Total starch assay kit

The seeds of 7, 14, 21, 28 and 35 DAF of mutant line and wild-type Gao 8901 were used to carry out starch content, amylose content and GBSS I activity analysis. Total starch and amylose contents were measured using the Total Starch Assay Kit and Amylose/Amylopectin Assay Kit (www.megazyme.com) according to manual instructions. GBSS I activity was measured using the Micro Bound Station amylosynthease Assay Kit (http://www.shkxbio.com). All tests were performed on three replicates. Student’s t-test assessed statistical differences between wild type and mutant line.

The total starch (TS) content was determined using Total Starch Assay Kit (amyloglucosidase/α–amylase method) suitable for food products (Megazyme, Ireland) according to manufacturer’s procedure [24 ], with slight modification–glucose content was determined based on DNS assay [25 (link)] using UV-Vis spectrophotometer (UV 1601PC, Shimadzu Co., Ltd., Kyoto, Japan). Starch was calculated as glucose × 0.9. Results were expressed in mg per 1 g DW of white bean paste.

The dry matter (DM, 135°C for 2 h) and chemical components of the diets and refusals were determined. The ether extract, Kjeldahl N, and crude ash values were determined according to Association of Official Analytical Chemists (AOAC) (2000 ; methods 920.39, 990.03, and 942.05, respectively). The organic matter (OM) was calculated as weight loss during ashing. The NDF was assayed with a heat‐stable amylase and sodium sulfite, and expressed exclusive of residual ash. This and acid detergent fiber (ADF) expressed exclusive of residual ash were analyzed according to the methods of van Soest et al. (1991 (link)) and the AOAC (2000 ; method 973.18). Starch was assayed using a commercial kit (Total Starch Assay Kit; Megazyme Ltd., Wicklow, Ireland). The gross energy was determined using an adiabatic bomb calorimeter (CA‐4PJ; Shimadzu, Kyoto, Japan). Short‐chain fatty acids (SCFAs) in the rumen fluid were determined using a gas chromatograph (6890; Hewlett‐Packard, Palo Alto, CA, USA) with a glass column packed with 5% Thermon 1000 and 0.5% H₃PO₄ on 80/100 mesh Chromosorb W (Wako Pure Chemical Ltd., Osaka, Japan). Milk samples were analyzed for fat, protein, and lactose concentrations by infrared spectroscopy (Milko‐Scan 133B; N. Foss Electric, Hillerød, Denmark).

The endosperm at different development of OsSUS3-OE and wild-type plants were used to measure the content of starch. The starch were collected and assayed by total starch assay kit (Megazyme, Bray, Co. Wicklow, Ireland).

Mature rice grains were air-dried and dehulled. Brown rice was further ground with a mortar and passed through a 0.15 mm sieve. The total starch content in the flour was determined with a total starch assay kit (Megazyme, Wicklow, Ireland). The amylose content was measured by using an iodine-potassium iodide colorimetric method as described previously [42 (link)]. The chain length distribution of amylopectin was analyzed using a fluorophore-assisted capillary electrophoresis (FACE) method according to the previous report [43 (link)]. The thermal properties and pasting properties of starch were measured by differential scanning calorimetry (DSC, 200-F3, Netzsch, Selb, Germany) and rapid visco analyzer (RVA, 3D, Newport Scientific, Narrabeen, Australia), respectively, following the method as described previously [44 (link)].

Chemical composition was assessed both on raw materials and dry pasta. Moisture was measured by a thermobalance (Sartorius MA 40, Goettingen, Germany) at 120 °C and all analytical data were expressed as dry weight (dw).
Total starch (TS) content was determined according to the Official Method 996.11 [28 ], by Total Starch Assay Kit (Megazyme, Bray, Ireland). Amylose content was determined using the Megazyme Amylose/Amylopectin assay kit. Resistant starch (RS) content was determined according to the Official Method 2002.02 [29 ], using Resistant Starch Assay Kit (Megazyme). Total dietary fiber (TDF) content was measured using the enzymatic kit Bioquant (Merck, Darmstadt, Germany) according to the Official Method 991.42 [30 ]. Ash content was determined according to the Official Method 08-01.01 [31 ]. Protein content was determined by micro-Kjeldhal nitrogen analysis, according to the ICC 105-2 method [32 ], using as conversion factor N × 6.25. Total antioxidant capacity (TAC) was determined according to Martini et al. [33 (link)].

Shepody potatoes were provided by the Dingbian Science and Technology Bureau of Shaanxi Province, China. Potato starch was isolated from Shepody potatoes containing 4.24% moisture, 95.76% starch, and 1.95% ash. Wheat gluten was purchased from Tian Long Wheat Flour Co., Ltd. (Henan, China). The wheat gluten contained 7.22% moisture, 89.34% protein, 9.82% starch, and 0.82% ash. The production way of the wheat gluten was as follows: Wheat flour was mixed with water and then separated by pumping the flour paste into a series of hydrocyclone. The obtained flow was then washed and sieved to get the wet gluten. After that, a drying process was applied before packing. Moisture, protein, and ash contents were determined using AOAC official methods (Association of Analytical Chemists, 2000), and starch content was determined using a total starch assay kit (Megazyme, K‐TSTA 04/2009). All these measurements were performed in triplicate.