Kinetics of in vitro digestion of starches monitored by time-resolved H nuclear magnetic resonance

A H NMR method is presented that monitors the initial and later stages of in vitro enzymatic digestion of starch suspensions. It allows, for the first time to our knowledge, the accurate analysis of the initial 5% of the extent of hydrolysis. This is significant because rapidly digested starch produces glucose that determines the blood glucose concentration immediately after ingestion of food. The two key hydrolytic enzymes, R-amylase and amyloglucosidase, showed clear systematic deviation from Michaelis-Menten kinetics as the starch or wheat flour substrate that was used changed its character during the reaction. Estimates of Michaelis-Menten parameters for amyloglucosidase and R-amylase were successfully found by analyzing two stages of digestion separately. The Michaelis-Menten constants for purified starch were (6.4 ( 0.8) and (1.1 ( 0.3) g dL-1 (% w/v), respectively; and the maximum velocities of glucose release by amyloglucosidase, and short oligoglucosides and glucose by R-amylase were (1.9 ( 0.4) × 10-2 and (1.6 ( 0.2) × 10-2 mmol L-1 s-1 for the first stage of digestion, and (9.0 ( 1.0) × 10-3 and (4.7 ( 1.4) × 10-3 mmol L-1 s-1 for the second stage, giving a ratio of the two Vmax values of 2.1 and 3.4, respectively.