Glycaemic potency reduction by coarse grain structure in breads is largely eliminated during normal ingestion.

The hypothesis that coarse grain particles in breads reduce glycaemic response only if the particles remain intact during ingestion was tested. Three breads were formulated: (1) White bread (WB - reference), (2) 75 % of kibbled purple wheat in 25 % white bread matrix (PB) and (3) a 1:1 mixture of 37·5 % kibbled soya beans and 37·5 % of kibble purple wheat in 25 % white bread matrix (SPB). Each bread was ingested in three forms: unchewed (U), as customarily consumed (C) and homogenised (H). Twelve participants ingested 40 g available carbohydrate portions of each bread in each form, with post-prandial blood glucose measured over 120 min. Glycaemic responses to WB were the same regardless of its form when ingested. Unchewed PB had significantly less glycaemic effect than WB, whereas the C and H forms were similar to WB. Based on a glycaemic index (GI) of 70 for WB, the GI values for the C, U and H breads, respectively, were WB: 70·0, 70 and 70, PB: 75, 42 and 61, SPB: 57, 48 and 55 (%) (Least significant difference = 17·43, P < 0·05, bold numbers significantly different from WB). The similar glycaemic response to the H and C forms of the breads, and their difference from the U form, showed that the glycaemia-moderating effect of grain structure on starch digestion was lost during customary ingestion of bread. We conclude that the kibbled-grain structure may not effectively retard starch digestion in breads as normally consumed because it is largely eliminated by ingestive processes including chewing.

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.

STARCH SYNTHASE 4 is required for normal starch granule initiation in amyloplasts of wheat endosperm.

Starch granule initiation is poorly understood at the molecular level. The glucosyltransferase, STARCH SYNTHASE 4 (SS4), plays a central role in granule initiation in Arabidopsis leaves, but its function in cereal endosperms is unknown. We investigated the role of SS4 in wheat, which has a distinct spatiotemporal pattern of granule initiation during grain development. We generated TILLING mutants in tetraploid wheat (Triticum turgidum) that are defective in both SS4 homoeologs. The morphology of endosperm starch was examined in developing and mature grains. SS4 deficiency led to severe alterations in endosperm starch granule morphology. During early grain development, while the wild-type initiated single 'A-type' granules per amyloplast, most amyloplasts in the mutant formed compound granules due to multiple initiations. This phenotype was similar to mutants deficient in B-GRANULE CONTENT 1 (BGC1). SS4 deficiency also reduced starch content in leaves and pollen grains. We propose that SS4 and BGC1 are required for the proper control of granule initiation during early grain development that leads to a single A-type granule per amyloplast. The absence of either protein results in a variable number of initiations per amyloplast and compound granule formation.

Cas9-mediated mutagenesis of potato starch-branching enzymes generates a range of tuber starch phenotypes.

We investigated whether Cas9-mediated mutagenesis of starch-branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium-mediated transformation or by PEG-mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low-SBE potato tubers. HPLC-SEC and 1 H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild-type starch. Mutants with strong reductions in SBE2 protein alone had near-normal amylopectin chain-length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 µm and granules with multiple hila. Thus, large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction in SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9-mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.

Salivary α-Amylase Activity and Starch-Related Sweet Taste Perception in Humans.

Starch-related sweet taste perception plays an important role as a part of the dietary nutrient sensing mechanisms in the oral cavity. However, the release of sugars from starchy foods eliciting sweetness has been less studied in humans than in laboratory rodents. Thus, 28 respondents were recruited and evaluated for their starch-related sweet taste perception, salivary alpha-amylase (sAA) activity, oral release of reducing sugars, and salivary leptin. The results demonstrated that a 2-min oral mastication of starchy chewing gum produced an oral concentration of maltose above the sweet taste threshold and revealed that the total amount of maltose equivalent reducing sugars produced was positively correlated with the sAA activity. In addition, respondents who consistently identified the starch-related sweet taste in two sessions (test and retest) generated a higher maltose equivalent reducing sugar concentration compared to respondents who could not detect starch-related sweet taste at all (51.52 ± 2.85 and 29.96 ± 15.58 mM, respectively). In our study, salivary leptin levels were not correlated with starch-related sweet taste perception. The data contribute to the overall understanding of oral nutrient sensing and potentially to the control of food intake in humans. The results provide insight on how starchy foods without added glucose can elicit variable sweet taste perception in humans after mastication as a result of the maltose generated. The data contribute to the overall understanding of oral sensing of simple and complex carbohydrates in humans.

High Molecular Weight Mixed-Linkage Glucan as a Mechanical and Hydration Modulator of Bacterial Cellulose: Characterization by Advanced NMR Spectroscopy.

Bacterial cellulose (BC) consists of a complex three-dimensional organization of ultrafine fibers which provide unique material properties such as softness, biocompatibility, and water-retention ability, of key importance for biomedical applications. However, there is a poor understanding of the molecular features modulating the macroscopic properties of BC gels. We have examined chemically pure BC hydrogels and composites with arabinoxylan (BC-AX), xyloglucan (BC-XG), and high molecular weight mixed-linkage glucan (BC-MLG). Atomic force microscopy showed that MLG greatly reduced the mechanical stiffness of BC gels, while XG and AX did not exert a significant effect. A combination of advanced solid-state NMR methods allowed us to characterize the structure of BC ribbons at ultra-high resolution and to monitor local mobility and water interactions. This has enabled us to unravel the effect of AX, XG, and MLG on the short-range order, mobility, and hydration of BC fibers. Results show that BC-XG hydrogels present BC fibrils of increased surface area, which allows BC-XG gels to hold higher amounts of bound water. We report for the first time that the presence of high molecular weight MLG reduces the density of clusters of BC fibrils and dramatically increases water interactions with BC. Our data supports two key molecular features determining the reduced stiffness of BC-MLG hydrogels, that is, (i) the adsorption of MLG on the surface of BC fibrils precluding the formation of a dense network and (ii) the preorganization of bound water by MLG. Hence, we have produced and fully characterized BC-MLG hydrogels with novel properties which could be potentially employed as renewable materials for applications requiring high water retention capacity (e.g. personal hygiene products).

Soil Organic Carbon Stabilization: Mapping Carbon Speciation from Intact Microaggregates.

The clearing of land for agricultural production depletes soil organic carbon (OC) reservoirs, yet despite their importance, the mechanisms by which C is stabilized in soils remain unclear. Using synchrotron-based infrared microspectroscopy, we have for the first time obtained in situ, laterally resolved data regarding the speciation of C within sections taken from intact free microaggregates from two contrasting soils (Vertisol and Oxisol, 0-20 cm depth) impacted upon by long-term (up to 79 y) agricultural production. There was no apparent gradient in the C concentration from the aggregate surface to the interior for any of the three forms of C examined (aliphatic C, aromatic C, and polysaccharide C). Rather, organo-mineral interactions were of critical importance in influencing overall C stability, particularly for aliphatic C, supporting the hypothesis that microaggregates form through organo-mineral interactions. However, long-term cropping substantially decreased the magnitude of the organo-mineral interactions for all three forms of C. Thus, although organo-mineral interactions are important for OC stability, C forms associated with the mineral phases are not entirely resistant to degradation. These results provide important insights into the underlying mechanisms by which microaggregates form and the factors influencing the persistence of OC in soils.

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.

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.

Stability of sugar solutions: a novel study of the epimerization kinetics of lactose in water.

This article reports on the stereochemical aspects of the chemical stability of lactose solutions stored between 25 and 60 °C. The lactose used for the preparation of the aqueous solutions was α-lactose monohydrate with an anomer purity of 96% α and 4% ß based on the supplied certificate of analysis (using a GC analytical protocol), which was further confirmed here by nuclear magnetic resonance (NMR) analysis. Aliquots of lactose solutions were collected at different time points after the solutions were prepared and freeze-dried to remove water and halt epimerization for subsequent analysis by NMR. Epimerization was also monitored by polarimetry and infrared spectroscopy using a specially adapted Fourier transform infrared attenuated total reflectance (FTIR-ATR) method. Hydrolysis was analyzed by ion chromatography. The three different analytical approaches unambiguously showed that the epimerization of lactose in aqueous solution follows first order reversible kinetics between 25 to 60 °C. The overall rate constant was 4.4 × 10(-4) s(-1) ± 0.9 (± standard deviation (SD)) at 25 °C. The forward rate constant was 1.6 times greater than the reverse rate constant, leading to an equilibrium constant of 1.6 ± 0.1 (±SD) at 25 °C. The rate of epimerization for lactose increased with temperature and an Arrhenius plot yielded an activation energy of +52.3 kJ/mol supporting the hypothesis that the mechanism of lactose epimerization involves the formation of extremely short-lived intermediate structures. The main mechanism affecting lactose stability is epimerization, as no permanent hydrolysis or chemical degradation was observed. When preparing aqueous solutions of lactose, immediate storage in an ice bath at 0 °C will allow approximately 3 min (180 s) of analysis time before the anomeric ratio alters significantly (greater than 1%) from the solid state composition of the starting material. In contrast a controlled anomeric composition (~38% α and ~62% ß) will be achieved if an aqueous solution is left to equilibrate for over 4 h at 25 °C, while increasing the temperature up to 60 °C rapidly reduces the required equilibration time.

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 ß 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.

Colonic in vitro fermentation of mycoprotein promotes shifts in gut microbiota, with enrichment of Bacteroides species.

Mycoprotein is a fungal-derived ingredient used for meat alternative products whose fungal cell walls are rich in dietary fibre (ß-glucans and chitin) and defines its structure. Several health benefits have been reported after mycoprotein consumption, however, little is known about the impact of mycoprotein fermentation on the gut microbiota. This study aims to identify changes in microbiome composition and microbial metabolites during colonic fermentation of mycoprotein following simulated upper gastrointestinal digestion. Changes in microbial populations and metabolites produced by the fermentation of mycoprotein fibre were investigated and compared to a plant (oat bran) and an animal (chicken) comparator. In this model fermentation system, mycoprotein and oat showed different but marked changes in the microbial population compared to chicken, which showed minimal differentiation. In particular, Bacteroides species known for degrading ß-glucans were found in abundance following fermentation of mycoprotein fibre. Mycoprotein fermentation resulted in short-chain fatty acid production comparable with oat and chicken at 72 h. Significantly higher branched-chain amino acids were observed following chicken fermentation. This study suggests that the colonic fermentation of mycoprotein can promote changes in the colonic microbial profile. These results highlight the impact that the unique structure of mycoprotein can have on digestive processes and the gut microbiota.

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.

The impact of the soluble protein fraction and kernel hardness on wheat flour starch digestibility.

Wheat is the staple crop for 35% of the world's population, providing a major source of calories, mainly in the form of starch. The digestibility of wheat starch varies between different flours and products. Wheat products that are rapidly digested elicit large post-prandial glucose peaks associated with metabolic disorders. We investigated the impact of protein on starch digestion in three commercial flours with different grain hardness. A soluble extract of wheat proteins reduced starch digestion, even following gastric proteolysis. This extract was enriched in proteinaceous α-amylase inhibitors which were partially degraded during gastric proteolysis. Starch digestion kinetic analysis was carried out for flour samples pre-treated with different pepsin activities. The rate of starch digestion was altered following pepsin pre-digestion, and the extent of starch digestion increased in response to pepsin pre-digestion. We conclude that soluble proteinaceous alpha-amylase inhibitors present in wheat can escape gastric digestion and significantly contribute to reducing starch digestion in the small intestine.

Mining the A.E. Watkins Wheat Landrace Collection for Variation in Starch Digestibility Using a New High-Throughput Assay.

Breeding for less digestible starch in wheat can improve the health impact of bread and other wheat foods. The application of forward genetic approaches has lately opened opportunities for the discovery of new genes that influence the digestibility of starch, without the burden of detrimental effects on yield or on pasta and bread-making quality. In this study we developed a high-throughput in vitro starch digestibility assay (HTA) for use in forward genetic approaches to screen wheat germplasm. The HTA was validated using standard maize and wheat starches. Using the HTA we measured starch digestibility in hydrothermally processed flour samples and found wide variation among 118 wheat landraces from the A. E. Watkins collection and among eight elite UK varieties (23.5 to 39.9% and 31.2 to 43.5% starch digested after 90 min, respectively). We further investigated starch digestibility in fractions of sieved wholemeal flour and purified starch in a subset of the Watkins lines and elite varieties and found that the matrix properties of flour rather than the intrinsic properties of starch granules conferred lower starch digestibility.

Starch hydrogels as targeted colonic drug delivery vehicles.

Targeted colonic drug delivery systems are needed for the treatment of endemic colorectal pathologies, such as Crohn's disease, ulcerative colitis, and colorectal cancer. These drug delivery vehicles are difficult to formulate, as they need to remain structurally intact whilst navigating a wide range of physiological conditions across the upper gastrointestinal tract. In this work we show how starch hydrogel bulk structural and molecular level parameters influence their properties as drug delivery platforms. The in vitro protocols mimic in vivo conditions, accounting for physiological concentrations of gastrointestinal hydrolytic enzymes and salts. The structural changes starch gels undergo along the entire length of the human gastrointestinal tract have been quantified, and related to the materials' drug release kinetics for three different drug molecules, and interactions with the large intestinal microbiota. It has been demonstrated how one can modify their choice of starch in order to fine tune its corresponding hydrogel's pharmacokinetic profile.

An allelic series of starch-branching enzyme mutants in pea (Pisum sativum L.) reveals complex relationships with seed starch phenotypes.

A set of mutant pea lines carrying induced mutations within the major seed-expressed starch-branching enzyme gene has been characterised at the molecular, chemical and agronomic levels. Eight of the induced mutations, three of which predicted a premature stop codon, were compared with the naturally occurring starch-branching enzyme mutation within the same genetic background. Starch, amylose and sugar measurements, coupled with analysis by ultra-high performance liquid chromatography-size exclusion chromatography of starches, identified a range of phenotypes which were grouped according to the nature of the mutation. Homology modelling of proteins supported the differences in phenotypes observed. Differences in field performance were evident for selected mutants, particularly in seed yield and mean seed weight traits for early compared with late spring sowings. The data show the potential of an allelic series of mutants at this locus for nutritional studies. CHEMICAL COMPOUNDS: starch, amylose, amylopectin, raffinose, stachyose, verbascose.

Loss of starch synthase IIIa changes starch molecular structure and granule morphology in grains of hexaploid bread wheat.

Starch synthase III plays a key role in starch biosynthesis and is highly expressed in developing wheat grains. To understand the contribution of SSIII to starch and grain properties, we developed wheat ssIIIa mutants in the elite cultivar Cadenza using in silico TILLING in a mutagenized population. SSIIIa protein was undetectable by immunoblot analysis in triple ssIIIa mutants carrying mutations in each homoeologous copy of ssIIIa (A, B and D). Loss of SSIIIa in triple mutants led to significant changes in starch phenotype including smaller A-type granules and altered granule morphology. Starch chain-length distributions of double and triple mutants indicated greater levels of amylose than sibling controls (33.8% of starch in triple mutants, and 29.3% in double mutants vs. 25.5% in sibling controls) and fewer long amylopectin chains. Wholemeal flour of triple mutants had more resistant starch (6.0% vs. 2.9% in sibling controls) and greater levels of non-starch polysaccharides; the grains appeared shrunken and weighed ~ 11% less than the sibling control which was partially explained by loss in starch content. Interestingly, our study revealed gene dosage effects which could be useful for fine-tuning starch properties in wheat breeding applications while minimizing impact on grain weight and quality.

Hybrid metagenome assemblies link carbohydrate structure with function in the human gut microbiome.

Complex carbohydrates that escape small intestinal digestion, are broken down in the large intestine by enzymes encoded by the gut microbiome. This is a symbiotic relationship between microbes and host, resulting in metabolic products that influence host health and are exploited by other microbes. However, the role of carbohydrate structure in directing microbiota community composition and the succession of carbohydrate-degrading microbes, is not fully understood. In this study we evaluate species-level compositional variation within a single microbiome in response to six structurally distinct carbohydrates in a controlled model gut using hybrid metagenome assemblies. We identified 509 high-quality metagenome-assembled genomes (MAGs) belonging to ten bacterial classes and 28 bacterial families. Bacterial species identified as carrying genes encoding starch binding modules increased in abundance in response to starches. The use of hybrid metagenomics has allowed identification of several uncultured species with the functional potential to degrade starch substrates for future study.