To begin, harvest the seeds of mutant and X134 plants, and allow them to dry naturally at room temperature until the moisture content reaches approximately 13 to 15%After activating the peeling machine, introduce 10 g of rice grains into the feeder to efficiently remove the glume shell. Transfer the peeled rice seeds into the rice mill and operate the machine for 60 seconds to eliminate the aleurone layer yielding polished rice. Next, place the polished rice into the tissue grinder.
Adjust the grinding frequency to 60 Hz and run the grinder for 15 seconds for two cycles to produce rice powder. Deposit the ground rice powder into a Petri dish and place it in a preheated oven set to 37 C for 12 hours. Then add 100 mg of rice powder into a 2 mL microcentrifuge tube, and gently tap the tube.
Add 180 L of purified water to the tube and boil the sample in a water bath for 20 minutes. After the sample cools down, introduce 4 mL of AMG containing pancreatic alpha amylase into the tube. Next, mix the contents on a vortex oscillator, and incubate the tube at 37 C for 16 hours with continuous agitation.
Now, add 4 mL of ethanol and mix the sample using a vortex. Then centrifuge the tube at 1, 500 G for 10 minutes. After decanting the supernatant, add 2 mL of 50%ethanol, followed by 6 mL of 50%IMS to the tube and mix.
After centrifugating the tube, carefully pour off the supernatant and invert the tube to drain excess liquid. Placing the tube in an ice bath, add 2 mL of 2 molar potassium hydroxide to it, and stir the sample for 20 minutes to re-suspend flock and dissolve resistant starch. Add 8 mL of 1.2 molar sodium acetate buffer adjusted to pH 3.8 to the tube and mix using a magnetic stirrer.
Then immediately introduce 0.1 mL of AMG into the mixture and mix thoroughly. Incubate the sample for 30 minutes at 50 C in a water bath with intermittent mixing using a vortex. After incubation, centrifuge the tube at 1, 500 G for 10 minutes.
Now transfer 0.1 mL of the supernatant to a fresh glass tube. Add 3 mL of glucose oxidase peroxidase reagent and incubate the sample at 50 C for 20 minutes. Carefully pipette 200 L of each blank sample solution and standard solution into a 96-well plate.
Measure the absorbance of each sample at 510 nm against the blank solution and calculate the resistant starch content using the formula. Finally, present the participants with 50 g of prepared rice with 200 mL of water. After the meal, collect venous blood samples at various time points to perform blood glucose analysis.
Resistant starch content was significantly higher in the SBEIIb mutant with 5.2%compared to the wild type. Consumption of the prepared rice led to a slower and reduced glucose response at 15 and 30 minutes with reductions of 9.7%and 3.7%respectively.
