Englyst modified Method ( based on [1] )
Measure 600 mg (db平行)starch sample and put the sample into COD tube. (Remember to do duplicate平行 样and record the exact sample amount you measured.)
Pipette移液管吸取 10.00 ml H2O into COD tube, then mix them well by vortexing涡旋 for 5 min. (Be sure to avoid lumps结块 and avoid flour from sticking to top of the tubes.)
Place the tubes in a boiling H2O bath. At the same time, shake the tubes and make sure to avoid the lumps. (The first few mins are the most important. Be sure to avoid the lumps or that part will never be cooked.)
Boiling bath for 30 min.
Place the cooked tubes in 37oC H2O bath until it is cooled
Put 5 glass beads玻璃珠 and 10.00 ml acetate 醋酸buffer (pH 5.2). Then immerse浸泡 it horizontally 水平in the shaking water bath at 37oC H2O bath with 160 strokes轻晃动 per min.
Incubate for 25-30 mins. (Do two things at the same time. 1. Prepare
Amylase/AMG enzyme mixture. 2. pipette 10ml of 66% alcohol solution to the centrifuge tubes. )
Start Starch digestion by adding 5.00 ml of enzyme solution. (Add 1 tube every 1 min interval. ) Cap the tube and immerse horizontally in the shaking water bath at 37oC H2O bath with 160 strokes per min.
Collect 1.00 ml hydrolysate水解产物 and put it to the alcohol solution in the centrifuge tube to stop the reaction after incubating孵化培养 for exact 20 min and 120 min. Vortex the tubes well.
Centrifuge the tubes at 1500g？？ for 10 min(4377rpm , r=7cm, for our centrifuge). G=1.118*10-5*r*v2
Transfer the supernanant 上清液to the 25 ml volumetic flask容量瓶 and make to 25 ml with water.
Pipette 1.00 ml sample, blank in duplicate into 25ml colorimetric tubes比色管 and then pipette 3.00 ml DNSreagent 3，5 -二硝基水杨酸（DNS）为显色剂 into each tubes.
Boiling bath for 15 min. Make to 25 ml with water until it is cooled. Determine glucose by spectrophotometry(read at 550nm).
Calculate starch digested at 20 min (RDS快消化淀粉), digested between 20 min and 120 min (SDS慢消化淀粉), and not digested at 120 min (> 120 min = RS抗性淀粉).
PS : Amylase (Pancreatin)/AMG (Amyloglucosidase) (for 4 samples)
a. 3.5775g pancteatin was weighted into 50 ml centrifuge tube and a magnetic stirring bar and 25 ml water was added. The pancreatin was suspended by vortex mixing and then mixed for 10 min on a magnetic stirrer. The tube was centrifuged at 1500g (3860rpm, r= 9cm) for 10 min.
b. pipette 20.00 ml supernatant from centrifuge tube and mix with 1.13 ml AMG, then vortex for 5 min. [1] ENGLYST K N, ENGLYST H N, HUDSON G J, et al. Rapidly available glucose in foods: an in vitro measurement that reflects the glycemic response [J]. American Journal of Clinical Nutrition, 1999, 69(3): 448-54.
体外消化方法测试Englyst modified Method
