Frederick J. Warren

Mechanisms of starch digestion by α-amylase—Structural basis for kinetic properties

ABSTRACT Recent studies of the mechanisms determining the rate and extent of starch digestion by α-amylase are reviewed in the light of current widely-used classifications for (a) the proportions of rapidly-digestible (RDS), slowly-digestible (SDS), and resistant starch (RS) based on in vitro digestibility, and (b) the types of resistant starch (RS 1,2,3,4…) based on physical and/or chemical form. Based on methodological advances and new mechanistic insights, it is proposed that both classification systems should be modified. Kinetic analysis of digestion profiles provides a robust set of parameters that should replace the classification of starch as a combination of RDS, SDS, and RS from a single enzyme digestion experiment. This should involve determination of the minimum number of kinetic processes needed to describe the full digestion profile, together with the proportion of starch involved in each process, and the kinetic properties of each process. The current classification of resistant starch types as RS1,2,3,4 should be replaced by one which recognizes the essential kinetic nature of RS (enzyme digestion rate vs. small intestinal passage rate), and that there are two fundamental origins for resistance based on (i) rate-determining access/binding of enzyme to substrate and (ii) rate-determining conversion of substrate to product once bound.

Inhibition of α-amylase activity by cellulose: Kinetic analysis and nutritional implications

We report on inhibition of α-amylase activity by cellulose based on in vitro experiments. The presence of cellulose in the hydrolysing medium reduced the initial velocity of starch hydrolysis in a concentration dependent manner. α-Amylase adsorption to cellulose was reversible, attaining equilibrium within 30min of incubation, and showed a higher affinity at 37°C compared to 20 and 0°C. The adsorption was almost unchanged in the presence of maltose (2.5-20mM) but was hindered in the presence of excess protein, suggesting non-specific adsorption of α-amylase to cellulose. Kinetic analyses of α-amylase hydrolysis of maize starch in the presence of cellulose showed that the inhibition is of a mixed type. The dissociation constant (Kic) of the EI complex was found to be ca. 3mg/mL. The observed inhibition of α-amylase activity suggests that cellulose in the diet can potentially attenuate starch hydrolysis.

Infrared spectroscopy as a tool to characterise starch ordered structure - A joint FTIR-ATR, NMR, XRD and DSC study

Starch has a heterogeneous, semi-crystalline granular structure and the degree of ordered structure can affect its behaviour in foods and bioplastics. A range of methodologies are employed to study starch structure; differential scanning calorimetry, (13)C nuclear magnetic resonance, X-ray diffraction and Fourier transform infrared spectroscopy (FTIR). Despite the appeal of FTIR as a rapid, non-destructive methodology, there is currently no systematically defined quantitative relationship between FTIR spectral features and other starch structural measures. Here, we subject 61 starch samples to structural analysis, and systematically correlate FTIR spectra with other measures of starch structure. A hydration dependent peak position shift in the FTIR spectra of starch is observed, resulting from increased molecular order, but with complex, non-linear behaviour. We demonstrate that FTIR is a tool that can quantitatively probe short range interactions in starch structure. However, the assumptions of linear relationships between starch ordered structure and peak ratios are overly simplistic.

Combined techniques for characterising pasta structure reveals how the gluten network slows enzymic digestion rate

The aim of the present study is to characterise the influence of gluten structure on the kinetics of starch hydrolysis in pasta. Spaghetti and powdered pasta were prepared from three different cultivars of durum semolina, and starch was also purified from each cultivar. Digestion kinetic parameters were obtained through logarithm-of-slope analysis, allowing identification of sequential digestion steps. Purified starch and semolina were digested following a single first-order rate constant, while pasta and powdered pasta followed two sequential first-order rate constants. Rate coefficients were altered by pepsin hydrolysis. Confocal microscopy revealed that, following cooking, starch granules were completely swollen for starch, semolina and pasta powder samples. In pasta, they were completely swollen in the external regions, partially swollen in the intermediate region and almost intact in the pasta strand centre. Gluten entrapment accounts for sequential kinetic steps in starch digestion of pasta; the compact microstructure of pasta also reduces digestion rates.

The interplay of α-amylase and amyloglucosidase activities on the digestion of starch in in vitro enzymic systems

In vitro hydrolysis assays are a key tool in understanding differences in rate and extent of digestion of starchy foods. They offer a greater degree of simplicity and flexibility than dynamic in vitro models or in vivo experiments for quantifiable, mechanistic exploration of starch digestion. In the present work the influence of α-amylase and amyloglucosidase activities on the digestion of maize and potato starch granules was measured using both glucose and reducing sugar assays. Data were analysed through initial rates of digestion, and by 1st order kinetics, utilising logarithm of slope (LOS) plots. The rate and extent of starch digestion was dependent on the activities of both enzymes and the type of starch used. Potato required more enzyme than maize to achieve logarithmic reaction curves, and complete digestion. The results allow targeted design of starch digestion experiments through a thorough understanding of the contributions of α-amylase and amyloglucosidase to digestion rates.

Amylase binding to starch granules under hydrolysing and non-hydrolysing conditions

Although considerable information is available about amylolysis rate, extent and pattern of granular starches, the underlying mechanisms of enzyme action and interactions are not fully understood, partly due to the lack of direct visualisation of enzyme binding and subsequent hydrolysis of starch granules. In the present study, α-amylase (AA) from porcine pancreas was labelled with either fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) fluorescent dye with maintenance of significant enzyme activity. The binding of FITC/TRITC-AA conjugate to the surface and interior of granules was studied under both non-hydrolysing (0 °C) and hydrolysing (37 °C) conditions with confocal microscopy. It was observed that enzyme binding to maize starch granules under both conditions was more homogenous compared with potato starch. Enzyme molecules appear to preferentially bind to the granules or part of granules that are more susceptible to enzymic degradation. The specificity is such that fresh enzyme added after a certain time of incubation binds at the same location as previously bound enzyme. By visualising the enzyme location during binding and hydrolysis, detailed information is provided regarding the heterogeneity of granular starch digestion.

Biodegradation of starch films: The roles of molecular and crystalline structure

The influences of molecular, crystalline and granular structures on the biodegradability of compression-molded starch films were investigated. Fungal α-amylase was used as model degradation agent. The substrates comprised varied starch structures obtained by different degrees of acid hydrolysis, different granular sizes using size fractionation, and different degrees of crystallinity by aging for different times (up to 14 days). Two stages are identified for unretrograded films by fitting degradation data using first-order kinetics. Starch films containing larger molecules were degraded faster, but the rate coefficient was independent of the granule size. Retrograded films were degraded much slower than unretrograded ones, with a similar rate coefficient to that in the second stage of unretrograded films. Although initially the smaller molecules or the easily accessible starch chains on the amorphous film surface were degraded faster, the more ordered structure (resistant starch) formed from retrogradation, either before or during enzymatic degradation, strongly inhibits film biodegradation.

Infrared Spectroscopy with Heated Attenuated Total Internal Reflectance Enabling Precise Measurement of Thermally Induced Transitions in Complex Biological Polymers

We report an improved tool for acquiring temperature-resolved fourier transform infrared (FT-IR) spectra of complex polymer systems undergoing thermal transitions, illustrated by application to several phenomena related to starch gelatinization that have proved difficult to study by other means. Starch suspensions from several botanical origins were gelatinized using a temperature-controlled attenuated total reflectance (ATR) crystal, with IR spectra collected every 0.25 °C. By following the 995/1022 cm(-1) peak ratio, clear transitions occurring between 59 and 70 °C were observed, for which the midpoints could be determined accurately by sigmoidal fits. The magnitude of the change in peak ratio was found to be strongly correlated to the enthalpy of gelatinization as measured by differential scanning calorimetry (DSC, R(2) = 0.988). An important advantage of the technique, compared to DSC, is that the signal-to-noise ratio is not reduced when measuring very broad transitions. This has the potential to allow more precise determination of the gelatinization parameters of high-amylose starches, for which gelatinization may take place over several tens of °C.

Acid Hydrolysis and Molecular Density of Phytoglycogen and Liver Glycogen Helps Understand the Bonding in Glycogen α (Composite) Particles

Phytoglycogen (from certain mutant plants) and animal glycogen are highly branched glucose polymers with similarities in structural features and molecular size range. Both appear to form composite α particles from smaller β particles. The molecular size distribution of liver glycogen is bimodal, with distinct α and β components, while that of phytoglycogen is monomodal. This study aims to enhance our understanding of the nature of the link between liver-glycogen β particles resulting in the formation of large α particles. It examines the time evolution of the size distribution of these molecules during acid hydrolysis, and the size dependence of the molecular density of both glucans. The monomodal distribution of phytoglycogen decreases uniformly in time with hydrolysis, while with glycogen, the large particles degrade significantly more quickly. The size dependence of the molecular density shows qualitatively different shapes for these two types of molecules. The data, combined with a quantitative model for the evolution of the distribution during degradation, suggest that the bonding between β into α particles is different between phytoglycogen and liver glycogen, with the formation of a glycosidic linkage for phytoglycogen and a covalent or strong non-covalent linkage, most probably involving a protein, for glycogen as most likely. This finding is of importance for diabetes, where α-particle structure is impaired.

Changes in glycogen structure over feeding cycle sheds new light on blood-glucose control

Liver glycogen, a highly branched polymer of glucose, is important for maintaining blood-glucose homeostasis. It was recently shown that db/db mice, a model for Type 2 diabetes, are unable to form the large composite glycogen α particles present in normal, healthy mice. In this study, the structure of healthy mouse-liver glycogen over the diurnal cycle was characterized using size exclusion chromatography and transmission electron microscopy. Glycogen was found to be formed as smaller β particles, and then only assembled into large α particles, with a broad size distribution, significantly after the time when glycogen content had reached a maximum. This pathway, missing in diabetic animals, is likely to give optimal blood-glucose control during the daily feeding cycle. Lack of this control may contribute to, or result from, diabetes. This discovery suggests novel approaches to diabetes management.