Sushil Dhital

Synergistic and antagonistic effects of α-Amylase and amyloglucosidase on starch digestion.

The influence of biomacromolecular physical structure on the kinetics of degradation with exo-acting or a mixture of endo- and exo-acting enzymes was studied using three physical forms of maize and potato starch, amyloglucosidase (exo-acting) and α-amylase (endo-acting) as exemplars. For starch in granular form, there was synergism between the enzymes in the production of glucose. In contrast, endo- and exo-acting enzymes showed antagonistic effects in digestion of cooked starches. Antagonism was ascribed to the rapid production of low molecular weight oligomers by α-amylase, which are less efficiently digested by amyloglucosidase than polymeric substrates. The rates of digestion of swollen granule ghosts cooked under low shear conditions were slower than starches cooked under high shear conditions that prevent granule ghost formation. There was also an enzyme-resistant fraction present in granule ghosts, in contrast to high shear cooked starches that were fully digested under the conditions used.

Molecular, mesoscopic and microscopic structure evolution during amylase digestion of maize starch granules

Cereal starch granules with high (>50%) amylose content are a promising source of nutritionally desirable resistant starch, i.e. starch that escapes digestion in the small intestine, but the structural features responsible are not fully understood. We report the effects of partial enzyme digestion of maize starch granules on amylopectin branch length profiles, double and single helix contents, gelatinisation properties, crystallinity and lamellar periodicity. Comparing results for three maize starches (27, 57, and 84% amylose) that differ in both structural features and amylase-sensitivity allows conclusions to be drawn concerning the rate-determining features operating under the digestion conditions used. All starches are found to be digested by a side-by-side mechanism in which there is no major preference during enzyme attack for amylopectin branch lengths, helix form, crystallinity or lamellar organisation. We conclude that the major factor controlling enzyme susceptibility is granule architecture, with shorter length scales not playing a major role as inferred from the largely invariant nature of numerous structural measures during the digestion process (XRD, NMR, SAXS, DSC, FACE). Results are consistent with digestion rates being controlled by restricted diffusion of enzymes within densely packed granular structures, with an effective surface area for enzyme attack determined by external dimensions (57 or 84% amylose - relatively slow) or internal channels and pores (27% amylose - relatively fast). Although the process of granule digestion is to a first approximation non-discriminatory with respect to structure at molecular and mesoscopic length scales, secondary effects noted include (i) partial crystallisation of V-type helices during digestion of 27% amylose starch, (ii) preferential hydrolysis of long amylopectin branches during the early stage hydrolysis of 27% and 57% but not 84% amylose starches, linked with disruption of lamellar repeating structure and (iii) partial B-type recrystallisation after prolonged enzyme incubation for 57% and 84% amylose starches but not 27% amylose starch.

Milling of rice grains: Effects of starch/flour structures on gelatinization and pasting properties

Starch gelatinization and flour pasting properties were determined and correlated with four different levels of starch structures in rice flour, i.e. flour particle size, degree of damaged starch granules, whole molecular size, and molecular branching structure. Onset starch-gelatinization temperatures were not significantly different among all flour samples, but peak and conclusion starch-gelatinization temperatures were significantly different and were strongly correlated with the flour particle size, indicating that rice flour with larger particle size has a greater barrier for heat transfer. There were slight differences in the enthalpy of starch gelatinization, which are likely associated with the disruption of crystalline structure in starch granules by the milling processes. Flours with volume-median diameter ≥56 μm did not show a defined peak viscosity in the RVA viscogram, possibly due to the presence of native protein and/or cell-wall structure stabilizing the swollen starch granules against the rupture caused by shear during heating. Furthermore, RVA final viscosity of flour was strongly correlated with the degree of damage to starch granules, suggesting the contribution of granular structure, possibly in swollen form. The results from this study allow the improvement in the manufacture and the selection criteria of rice flour with desirable gelatinization and pasting properties.

Physicochemical and structural properties of maize and potato starches as a function of granule size

Chemical composition, molecular structure and organization, and thermal and pasting properties of maize and potato starches fractionated on the basis of granule size were investigated to understand heterogeneity within granule populations. For both starches, lipid, protein, and mineral contents decreased and apparent amylose contents increased with granule size. Fully branched (whole) and debranched molecular size distributions in maize starch fractions were invariant with granule size. Higher amylose contents and amylopectin hydrodynamic sizes were found for larger potato starch granules, although debranched molecular size distributions did not vary. Larger granules had higher degrees of crystallinity and greater amounts of double and single helical structures. Systematic differences in pasting and thermal properties were observed with granule size. Results suggest that branch length distributions in both amylose and amylopectin fractions are under tighter biosynthetic control in potato starch than either molecular size or amylose/amylopectin ratio, whereas all three parameters are controlled during the biosynthesis of maize starch.

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.

Freeze-drying changes the structure and digestibility of B-polymorphic starches.

Starch granules both isolated from plants and used in foods or other products have typically been dried. Common food laboratory and industry practices include oven (heat), freeze, and ethanol (solvent-exchange) drying. Starch granules isolated from maize (A-type polymorph) and potato (B-type polymorph) were used to understand the effects of different dehydration methods on starch structure and in vitro digestion kinetics. Oven and ethanol drying do not significantly affect the digestion properties of starches compared with their counterparts that have never been dried. However, freeze-drying results in a significant increase in the digestion rate of potato starch but not maize starch. The structural and conformational changes of starch granules after drying were investigated at various length scales using scanning electron microscopy, confocal laser scanning microscopy, X-ray diffraction, FTIR spectroscopy, and NMR spectroscopy. Freeze-drying not only disrupts the surface morphology of potato starch granules (B-type polymorph), but also degrades both short- and long-range molecular order of the amylopectin, each of which can cause an increase in the digestion rate. In contrast to A-polymorphic starches, B-polymorphic starches are more disrupted by freeze-drying, with reductions of both short- and long-range molecular order. We propose that the low temperatures involved in freeze-drying compared with oven drying result in greater chain rigidity and lead to structural disorganization during water removal at both nanometer and micrometer length scales in B-type polymorphic starch granules, because of the different distribution of water within crystallites and the lack of pores and channels compared with A-type polymorphic starch granules.

Enzymatic hydrolysis of starch in the presence of cereal soluble fibre polysaccharides

The in vitro amylolysis of both granular and cooked maize starch and the diffusion of glucose in the presence of 1% and 2% cereal soluble fibre polysaccharides (arabinoxylan and mixed linkage beta-glucan) were studied at various levels of shear mixing in order to identify potential molecular mechanisms underlying observed glycemia-reducing effects of soluble fibres in vivo. The presence of soluble fibres increased viscosity by ca. 10× and 100× for 1% and 2% concentrations respectively. Despite this large difference in viscosity, measured digestion and mass transfer coefficients were only reduced by a factor of 1.5 to 2.5 at the same mixing speed. In contrast, introduction of mixing in the digesting and diffusing medium significantly increased the rate of amylolytic starch digestion and mass transfer of glucose. This effect is such that mixing at high speeds negates the hindering effect of the 100× increased viscosity imparted by the presence of 2% soluble fibre; this is essentially captured by the Reynolds number (the ratio of inertial and viscous forces) that defines the flow kinematics. The modest reduction of in vitro starch hydrolysis and glucose diffusion at increased viscosity suggests that the established benefits of soluble fibres on post-prandial glycaemia, in terms of attenuation of the overall rate and extent of dietary starch conversion to blood glucose, are not primarily due to a direct effect of viscosity. Alternative hypotheses are proposed based on gastric emptying, restriction of turbulent flow, and/or stimulation of mucus turnover.

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.

Rice starch granule amylolysis--differentiating effects of particle size, morphology, thermal properties and crystalline polymorph.

The underlying mechanism of amylolysis of rice starch granules was investigated using isolated starch granules from wild-type, as well as SBEIIb mutant and down-regulated lines. Fused granule agglomerates isolated from mutant and transgenic lines were hydrolysed at similar rates by amylases, and had similar crystalline patterns and thermal properties as individual granules. Surface pores, a feature previously only reported for A-polymorphic starch granules, were also observed in B- and C-polymorphic rice starch granules. Although the microscopic patterns of hydrolysis among granules with different crystalline polymorphs were qualitatively similar, the extent and the rate of amylolysis were different, suggesting that B-type crystalline polymorphs are intrinsically more resistant to enzymatic hydrolysis than A-type in rice starch granules. It is proposed that the slightly longer branch lengths of amylopectin which leads to the formation of more stable B-type double helical structures compared to their A-type counterparts is the major parameter, with other factors such as granule size, surface pores and interior channels having secondary roles, in determining the rate of enzymatic hydrolysis of rice starch granules.