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.

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.

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.

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.

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.

The impact of oat structure and ß-glucan on in vitro lipid digestion.

Oat ß-glucan has been shown to play a positive role in influencing lipid and cholesterol metabolism. However, the mechanisms behind these beneficial effects are not fully understood. The purpose of the current work was to investigate some of the possible mechanisms behind the cholesterol lowering effect of oat ß-glucan, and how processing of oat modulates lipolysis. ß-Glucan release, and the rate and extent of lipolysis measured in the presence of different sources of oat ß-glucan, were investigated during gastrointestinal digestion. Only a fraction of the original ß-glucan content was released during digestion. Oat flakes and flour appeared to have a more significant effect on lipolysis than purified ß-glucan. These findings show that the positive action of ß-glucan is likely to involve complex processes and interactions with the food matrix. This work also highlights the importance of considering the structure and physicochemical properties of foods, and not just the nutrient content.

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.

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.

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.

The role of plant cell wall encapsulation and porosity in regulating lipolysis during the digestion of almond seeds.

Previous studies have provided evidence that the physical encapsulation of intracellular nutrients by cell walls of plant foods (i.e. dietary fibre) plays a predominant role in influencing macronutrient bioaccessibility (release) from plant foods during human digestion. One unexplored aspect of this is the extent to which digestive enzymes can pass through the cell-wall barrier and hydrolyse the intracellular lipid in almond seeds. The purpose of the present study was to assess the role played by cell walls in influencing the bioaccessibility and digestibility of almond lipid using a range of techniques. Digestibility experiments were performed on raw and roasted almond cells as well as isolated almond oil bodies using in vitro gastric and duodenal digestion models. Residual triacylglycerols and lipolysis products were extracted after 1 h of incubation and analysed by thin layer chromatography. The lipolysis kinetics of almond cells and oil bodies were also investigated using the pH-stat technique. Finally, the potential penetration of pancreatic lipase through the cell wall matrix was investigated using confocal microscopy. Differences in the rates and extent of lipolysis were clearly seen between almond cells and oil bodies, and these differences were observed regardless of the lipase(s) used. These results also showed that almond cell walls that are completely intact limit lipid digestibility, due to an encapsulation mechanism that hinders the diffusion of lipase into the intracellular environment and lipolysis products out of the cells.

Manipulation of starch bioaccessibility in wheat endosperm to regulate starch digestion, postprandial glycemia, insulinemia, and gut hormone responses: a randomized controlled trial in healthy ileostomy participants.

BACKGROUND: Cereal crops, particularly wheat, are a major dietary source of starch, and the bioaccessibility of starch has implications for postprandial glycemia. The structure and properties of plant foods have been identified as critical factors in influencing nutrient bioaccessibility; however, the physical and biochemical disassembly of cereal food during digestion has not been widely studied. OBJECTIVES: The aims of this study were to compare the effects of 2 porridge meals prepared from wheat endosperm with different degrees of starch bioaccessibility on postprandial metabolism (e.g., glycemia) and to gain insight into the structural and biochemical breakdown of the test meals during gastroileal transit. DESIGN: A randomized crossover trial in 9 healthy ileostomy participants was designed to compare the effects of 55 g starch, provided as coarse (2-mm particles) or smooth (<0.2-mm particles) wheat porridge, on postprandial changes in blood glucose, insulin, C-peptide, lipids, and gut hormones and on the resistant starch (RS) content of ileal effluent. Undigested food in the ileal output was examined microscopically to identify cell walls and encapsulated starch. RESULTS: Blood glucose, insulin, C-peptide, and glucose-dependent insulinotropic polypeptide concentrations were significantly lower (i.e., 33%, 43%, 40%, and 50% lower 120-min incremental AUC, respectively) after consumption of the coarse porridge than after the smooth porridge (P < 0.01). In vitro, starch digestion was slower in the coarse porridge than in the smooth porridge (33% less starch digested at 90 min, P < 0.05, paired t test). In vivo, the structural integrity of coarse particles (∼2 mm) of wheat endosperm was retained during gastroileal transit. Microscopic examination revealed a progressive loss of starch from the periphery toward the particle core. The structure of the test meal had no effect on the amount or pattern of RS output. CONCLUSION: The structural integrity of wheat endosperm is largely retained during gastroileal digestion and has a primary role in influencing the rate of starch amylolysis and, consequently, postprandial metabolism. This trial was registered at isrctn.org as ISRCTN40517475.

A study of starch gelatinisation behaviour in hydrothermally-processed plant food tissues and implications for in vitro digestibility.

The aim of this study was to investigate the role of the plant food matrix in influencing the extent of starch gelatinisation during hydrothermal processing, and its implications for starch digestibility. Differential scanning calorimetry (DSC) was used to provide a detailed examination of the gelatinisation behaviour of five distinct size fractions (diameters <0.21 to 2.58 mm) of milled chickpea and durum wheat. Gelatinisation parameters were obtained from the DSC thermograms and concomitant microscopy analyses were performed. The estimated terminal extent of gelatinisation (TEG) was compared with our previously published data for in vitro starch digestibility of the same food materials. We observed clear differences in the gelatinisation behaviour of matched size-fractions of chickpeas and durum wheat. In chickpea materials, the TEG values (34-100%) were inversely related to particle size, whereas in durum wheat, no size-dependent limitations on TEG were observed. The TEG values were completely consistent with the extent of starch amylolysis in all size fractions of both durum wheat and chickpea. Microstructural analysis following hydrothermal processing confirmed the presence of some partially gelatinised birefringent starch within intact chickpea cells. Birefringent starch granules were not present in any of the processed fractions of durum wheat. The differences in gelatinisation behaviour of these plant species seem to reflect the individual cell wall properties of these materials. These findings demonstrate the applicability of DSC to real food materials to provide insight into the mechanisms by which the food matrix (particularly the plant cell walls) influences gelatinisation, and consequently, starch amylolysis.

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.

Impact of cell wall encapsulation of almonds on in vitro duodenal lipolysis.

Although almonds have a high lipid content, their consumption is associated with reduced risk of cardiovascular disease. One explanation for this paradox could be limited bioaccessibility of almond lipids due to the cell wall matrix acting as a physical barrier to digestion in the upper gastrointestinal tract. We aimed to measure the rate and extent of lipolysis in an in vitro duodenum digestion model, using raw and roasted almond materials with potentially different degrees of bioaccessibility. The results revealed that a decrease in particle size led to an increased rate and extent of lipolysis. Particle size had a crucial impact on lipid bioaccessibility, since it is an indicator of the proportion of ruptured cells in the almond tissue. Separated almond cells with intact cell walls showed the lowest levels of digestibility. This study underlines the importance of the cell wall for modulating lipid uptake and hence the positive health benefits underlying almond consumption.

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.

Effect of mastication on lipid bioaccessibility of almonds in a randomized human study and its implications for digestion kinetics, metabolizable energy, and postprandial lipemia.

BACKGROUND: The particle size and structure of masticated almonds have a significant impact on nutrient release (bioaccessibility) and digestion kinetics. OBJECTIVES: The goals of this study were to quantify the effects of mastication on the bioaccessibility of intracellular lipid of almond tissue and examine microstructural characteristics of masticated almonds. DESIGN: In a randomized, subject-blind, crossover trial, 17 healthy subjects chewed natural almonds (NAs) or roasted almonds (RAs) in 4 separate mastication sessions. Particle size distributions (PSDs) of the expectorated boluses were measured by using mechanical sieving and laser diffraction (primary outcome). The microstructure of masticated almonds, including the structural integrity of the cell walls (i.e., dietary fiber), was examined with microscopy. Lipid bioaccessibility was predicted by using a theoretical model, based on almond particle size and cell dimensions, and then compared with empirically derived release data. RESULTS: Intersubject variations (n = 15; 2 subjects withdrew) in PSDs of both NA and RA samples were small (e.g., laser diffraction; CV: 12% and 9%, respectively). Significant differences in PSDs were found between these 2 almond forms (P < 0.05). A small proportion of lipid was released from ruptured cells on fractured surfaces of masticated particles, as predicted by using the mathematical model (8.5% and 11.3% for NAs and RAs, respectively). This low percentage of lipid bioaccessibility is attributable to the high proportion (35-40%) of large particles (>500 µm) in masticated almonds. Microstructural examination of the almonds indicated that most intracellular lipid remained undisturbed in intact cells after mastication. No adverse events were recorded. CONCLUSIONS: Following mastication, most of the almond cells remained intact with lipid encapsulated by cell walls. Thus, most of the lipid in masticated almonds is not immediately bioaccessible and remains unavailable for early stages of digestion. The lipid encapsulation mechanism provides a convincing explanation for why almonds have a low metabolizable energy content and an attenuated impact on postprandial lipemia.

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.

A mechanistic approach to studies of the possible digestion of retrograded starch by α-amylase revealed using a log of slope (LOS) plot.

The rate and extent of digestibility of starch were analysed using the logarithm of the slope (LOS) method. Digestibility curves with α-amylase were obtained for starches in their native, gelatinised and 24h retrograded form. A LOS plot of the digestibility curves was then constructed, which allowed the rate constant (k) and the concentration of the product at the end of the reaction (C∞) to be calculated. It also allowed the identification of rapid and slow phases in starch digestion. Upon gelatinisation, both k and C∞ increased with dramatic changes notably in C∞; however after starch samples had been stored for 24h at room temperature, k was not affected but C∞ decreased. This suggests that retrograded starch is virtually inert to amylase action. Both k and C∞ were strongly related to the increase in degree of order of the α-glucan chains, monitored by FTIR-ATR spectroscopy, in retrograded starch.