The Physical Adsorption of Gelatinized Starch with Tannic Acid Decreases the Inhibitory Activity of the Polyphenol against α-Amylase

Abstract

The effects of mixing orders of tannic acid (TA), starch, and α-amylase on the enzyme inhibition of TA were studied, including mixing TA with α-amylase before starch addition (order 1), mixing TA with pre-gelatinized starch before α-amylase addition (order 2) and co-gelatinizing TA with starch before α-amylase addition (order 3). It was found that the enzyme inhibition was always highest for order 1 because TA could bind with the enzyme active site thoroughly before digestion occurred. Both order 2 and 3 reduced α-amylase inhibition through decreasing binding of TA with the enzyme, which resulted from the non-covalent physical adsorption of TA with gelatinized starch. Interestingly, at low TA concentration, α-amylase inhibition for order 2 was higher than order 3, while at high TA concentration, the inhibition was shown with the opposite trend, which arose from the difference in the adsorption property between the pre-gelatinized and co-gelatinized starch at the corresponding TA concentrations. Moreover, both the crystalline structures and apparent morphology of starch were not significantly altered by TA addition for order 2 and 3. Conclusively, although a polyphenol has an acceptable inhibitory activity in vitro, the actual effect may not reach the expected one when taking processing procedures into account.

Keywords: adsorption; binding interactions; mixing order; tannic acid; α-amylase inhibition.

Figure 1

The contents of reducing sugars (maltose equivalents) produced along with starch digestion at a time interval of 4 min in the absence and presence of TA for three mixing procedures, and the initial reaction velocity was obtained from the slope of plot of reducing sugar contents against digestion time (A). Based on this, the enzyme inhibition of TA for three mixing procedures at the low (10 mg/mL) and high (20 and 30 mg/mL) TA concentrations were obtained (B). The time course of starch digestion in the absence and presence of TA for three mixing procedures (C). The logarithm of slope (LOS) analysis for the fraction of digested starch along with digestion time (D), from which the digestion rate constants k in each digestion phases were obtained.

Figure 2

The mechanism in α-amylase inhibition of TA. α-Amylase inhibition at a series of TA concentrations determined by use of an EnzCheck^TM ultra amylase assay kit, and the curve was fitted according to the IC₅₀ value calculation equation (A). Inhibition kinetics of TA for order 2 (B) and order 3 (C), in which Dixon and Cornish-Bowden (inserted) plots were described to obtain the competitive inhibition constant, K_ic; The quenching effect of TA on α-amylase fluorescence (D) and the fluorescence quenching constant K_FQ was obtained from Stern-Volmer equation (inserted).

Figure 3

The dialysis scheme of TA for the control (the upper one) and for the TA-starch gelatinized systems (the below one, including two mixing procedures, i.e., mixing TA with pre-gelatinized starch, and co-gelatinizing TA with starch) (A). The adsorption capacity (adsorption amount of TA per mass of starch) of starch for two mixing procedures at the dialysis time of 20 h at the low (2 mg/mL) and high (4, 6, and 8 mg/mL) initial TA concentrations in the dialysis bag (B). The concentrations of dialyzed TA outside the dialysis bag along with time at 2 (C), 4 (E), and 6 (G) mg/mL of initial TA concentrations inside the bag for two mixing procedures; The logarithm of slope analysis for the fraction of dialyzed TA along with time at 2 (D), 4 (F), and 6 (H) mg/mL of initial TA concentrations inside the bag, from which the transport rate constants k_t that reflect the dialysis velocity were obtained.

Figure 4

The FTIR spectra of lyophilized gelatinized starch in the absence and presence of TA for mixing TA with pre-gelatinized starch (A), and co-gelatinizing TA with starch (B). The XRD profiles of lyophilized gelatinized starch in the absence and presence of TA for mixing TA with pre-gelatinized starch (C), and co-gelatinizing TA with starch (D). The SEM profiles of lyophilized gelatinized starch in the absence (E) and presence of TA for mixing TA with pre-gelatinized starch (F), and co-gelatinizing TA with starch (G) at the magnification of 4000 times.

Figure 5

The scheme of effects of mixing orders (procedures) on TA-amylase binding interactions. For order 1, TA was firstly mixed with α-amylase before starch addition; therefore, TA could bind with the active site (in a competitive inhibition manner) of the enzyme thoroughly. Notably, in the binding equilibrium, K_ic indicates the dissociation constant of TA-amylase complex (reforming individual TA and α-amylase); therefore, 1/K_ic suggests the binding constant of TA with the enzyme active site. For order 2, TA was firstly mixed with the pre-gelatinized starch before α-amylase addition. In this order, TA molecules were adsorbed onto the unfolded starch chains in a disordered manner, decreasing the amount of free TA molecules that could bind with α-amylase. Because the inhibition kinetics of TA for this order could still be well-fitted with the competitive Dixon and Cornish-Bowden equations that were performed at a series of starch concentrations (also a series of mass ratios of TA to starch), the adsorption of TA with pre-gelatinized starch at the low and high TA concentrations were suggested to have a similar property. For order 3, TA was mixed with raw starch and then co-gelatinized before α-amylase addition. In this order, TA had a longer contacting process and a higher interacting temperature (than order 2) with starch from the beginning of gelatinization process. Therefore, TA molecules interacted with starch chains more thoroughly (than order 2) along with the swelling of starch granules and unfolding of ordered structures, tending to form a network where TA(s) are included inside acting as a ‘bridge’ linkage of swollen starch. This decreased the binding of TA with α-amylase and the decreasing effect was higher than order 2, specially at a low TA concentration. However, with the TA concentration increasing, the incorporated TA in the network gradually reached saturation, and the entering of additional TA was retarded. By this way, the amount of unbound TA that could bind with α-amylase for this order was more than that for order 2 at a high TA concentration.