Pullulanase

The sample preparation was performed according to a previously described method [2 (link)]. Briefly, 50 mg of each rice starch sample was dispersed in 3.8 mL of double distilled water and then boiled in a water bath at 100 °C for 1 h. The mixtures were incubated with 1 mL of the enzyme cocktail (3.6 U of pullulanase (Megazyme International Ireland Ltd., Wicklow, Ireland) and 0.5 U of isoamylase (Megazyme International Ireland Ltd., Wicklow, Ireland) in 100 mM sodium acetate buffer pH 5.0) at 40 °C for 48 h. To inactivate the enzymes, the debranched samples were augmented with 0.2 mL of 100 mM sodium hydroxide and 5 mL of deionized water, respectively. The mixtures were then centrifuged at 5000× g for 5 min at 25 °C and filtered through a 0.45 μm nylon filter. Finally, 1 mg/mL of the debranched amylopectin was used to analyze and determine the amylopectin CLD using a high-performance anion-exchange chromatography system equipped with a pulsed amperometric detector (HPAEC-PAD) (ICS-5000, Dionex Co., Ltd., Sunnyvale, CA, USA), according to the protocol of Lee et al. [30 (link)].

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.

The selected leaves were processed for alcohol insoluble residue (AIR) preparations as described in Nguema-Ona et al. [34 (link),39 (link)]. Leaf material was plunge frozen with liquid nitrogen and then ground to a fine powder using a Retsch Mixer Mill (30 rounds per minute, 60 s, Retsch, Haan, Germany). After boiling the tissue powder for 20 min in 80% aqueous ethanol (reagent grade) to deactivate enzymes, the insoluble material was washed with methanol for two hours on a rotating wheel. After centrifugation at 3000 rpm for 3 min, the pellets were sequentially washed twice with a (1:1) methanol/chloroform solution for 2 h to remove plant oils/lipids and then rinsed with acetone for another 2 h before being air-dried. The extracted material was resuspended in distilled water and freeze-dried to obtain cell wall AIR. Samples were destarched using a combination of thermostable alpha-amylase, amyloglucosidase and pullulanase (from Megazyme). The AIR material was used for all further analytical experiments. All solvents used were from (Sigma-Aldrich, MO, USA) at reagent grade.