)]. The crude β-gal was used for the production of GalOS through bioconversion of lactose by following method of Splechtna, Nguyen, Steinböck, Kulbe, Lorenz and Haltrich [48 (link)]. The transgalactosylated mixture were analyzed for glucose using GOPOD assay kit (K-GLUC) however galactose and lactose were measured using Lactose/Galactose assay kit (K-LACGAR) from Megazyme (Wicklow, Ireland) by following protocol given in the provided documents. The main glycosidic linkage in present prebiotic GalOS was β-1,6 with main products β-d-Galp-(1–6)-d-Glc, β- d-Galp-(1–6)-d-Gal and β-d-Galp-(1–6)-Lac, followed by β-1,3 linked products, i.e., β- d-Galp-(1–3)-d -Gal and β-d-Galp-(1–3)-Lac. While no product with β1–4 linkage was identified [47 (link),48 (link)] as composition determined by High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and capillary electrophoresis.

Three millet cultivars harvested from different areas in inner Mongolia Chifeng, Ordos, and Tongliao, respectivey, with a known starch content of 70.7%, 69.0%, and 79.1%, respectively, were used and denoted as M1, M2, and M3, respectively. Dimethyl sulfoxide (DMSO, HPLC grade for analysis) was obtained from Merck Co. Inc. (Darmstad, Germany). Protease (type XIV, from Streptomyces griseus), Pepsin (672 U/mg, from porcine gastric mucosa), α-amylase (≥5 U/mg, from porcine pancreas), and amyloglucosidase (≥260 U/mL, from Aspergillus niger) were purchased from Sigma-Aldrich Pty (St. Louis, MO, USA). Isoamylase (200 U/mL) and GOPOD assay kit were purchased from Megazyme Ltd. (Wicklow, Ireland). All other reagents were of analytical grade.

The digestibility of starch in the flour samples studied was determined in uncooked states according to the procedures of Englyst et al. [31 ] with slight modifications. The flours (0.80 g, dry basis), guar gum (0.05 g), and sodium acetate buffer (0.25 M, pH 5.2) (20 mL) were mixed with an enzyme solution of pancreatin and amyloglucosidase (5 mL) thoroughly. The mixture was incubated at 37 °C in a water bath with shaking for 2 h. During incubation, aliquots (0.25 mL) of the mixture were taken at different points in time (20, 60, 90, and 120 min). These aliquots were then mixed with ethanol (66% v/v, 10 mL) to terminate the enzymatic reaction and then centrifuged at 1500× g for 10 min. The supernatant was used to analyze the amount of glucose released using the GOPOD assay kit (Megazyme International Ireland Ltd., Wicklow, Ireland). The percentage of starch hydrolysis at different hydrolysis times and starch fractions (RDS, SDS, and RS) were calculated as follows:



where G_ht is the glucose content released in each hydrolysis time; G20 and G120 are the glucose content released at 20 and 120 min of hydrolysis, respectively; and TS is total starch content.