Glucose oxidase peroxidase gopod reagent

The total starch content of feed and digesta samples was determined according to the AOAC Method 996.11 (KOH format) [36 (link)] using the Total Starch Assay Kit (Megazyme, Bray, Ireland) as modified by Martens et al. (2018) [37 (link)]. In brief, 25 μL supernatant of enzymatically treated samples was transferred in the wells of a 96 well plate followed by the addition of 225 μL glucose oxidase peroxidase (GOPOD) reagent (Megazyme). The reaction was performed in a shaking incubator at 50 °C for 20 min, and the absorbance at 520 nm was read against reagent blank using a Tecan Infinite^® F500 (Tecan Group Ltd., Männedorf, Switzerland) spectrophotometer. The glucose (Glc) content was determined using a Glc calibration curve (0.1–0.6 mg/mL).

Wheat flour, whole wheat flour, and drinking water were purchased from local producers in Turkey (Akınsoy Food Industry and Pınar Water and Beverage Industry, respectively). Invertase (from yeast, 300 U/mL, E-INVPD-2G), thermostable α-amylase (from Bacillus licheniformis, 3000 U/mL, E-BLAAM-10ML), amyloglucosidase (from Aspergillus niger, 3330 U/mL, E-AMGDF-10ML), and glucose oxidase-peroxidase (GOPOD) reagent were purchased from Megazyme (Wicklow, Ireland). Pepsin (from porcine gastric mucosa, 250 U/mL, P7000-100G), pancreatin (from porcine pancreas, 8 × USP specifications, P7545-100G), and guar gum were obtained from Sigma–Aldrich Co., LLC. (St. Louis, MO, USA).

Waxy, medium-amylose, and high-amylose black rice (rough rice, varieties of Xuenuomi, Heizhenzhu, and Haishuidao) were supplied by a grain trader (Hubei, China). Total starch assay kit and glucose oxidase-peroxidase (GOPOD) reagent were obtained from Megazyme International Ireland Ltd. (Bray Co., Wicklow, Ireland). Pancreatic α-amylase (P7545), pepsin (P7000), and amyloglucosidase (A7095) were purchased from Sigma Chemical Co. (St. Louis, MO., U.S.A.). All other chemicals were of analytical grade unless otherwise stated.