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Title: Molecular mechanisms of α-amylase action on retrograded starch : the relationship between digestion kinetics and physicochemical characteristics
Author: Patel, Hamung
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2015
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Depending upon the physicochemical structure and properties of starch, the rate and extent of starch digestion can vary significantly from being very rapid to a much slower process and in some cases being virtually indigestible. The purpose of this mechanistic research project is to produce kinetic and structural data that provides an improved understanding of the interaction between porcine pancreatic α-amylase (PPA) and starch structures. Native, gelatinised and retrograded starch samples (stored between 0-96h) were digested with PPA to produce digestibility plots from which Michaelis-Menten kinetic parameters were determined. Logarithm of slope (LOS) plots of digestibility curves for prolonged incubations were also constructed to allow the rate constant (k) and the total digestible starch (C∞) to be calculated. Following gelatinisation, the kcat/Km ratio increased drastically compared with native samples. For retrograded starches, the kcat/Km decreased relative to the non-retrograded samples, with the biggest difference being noticed in wheat, potato and high amylose maize starches. Surprisingly, the binding facility of α-amylase to starch did not change over 96h, indicated by the Km values. The LOS plots showed no change in k but a decrease in C∞ when gelatinised starch was stored at room temperature. The LOS plots also revealed a single rate constant for processed starches with only native starches being digested in distinct rapid and slower phases. Evidence for the structural changes that occur during starch processing was obtained using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The results showed that the degree of crystalline/ordered structure increased upon storage. Isolated retrograded starch from high amylose maize was harvested and physically and chemically characterised. The results indicate that retrograded starch material is mainly amorphous with small amounts of crystallinity. Upon digestion with α-amylase however, no starch products were detected indicating that the mainly amorphous retrograded starch material is resistant to digestion. The inhibition studies showed that retrograded starch acts as a non-competitive inhibitor of PPA and can therefore still bind to the enzyme but no amylolysis occurs.
Supervisor: Ellis, Peter Rory ; Butterworth, P. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available