α amylase

Dao Huaxiang rice grains (Wuyoudao No.4, one variety of non-pigmented rice, long grain) were cultivated in the year 2021 in Wuchang district (Heilongjiang province, China). Brown rice is a rice grain prepared after removing the rice husks with a rice huller, including the cortex, aleurone layer and endosperm. The rice grain obtained after removing about 90% of the rice bran in the brown rice is white rice.
Lactobacillus plantarum (CICC No. 22696) was purchased from the China Industrial Culture Collection Center (Beijing, China). α-Amylase (enzyme activity [EA] ≥ 150,000 U/mL), amyloglucosidase (EA ≥ 110,000 U/g) and protease (EA ≥ 500,000 U/g) were supplied by Macklin Biochemical Co., Ltd. (Shanghai, China). Dietary fiber assay kit and antioxidant capacity assay kit (T-AOC) were purchased from Megazyme Co. (Bray, Wicklow, Ireland) and Jiancheng Bioengineering Institute (Nanjing, China). Other reagents used in this study were of analytical grade and originated from Chemical Co. (Beijing, China).

The fractionation of cell walls was conducted as previously described [62 (link)]. Separated tissues were fixed for 10 min in 15 mL of boiling methanol. The methanol-fixed tissues were rehydrated with water, then homogenized in water with a mortar and pestle. The residue obtained by centrifugation was washed with water, acetone, and a methanol:chloroform mixture (1:1, v/v) and air-dried. The washed residue was dried overnight at 40 °C and then treated with 2 units/mL α-amylase (Macklin, Shanghai, China) in 100 mM MOPS (Coolaber, Beijing, China) buffer (pH 7.3) for 0.5 h at 80 °C, then with 1 unit/mL pullulanase (Coolaber, Beijing, China) and 3 units/mL amyloglucosidase (Coolaber, Beijing, China) in sodium acetate buffer for 3 h at 50 °C, to remove starch. Hemicellulose was extracted for 18 h with 17.5% NaOH containing 0.02% NaBH4. The hemicellulosic fraction was neutralized with glacial acetic acid in an ice-cold water bath, then dialyzed against water. The dialyzed hemiceiiuiosic fraction was centrifuged for 20 min at 10,000× g, and dried. The xyloglucan content was determined using the iodine-staining method [62 (link)].

Starch digestibility was determined as the previous report (20 (link), 23 (link)–26 (link)) with modifications. α-Amylase (6 g, 35 U/mg) (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) and 40 mL of deionized water were mixed followed by stirring for 10 min. Then, the mixed solution was centrifuged at 4,000 rpm for 15 min. Finally, 30 mL of the supernatant was mixed with 2 mL of amyloglucosidase (from Aspergillus niger, 1 × 10⁵ U/mL) (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) and 3 mL of deionized water to prepare digestive enzyme solution. Brown rice powders (500 mg) was mixed with 14 mL of deionized water and boiled in a water bath for 5 min. Then, 1 mL of digestive enzyme solution was added, shaken, and incubated at 37°C. Sample solutions of 0.2 mL were taken out after 20, 60, 120, 180, and 240 min and added 80% ethanol solution (5 mL) immediately.