Enzyme kinetic approach for mechanistic insight and predictions of in vivo starch digestibility and the glycaemic index of foods.

BACKGROUND: Starch is a principal dietary source of digestible carbohydrate and energy. Glycaemic and insulinaemic responses to foods containing starch vary considerably and glucose responses to starchy foods are often described by the glycaemic index (GI) and/or glycaemic load (GL). Low GI/GL foods are beneficial in the management of cardiometabolic disorders (e.g., type 2 diabetes, cardiovascular disease). Differences in rates and extents of digestion of starch-containing foods will affect postprandial glycaemia. SCOPE AND APPROACH: Amylolysis kinetics are influenced by structural properties of the food matrix and of starch itself. Native (raw) semi-crystalline starch is digested slowly but hydrothermal processing (cooking) gelatinises the starch and greatly increases its digestibility. In plants, starch granules are contained within cells and intact cell walls can limit accessibility of water and digestive enzymes hindering gelatinisation and digestibility. In vitro studies of starch digestion by α-amylase model early stages in digestion and can suggest likely rates of digestion in vivo and expected glycaemic responses. Reports that metabolic responses to dietary starch are influenced by α-amylase gene copy number, heightens interest in amylolysis. KEY FINDINGS AND CONCLUSIONS: This review shows how enzyme kinetic strategies can provide explanations for differences in digestion rate of different starchy foods. Michaelis-Menten and Log of Slope analyses provide kinetic parameters (e.g., K m and k cat /K m ) for evaluating catalytic efficiency and ease of digestibility of starch by α-amylase. Suitable kinetic methods maximise the information that can be obtained from in vitro work for predictions of starch digestion and glycaemic responses in vivo.

Inhibition of the facilitative sugar transporters (GLUTs) by tea extracts and catechins.

Tea polyphenolics have been suggested to possess blood glucose lowering properties by inhibiting sugar transporters in the small intestine and improving insulin sensitivity. In this report, we studied the effects of teas and tea catechins on the small intestinal sugar transporters, SGLT1 and GLUTs (GLUT1, 2 and 5). Green tea extract (GT), oolong tea extract (OT), and black tea extract (BT) inhibited glucose uptake into the intestinal Caco-2 cells with GT being the most potent inhibitor (IC50 : 0.077 mg/mL), followed by OT (IC50 : 0.136 mg/mL) and BT (IC50 : 0.56 mg/mL). GT and OT inhibition of glucose uptake was partial non-competitive, with an inhibitor constant (Ki ) = 0.0317 and 0.0571 mg/mL, respectively, whereas BT was pure non-competitive, Ki  = 0.36 mg/mL. Oocytes injected to express small intestinal GLUTs were inhibited by teas, but SGLT1 was not. Furthermore, catechins present in teas were the predominant inhibitor of glucose uptake into Caco-2 cells, and gallated catechins the most potent: CG > ECG > EGCG ≥ GCG when compared to the non-gallated catechins (C, EC, GC, and EGC). In Caco-2 cells, individual tea catechins reduced the SGLT1 gene, but not protein expression levels. In contrast, GLUT2 gene and protein expression levels were reduced after 2 hours exposure to catechins but increased after 24 hours. These in vitro studies suggest teas containing catechins may be useful dietary supplements capable of blunting postprandial glycaemia in humans, including those with or at risk to Type 2 diabetes mellitus.

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

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.

Infrared microspectroscopic imaging of plant tissues: spectral visualization of Triticum aestivum kernel and Arabidopsis leaf microstructure.

Infrared microspectroscopy is a tool with potential for studies of the microstructure, chemical composition and functionality of plants at a subcellular level. Here we present the use of high-resolution bench top-based infrared microspectroscopy to investigate the microstructure of Triticum aestivum L. (wheat) kernels and Arabidopsis leaves. Images of isolated wheat kernel tissues and whole wheat kernels following hydrothermal processing and simulated gastric and duodenal digestion were generated, as well as images of Arabidopsis leaves at different points during a diurnal cycle. Individual cells and cell walls were resolved, and large structures within cells, such as starch granules and protein bodies, were clearly identified. Contrast was provided by converting the hyperspectral image cubes into false-colour images using either principal component analysis (PCA) overlays or by correlation analysis. The unsupervised PCA approach provided a clear view of the sample microstructure, whereas the correlation analysis was used to confirm the identity of different anatomical structures using the spectra from isolated components. It was then demonstrated that gelatinized and native starch within cells could be distinguished, and that the loss of starch during wheat digestion could be observed, as well as the accumulation of starch in leaves during a diurnal period.

Re-evaluation of the mechanisms of dietary fibre and implications for macronutrient bioaccessibility, digestion and postprandial metabolism.

The positive effects of dietary fibre on health are now widely recognised; however, our understanding of the mechanisms involved in producing such benefits remains unclear. There are even uncertainties about how dietary fibre in plant foods should be defined and analysed. This review attempts to clarify the confusion regarding the mechanisms of action of dietary fibre and deals with current knowledge on the wide variety of dietary fibre materials, comprising mainly of NSP that are not digested by enzymes of the gastrointestinal (GI) tract. These non-digestible materials range from intact cell walls of plant tissues to individual polysaccharide solutions often used in mechanistic studies. We discuss how the structure and properties of fibre are affected during food processing and how this can impact on nutrient digestibility. Dietary fibre can have multiple effects on GI function, including GI transit time and increased digesta viscosity, thereby affecting flow and mixing behaviour. Moreover, cell wall encapsulation influences macronutrient digestibility through limited access to digestive enzymes and/or substrate and product release. Moreover, encapsulation of starch can limit the extent of gelatinisation during hydrothermal processing of plant foods. Emphasis is placed on the effects of diverse forms of fibre on rates and extents of starch and lipid digestion, and how it is important that a better understanding of such interactions with respect to the physiology and biochemistry of digestion is needed. In conclusion, we point to areas of further investigation that are expected to contribute to realisation of the full potential of dietary fibre on health and well-being of humans.

Investigating the mechanisms of amylolysis of starch granules by solution-state NMR.

Starch is a prominent component of the human diet and is hydrolyzed by α-amylase post-ingestion. Probing the mechanism of this process has proven challenging, due to the intrinsic heterogeneity of individual starch granules. By means of solution-state NMR, we demonstrate that flexible polysaccharide chains protruding from the solvent-exposed surfaces of waxy rice starch granules are highly mobile and that during hydrothermal treatment, when the granules swell, the number of flexible residues on the exposed surfaces increases by a factor of 15. Moreover, we show that these flexible chains are the primary substrates for α-amylase, being cleaved in the initial stages of hydrolysis. These findings allow us to conclude that the quantity of flexible α-glucan chains protruding from the granule surface will greatly influence the rate of energy acquisition from digestion of starch.

The surface structure of a complex substrate revealed by enzyme kinetics and Freundlich constants for α-amylase interaction with the surface of starch.

BACKGROUND: Starch is a main source of carbohydrate in human diets, but differences are observed in postprandial glycaemia following ingestion of different foods containing identical starch contents. Such differences reflect variations in rates at which different starches are digested in the intestine. In seeking explanations for these differences, we have studied the interaction of α-amylase with starch granules. Understanding this key step in digestion should help with a molecular understanding for observed differences in starch digestion rates. METHODS: For enzymes acting upon solid substrates, a Freundlich equation relates reaction rate to enzyme adsorption at the surface. The Freundlich exponent (n) equals 2/3 for a liquid-smooth surface interface, 1/3 for adsorption to exposed edges of ordered structures and 1.0 for solution-solution interfaces. The topography of a number of different starch granules, revealed by Freundlich exponents, was compared with structural data obtained by differential scanning calorimetry and Fourier transform infrared spectroscopy with attenuated total internal reflectance (FTIR-ATR). RESULTS: Enzyme binding rate and FTIR-ATR peak ratio were directly proportional to n and ΔgelH was inversely related to n. Amylase binds fastest to solubilised starch and to granules possessing smooth surfaces at the solid-liquid interface and slowest to granules possessing ordered crystalline surfaces. CONCLUSIONS: Freundlich exponents provide information about surface blocklet structures of starch that supplements knowledge obtained from physical methods. GENERAL SIGNIFICANCE: Nanoscale structures at the surface of starch granules influence hydrolysis by α-amylase. This can be important in understanding how dietary starch is digested with relevance to diabetes, cardiovascular health and cancer.

The effects of processing and mastication on almond lipid bioaccessibility using novel methods of in vitro digestion modelling and micro-structural analysis.

A number of studies have demonstrated that consuming almonds increases satiety but does not result in weight gain, despite their high energy and lipid content. To understand the mechanism of almond digestion, in the present study, we investigated the bioaccessibility of lipids from masticated almonds during in vitro simulated human digestion, and determined the associated changes in cell-wall composition and cellular microstructure. The influence of processing on lipid release was assessed by using natural raw almonds (NA) and roasted almonds (RA). Masticated samples from four healthy adults (two females, two males) were exposed to a dynamic gastric model of digestion followed by simulated duodenal digestion. Between 7·8 and 11·1 % of the total lipid was released as a result of mastication, with no significant differences between the NA and RA samples. Significant digestion occurred during the in vitro gastric phase (16·4 and 15·9 %) and the in vitro duodenal phase (32·2 and 32·7 %) for the NA and RA samples, respectively. Roasting produced a smaller average particle size distribution post-mastication; however, this was not significant in terms of lipid release. Light microscopy showed major changes that occurred in the distribution of lipid in all cells after the roasting process. Further changes were observed in the surface cells of almond fragments and in fractured cells after exposure to the duodenal environment. Almond cell walls prevented lipid release from intact cells, providing a mechanism for incomplete nutrient absorption in the gut. The composition of almond cell walls was not affected by processing or simulated digestion.

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.

Study of the structure and properties of native and hydrothermally processed wild-type, lam and r variant pea starches that affect amylolysis of these starches.

Starches from WT, lam, and r pea mutants differing in amylopectin/amylose contents (70, 90, and 28% amylopectin, respectively) were used in kinetic studies of pancreatic α-amylase action at 37 °C and for investigations of their supramolecular structure and physicochemical properties during heating. For WT and lam starches, amylase accessibility and catalytic efficiencies (CE) increased following hydrothermal processing up to 100 °C. Accessibility changed relatively less in r during heating with increasing K(m) between 60-90 °C. Limiting values of K(m) after gelatinization were very similar for all three mutants, indicating that relative proportions of amylose/amylopectin have little influence on amylase accessibility once ordered structures are lost. For WT and lam, increases in enzyme accessibility and CE paralleled a rise in amorphous content. It is suggested that the complex behavior for r resulted from amylose gel formation between 60-90 °C. Amorphous amylopectin seems a better substrate for amylase than amorphous amylose.

Dietary vitamin C and E modulates antioxidant levels in blood, brain, liver, muscle, and testes in diabetic aged rats.

While tissue dysfunction is a well-recognized consequence of diabetes mellitus in aged people, the underlying mechanisms are poorly understood. Daily (VCE) supplementation of vitamins C and E can be beneficial to diabetic aged animals in reducing free radical production. The aim of this study was to investigate whether dietary VCE supplementation modulates oxidative stress and antioxidant redox systems in streptozotocin (STZ)-induced aged diabetic rats. Thirty aged rats (18 - 20 months) were randomly divided into three groups. The first group acted as a control and the second group was diabetic. VCE-supplemented feed was given to aged, diabetic rats, constituting the third group. Diabetes was induced using a single dose of intraperitoneal STZ. On the 21(st) day after STZ dosage, blood and tissue samples were taken from all animals. Glutathione peroxidase activity in liver, erythrocytes, muscle, and testes; catalase activity in plasma and erythrocytes; reduced glutathione levels in plasma; vitamin E concentration in plasma, liver, and muscle; b-carotene concentration in brain; and high-density lipoprotein (HDL)-cholesterol levels in plasma were lower in the diabetic group than in the control group. Lipid peroxidation (LP) levels in plasma, liver, brain, and muscle, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), triacyglycerols, and total and low-density lipoprotein (LDL)-cholesterol values in plasma were higher in the diabetic group than in the control group. The LP, enzyme, vitamin, and lipid profile values levels were mostly restored by VCE treatment. Liver and testis weights did not change by diabetic status and VCE supplementation, although body weight was lower in the diabetic group than in the control group. In conclusion, brain, liver, and testes tissues seem most sensitive in aged diabetic rats to oxidative stress. We observed that VCE supplementation relieves oxidative stress in the blood and tissues of diabetic aged rats by modulating the antioxidant system and lipid profile.

Structure-function studies of chickpea and durum wheat uncover mechanisms by which cell wall properties influence starch bioaccessibility.

Positive health effects of dietary fibre have been established; however, the underpinning mechanisms are not well understood. Plant cell walls are the predominant source of fibre in the diet. They encapsulate intracellular starch and delay digestive enzyme ingress, but food processing can disrupt the structure. Here we compare digestion kinetics of chickpea (cotyledon) and durum wheat (endosperm), which have contrasting cell wall structures (Type I and II, respectively), to investigate a 'cell-wall barrier' mechanism that may underpin the health effects of dietary fibre. Using in vitro models, including the Dynamic Gastric Model, to simulate human digestion together with microscopy, we show that starch bioaccessibility is limited from intact plant cells and that processing treatments can have different effects on cell integrity and digestion kinetics when applied to tissues with contrasting cell wall properties. This new understanding of dietary fibre structure is important for effective fibre supplementation to benefit human health.

α-Amylase action on starch in chickpea flour following hydrothermal processing and different drying, cooling and storage conditions.

Starch is present in many prepared 'ready-meals' that have undergone processing and/or storage in frozen or chilled state. Hydrothermal processing greatly increases starch digestibility and postprandial glycaemia. Effects of different heating/drying and cooling regimes on amylolysis have received little attention. Hence, we examined the effects of different processing treatments on in vitro digestibility of starch in chickpea flour. Solid-state 13C NMR was used to estimate ordered double-helical structure in the starch. Native starch with 25 % double-helical content was the most resistant to digestion but hydrothermal processing (gelatinisation) resulted in >95 % loss of order and a large increase in starch digestibility. Air-drying of pre-treated flour produced slowly-digestible starch (C∞, 55.9 %). Refrigeration of gelatinised samples decreased ease of amylolysis coincident with increase in double-helical content. Freezing maintained the same degree of digestibility as freshly gelatinised material and produced negligible retrogradation. Chilling may be exploited to produce ready-meals with a lower glycaemic response.

Oral vitamin C and E combination modulates blood lipid peroxidation and antioxidant vitamin levels in maximal exercising basketball players.

Oxidative stress occurs during maximal exercise, perhaps as a result of increased consumption of oxygen. Vitamins C and E can overcome the effects of antioxidants in exercise. We investigated the effects of supplementation with a combination of vitamin C and E (VCE) on blood lipid peroxidation (LP) and antioxidant levels following maximal training in basketball players.Blood samples were taken from 14 players (group A) and divided into two subgroups namely maximal training (group B) and maximal training plus VCE groups (group C). Group B maximally exercised for 35 days. VCE was supplemented to group C for 35 days and blood samples were taken from group B and C. Plasma and hemolyzed erythrocyte samples were obtained from the players.Erythrocyte glutathione peroxidase (GSH-Px) activity and plasma vitamin E concentration were lower in group B than in group A, whereas plasma and erythrocyte LP levels were higher in group B than in group A. Plasma vitamin A, vitamin E, erythrocyte GSH-Px, and reduced glutathione (GSH) values were higher in group C than in groups A and B although LP levels in plasma and erythrocytes were lower in group C than in group A and B. beta-Carotene values did not change in the three groups.In conclusion, VCE supplementation in maximal exercising basketball players may strengthen the antioxidant defense system by decreasing reactive oxygen species (ROS).

Incorporation of a novel leguminous ingredient into savoury biscuits reduces their starch digestibility: Implications for lowering the Glycaemic Index of cereal products.

Many carbohydrate foods contain starch that is rapidly digested and elicits a high Glycaemic Index. A legume ingredient (PulseON®) rich in Type 1 resistant starch (RS1) was recently developed; however, its potential as a functional ingredient when processed into a food product required assessment. PulseON® was used to replace 0, 25, 50, 75, and 100% of the wheat flour in a savoury biscuit recipe. In vitro starch digestion kinetics of biscuits and water-holding properties of ingredients were assessed. The RS1 in PulseON® did not appear to be structurally compromised during biscuit making. Replacing 50% wheat flour with PulseON® reduced the starch hydrolysis index of biscuits by nearly 60%. This seems to result from the ingredients' impact on water availability for starch gelatinisation. Overall, these findings highlight the potential of using biscuits as a food vehicle for PulseON® to increase consumer intakes of legume protein, dietary fibre, and potentially low glycaemic starch.

Topiramate and vitamin e modulate the electroencephalographic records, brain microsomal and blood antioxidant redox system in pentylentetrazol-induced seizure of rats.

We investigated the effects of vitamin E and topiramate (TPM) administrations on pentylentetrazol (PTZ)-induced blood and brain toxicity in rats. Forty rats were randomly divided into five equal groups. The first and second groups were used for the control and PTZ groups, respectively. Fifty or 100 mg TPM were administered to rats constituting the third and fourth groups for 7 days, respectively. The TPM and vitamin E combination was given to animals in the fifth group. At the end of 7 days, all groups except the first received a single dose of PTZ. Blood and brain samples were taken at 3 hrs after PTZ administration. Lipid peroxidation levels of plasma, erythrocyte, brain cortex and brain microsomal fraction; nitric oxide levels of serum; and the number of spikes and epileptiform discharges of the EEG were increased by PTZ administration. Plasma and brain vitamin E concentration, erythrocyte glutathione peroxidase (GSH-Px) activity and latency to first spike of the EEG were decreased by PTZ. Plasma lipid peroxidation levels in the third group and plasma and erythrocyte lipid peroxidation levels in the fifth group were decreased compared to the second group, whereas brain vitamin C, vitamin E, erythrocyte GSH-Px and reduced glutathione (GSH) values increased in the fifth group. Brain microsomal GSH levels and EEG records in the third, fourth and fifth groups were restored by the TPM and vitamin E treatment. In conclusion, TPM and vitamin E seems to have protective effects on PTZ-induced blood and brain toxicity by inhibiting free radicals and supporting the antioxidant redox system.

Use of the Extended Fujita method for representing the molecular weight and molecular weight distributions of native and processed oat beta-glucans.

Beta 1-3, 1-4 glucans ("beta-glucans") are one of the key components of the cell wall of cereals, complementing the main structural component cellulose. Beta-glucans are also an important source of soluble fibre in foods containing oats with claims of other beneficial nutritional properties such as plasma cholesterol lowering in humans. Key to the function of beta-glucans is their molecular weight and because of their high polydispersity - molecular weight distribution. Analytical ultracentrifugation provides a matrix-free approach (not requiring separation columns or media) to polymer molecular weight distribution determination. The sedimentation coefficient distribution is converted to a molecular weight distribution via a power law relation using an established procedure known as the Extended Fujita approach. We establish and apply the power law relation and Extended Fujita method for the first time to a series of native and processed oat beta-glucans. The application of this approach to beta-glucans from other sources is considered.

Impact of hydrothermal and mechanical processing on dissolution kinetics and rheology of oat ß-glucan.

Oat mixed-linkage ß-glucan has been shown to lower fasting blood cholesterol concentrations due notably to an increase in digesta viscosity in the proximal gut. To exert its action, the polysaccharide has to be released from the food matrix and hydrated. The dissolution kinetics of ß-glucan from three oat materials, varying in their structure, composition and degree of processing, was investigated by incubating the oats at 37°C over multiple time points (up to 72h). The samples were analysed for ß-glucan content, weight-average molecular weight and rheological behaviour. Regardless of the materials studied and the processing applied, the solubilisation of ß-glucan was not complete. Mechanical and hydrothermal processing led to differences in the viscosity flow curves of the recovered solutions, with the presence of particulates having a marked effect. This study revealed that the structure and processing methods applied to oat materials resulted in varied and complex rheological properties, especially when particulates are present.

Structural and enzyme kinetic studies of retrograded starch: Inhibition of α-amylase and consequences for intestinal digestion of starch.

Retrograded starch is known to be resistant to digestion. We used enzyme kinetic experiments to examine how retrogradation of starch affects amylolysis catalysed by porcine pancreatic amylase. Parallel studies employing differential scanning calorimetry, infra red spectroscopy, X-ray diffraction and NMR spectroscopy were performed to monitor changes in supramolecular structure of gelatinised starch as it becomes retrograded. The total digestible starch and the catalytic efficiency of amylase were both decreased with increasing evidence of retrogradation. A purified sample of retrograded high amylose starch inhibited amylase directly. These new findings demonstrate that amylase binds to retrograded starch. Therefore consumption of retrograded starch may not only be beneficial to health through depletion of total digestible starch, and therefore the metabolisable energy, but may also slow the rate of intestinal digestion through direct inhibition of α-amylase. Such physiological effects have important implications for the prevention and management of type 2 diabetes and cardiovascular disease.