α amylase

The dry matter content of each ration was detected after drying at 135°C for 3 h, and the ash content was detected following combustion at 550°C for 6 h according to the protocols of the AOAC [26 ]. The Kjeldahl method [27 (link)] was used to determine the levels of crude protein (CP, 6.25×N) using an automated nitrogen analyzer (FOSS, DK-3400 Hillerød, Denmark). The total starch content in the TMR was determined via an enzymatic method (α-amylase and amyloglucosidase) using a commercial kit (Megazyme, Megazyme International Ireland Ltd., Bray, Ireland). To analyze the neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents, heat-stable α-amylase (Sigma A3306; Sigma–Aldrich, Shanghai, China) and sodium sulfite were used according to the methods previously established by Van Soest et al. [27 (link)]. The total calcium (Ca) and phosphorus (P) levels were determined according to the National Measurement Principles of China (GB/T 5009.92–2003 and GB/T 5009.87–2003, respectively). The dietary lysine to methionine ratio (Lys: Met) and the net energy for lactation (NE_L) of each ration were estimated based on the included ingredients using the Cornell-Penn-Miner Dairy (CPM-Dairy, version 3.0.8.1) software.

Extrusion trials were performed using commercial chokeberry (Aronia melanocarpa) pomace powder (CPP) (Aronia Original Naturprodukte GmbH, Dresden, Germany) [20 (link)]. Chemicals and reagents of analytical purity grade were obtained from Merck KGaA (Darmstadt, Germany), unless stated otherwise. Amyloglucosidase (E-AMGDFPD, from Aspergillus niger, 36,000 U/g), α-amylase (E-PANAA, from pig pancreas 100,000 U/g), protease (E-BSPRPD from Bacillus licheniformis 9000 U/g), Celite 545 and endo-arabinanase (EC 3.2.1.99, from A. niger, 9 U/mg) were from Megazyme (Bray, Ireland) and Amberlite FPA53 and Ambersep 200 from Rohm and Haas Europe (Frankfurt, Germany). Thermostable α-amylase (Thermamyl 120 L, EC 3.2.1.1, from B. licheniformis, 120 KNU/g), protease (Alcalase 2.5 L, EC 3.4.21.62, from B. licheniformis, 2.5 AU/g), and Amyloglucosidase (AMG 300 L, EC 3.2.1.3, from A. niger, 300 AGU/g) were a gift from Novozymes, (Bagsværd, Denmark). Rotihydroquant C5 and D used for Karl Fischer titration as well as cyanidin-3-O-glucoside (≥96%), cyanidin chloride (≥97%), 5-caffeoylquinic acid (≥97%), quercetin-3-O-glucoside (≥99%), and quercetin dihydrate (≥99%) used as PP standards were obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Ultrapure water was used for all experiments.

Alcohol insoluble residue (AIR) was prepared as described by Fry (1988) with modifications. A total of 100 mg of ground stem material was incubated in phenol for 30 min at room temperature while shaking, followed by centrifugation at 3000 g for 10 min at 4°C. The supernatant was removed and the pellet was washed with the following solutions: twice with chloroform: methanol (1 : 1, v/v), twice with 80% (v/v) methanol, and once with 100% methanol. The pellets were left to dry overnight at room temperature. The samples were destarched by amylase treatment and 20 mg were suspended in 2 ml of 10 mM potassium phosphate buffer (pH 6.5), 1 mM CaCl₂ and 0.05% NaN₃. This suspension was heated at 95°C and the starch was allowed to gelatinize for 30 s before 1 U ml⁻¹ thermostable α‐amylase (Megazyme, Leinster, Ireland). The suspension was incubated at 85°C for 15 min then cooled to 25°C before 10 U ml⁻¹ amyloglucosidase and 1 U ml⁻¹ pullulanase (Megazyme) were added. This solution was incubated for 16 h at 25°C with continuous shaking at 500 rpm. The suspension was centrifuged for 10 min at 6000 g and the supernatant was removed. The pellet was washed with 2 ml 10 mM potassium phosphate (pH 6.5), 1 mM CaCl₂, 0.05% NaN₃, centrifuged at 6000 g and the supernatant was discarded.

Starch was measured according to do Amaral et al. (2007) and Arenque et al. (2014) (link). AIR was treated with 120 U⋅ mL^–1 of α-amylase (E.C. 3.2.1.1) of Bacillus licheniformis (Megazyme^® Inc., Australia) diluted in 10 mM MOPS buffer pH 6.5 at 75°C for 1 h. Incubation was followed by the addition of 30 U⋅ mL^–1 of amyloglucosidase (E.C. 3.2.1.3) of Aspergillus niger (Megazyme^® Inc., Australia) diluted in 100 mM sodium acetate buffer pH 4.5 at 50°C for 1 h. For starch determination, 50 μL of each sample was added to a 250 μL of a mixture containing glucose oxidase (1,100 U⋅ mL^–1), peroxidase (700 U⋅ mL^–1), 4-aminoantipirin (290 μmol⋅ L^–1), and 50 mM phenol at pH 7.5. The plates were incubated for 15 min at 30°C, and the absorbance was measured at 490 nm. The standard curve was performed with commercial glucose (Sigma^®).

Water caltrops (Trapa taiwanensis Nakai) were cultivated and harvested in Tainan, Taiwan. The α-amylase, amyloglucosidase, amylose kit, and damaged starch kit were purchased from Megazyme (Megazyme International Ireland, Co. Wicklow, Ireland). All chemical substances applied in this study were of analytical grade.

Total starch content was determined according to AACC approved method 76−13.01 [54 ] using a kit purchased from Megazyme International (Bray, Wicklow, Ireland) (100/100 tests per kit). Each of the wheat flour samples (~100 mg, accurately weighed), over which 0.2 mL of 80% (v/v) ethanol aqueous solution was added in order to moisten the samples and to help upon dispersal, were transferred to culture tubes. The tubes were agitated using a vortex mixer. Immediately, the samples were treated with sodium acetate buffer solution (100 mM, pH 5.0) and 3 mL each of thermostable α-amylase from Megazyme. The tubes were incubated in boiling water for 6 min, and then vigorously shaken after 2, 4 and 6 min to ensure complete homogeneity of the suspensions. Tubes containing 0.1 mL of amyloglucosidase (20 U) from the Megazyme kit were incubated in a water bath at 50 °C for 30 min. The transferred samples were diluted to 100 mL with distilled water and centrifuged for 10 min at 3000 rpm, and then the supernatant (clear, undiluted filtrate) was retained for analysis. To determine D-glucose, samples were treated with a glucose oxidase assay kit (GOPOD) from Megazyme and the absorbance was recorded at 340 nm on a UV-vis spectrophotometer (Shimadzu 3600, Tokyo, Japan).

Two white rice cultivars, Jasmin rice grains (KDML105 cultivar with 16.9 % amylose content) and Chai-Nat1 rice grains (CN1 cultivar with 29.35% amylose content), were obtained from Upon Ratchathani province, Thailand. The crude protein, fat, and total starch of KDML105 rice were 7.29, 0.89, and 73.76% (w/w), respectively, while CN1 has a corresponding crude protein, fat, and total starch of 7.39, 1.25, and 71.44% (w/w). α-amylase (EC 3.2.1.1., 3,000 U/g), amyl glucosidase (EC 3.2.1.3., 3,300 U/mL) and glucose oxidase peroxidase (GOPOD) kit were obtained from Megazyme International, Ireland Ltd. Other chemicals used were analytical grade.