Mechanism of reduction in starch digestion rate of Durum Wheat by protein

Zou, Wei (2017). Mechanism of reduction in starch digestion rate of Durum Wheat by protein PhD Thesis, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland. doi:10.14264/uql.2017.359

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Author Zou, Wei
Thesis Title Mechanism of reduction in starch digestion rate of Durum Wheat by protein
School, Centre or Institute Queensland Alliance for Agriculture and Food Innovation
Institution The University of Queensland
DOI 10.14264/uql.2017.359
Publication date 2017-02-28
Thesis type PhD Thesis
Supervisor Robert G. Gilbert
Michael J. Gidley
Frederick J. Warren
Total pages 183
Language eng
Subjects 0908 Food Sciences
060107 Enzymes
030406 Proteins and Peptides
Formatted abstract
The aim of the project is to explore the mechanism of durum wheat proteins in slowing starch digestion; this reduction in digestion rate is nutritionally advantageous. The grains of three commercial durum wheat varieties (Jandaroi, Caparoi and Yawa) were employed, from which a range of pasta-derived cooked substrates were prepared: semolina (SE), whole pasta (spaghetti) (WP), powdered pasta (PP) and extracted starch (ST). SE contains inherent protein components; WP has an intact compact structure and gluten network formed by kneading and extruding the SE proteins; PP was ground from WP, thus breaking up the intact compact structure while the gluten network remained intact; and ST was extracted from SE with removal of proteins. To understand how pasta compact structure and proteins influence starch digestion, all these starch-containing samples with different protein composition and structure were subjected to in vitro digestion with various combinations of treatments mimicking gastric conditions with acid and pepsin, before the starch was digested with porcine α-amylase or pancreatin. After plotting the percentage of starch digested vs. time, first-order kinetics characterization through logarithm-of-slope analysis and morphological characterization by confocal microscopy were combined to reveal how the pasta compact structure and gluten network together slow starch digestion rate. Digested samples were collected at different times to characterize the weight distributions of branched starch molecules (wbr (logRh), Rh being hydrodynamic radius) using size-exclusion chromatography (SEC, also termed GPC). These distributions, together with the measured activity of α-amylase in the digestive solution, were used to explore the role that the compact structure and proteins in pasta play in retarding the evolution of starch molecular structure during digestion. Gluten powder (GP) extracted from SE was cooked and centrifuged to separate supernatant from gluten as a pellet, followed by the addition of α-amylase, to characterize to what extent α-amylase interacted with wheat proteins by measuring the activity of α-amylase, to elucidate if the protein components are capable of reducing enzymatic activity; the analytical technique used was high performance liquid chromatography-mass spectrometry (LC-MS).

The results showed that ST and SE were digested following simple first-order kinetics, while WP and PP followed two sequential first-order steps. The rate coefficients for these various steps were altered by pepsin hydrolysis. Confocal microscopy revealed that, following cooking, starch granules were completely swollen for ST, SE and PP samples. In WP, the granules were completely swollen in the external regions, partially swollen in the intermediate region and almost intact in the WP strand center. Gluten entrapment accounts for the sequential kinetic steps in the digestion of pasta starch; the compact microstructure of pasta also reduces digestion rates. A reduced activity of porcine α-amylase and retarded digestion for branched starch molecules of intermediate/small sizes were seen for samples which contain soluble proteins in the digestive solution but rapid digestion for branched starch molecules of small/intermediate/large sizes was seen for samples where these proteins were removed. The combined observations support the hypothesis that soluble protein(s) present in cooked SE, PP and WP interact with α-amylase to reduce its enzymatic activity, and thus retard the digestive evolution of branched starch molecules. The data also suggest that this enzyme/soluble protein interaction is a physical one, probably non-covalent (e.g. entanglement or H bonding), because the enzyme activity could be restored. The compact structure of WP protects the inner region of a pasta fragment from protein-degrading and starch-degrading enzymes, while the remaining soluble protein(s) reduces the activity of α-amylase. Additionally, the residual gluten network may be able to prevent the leaching of large amylopectin molecules. All these factors reduce the rate of enzymatic degradation of the starch, especially for larger molecules with Rh >100 nm. Further study indicated that cooked GP released nearly all these active soluble proteins capable of reducing the activity of α-amylase into the aqueous solution, as only the supernatant was observed to be capable of significantly reducing the activity of α-amylase. Protein compositional analysis reveals that the added α-amylase mainly existed in the supernatant, with little in the gluten pellet. Moreover, less than ~16% of protein became soluble in the supernatant, of which there was a notable abundance of inherent α-amylase inhibitors and glutenin subunits but almost no gliadin. In contrast, the gluten pellet contained the most proteins, which comprised mainly gliadin and glutenin subunits, while few α-amylase inhibitors were present. Taken together, these results suggest that the inherent α-amylase inhibitors are probably the active soluble protein components that are capable of reducing the activity of α-amylase, whereas the gliadin and glutenin subunits (as the main protein components) are not capable of reducing the activity of α-amylase.

The research findings for the whole project have been used to further clarify the roles wheat proteins play in slowing the starch digestion of pasta products. In essence, wheat proteins firstly form the gluten network as a backbone, supporting the compact and dense structure which both protects inner starch granules from being gelatinized, swollen and accessed by the penetrative enzymes. In addition, the digestion is slowed for starch entrapped by the gluten network, which contains protein α-amylase inhibitors with the capacity of reducing the activity of the penetrative α-amylase. These two structural features created by wheat proteins combine to lead to a slowed starch digestion for pasta products processed from wheat varieties.
Keyword Starch digestion
Compact structure
Gluten network
Digestive evolution of starch molecules
Protein α-amylase inhibitors

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Created: Wed, 08 Feb 2017, 10:49:32 EST by Wei Zou on behalf of Learning and Research Services (UQ Library)