α-Amylase interaction with soluble fibre: insights from diffusion experiment using fluorescence recovery after photobleaching (FRAP) and permeation experiment using ultrafiltration membrane

Haiteng Li, Sushil Dhital

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Abstract

The modulation of starch hydrolysis by soluble fibres has been reported in both in vitro and in vivo conditions. Several mechanisms have been put forward including increased gastric retention time, and altered gastric digesta rheology affecting overall mass transfer (enzyme, substrate and hydrolysed products). The direct inhibition of amylase activity by soluble fibres has also been reported but little is known on the mechanisms. Herein we studied the interaction of porcine α-amylase with soluble fibre at low concentrations (to reduce the effect of viscosity). Two methods were performed, free diffusion of α-amylase in fibre solution using fluorescence recovery after photobleaching (FRAP) and forced elution of α-amylase in fibre solution through ultrafiltration membrane. The results provided the evidence that α-amylase potentially binds with soluble fibre. The degree of diffusion (D/D0 from FRAP experiments) by arabinoxylan and β-glucan at the concentration level of 0.05% and 0.5% w/v is significantly lower than that of pectin. The permeation experiment using ultrafilter membrane showed that the effect of arabinoxylan on intensity retention of fluorescence-labelled α-amylase was found to be in between β-glucan and pectin; pectin has a higher level of intensity retention than arabinoxylan and β-glucan. The results suggested that the interaction (primarily due to binding) could be more pronounced in neutral fibre (β-glucan and arabinoxylan) than the charged polymer (pectin). Overall, this short communication provided a new perspective of soluble fibre-enzyme interactions and experimental approaches for further research.

Original languageEnglish
Article number100319
Number of pages5
JournalBioactive Carbohydrates and Dietary Fibre
Volume28
DOIs
Publication statusPublished - Nov 2022

Keywords

  • Amylase
  • Binding
  • Diffusion
  • FRAP
  • Soluble fibre

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