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

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

A comparison of the kinetics of in vitro starch digestion in smooth and wrinkled peas by porcine pancreatic alpha-amylase

This study describes the impact of crop genetics and processing in two pea lines (Pisum sativum L.) on starch digestion kinetics. Mutation at the rugosus (r) locus leads to wrinkled pea seeds, a reduction in starch content and a lower extent of in vitro starch digestibility. The Logarithm of Slope (LOS) kinetic model was used to analyse digestion curves obtained using porcine pancreatic α-amylase for a range of particle size fractions. Changes in starch structure induced by the r mutation led to clear differences in starch digestion kinetics for purified starches and pea flours. Larger particle size fractions showed slowed starch digestion relative to the purified starch, but significant differences still existed between r and wild type pea lines. It is expected that this work will help inform the design of future studies where both starch structure and food structure are important determinants of digestion behavior

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 orin 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 starchgranules was measured using both glucose and reducing sugar assays. Data were analysed through initialrates 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. Potatorequired 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

Compact structure and proteins of pasta retard in vitro digestive evolution of branched starch molecular structure

The roles that the compact structure and proteins in pasta play in retarding evolution of starch molecular structure during in vitro digestion are explored, using four types of cooked samples: whole pasta, pasta powder, semolina (with proteins) and extracted starch without proteins. These were subjected to in vitro digestion with porcine alpha-amylase, collecting samples at different times and characterizing the weight distribution of branched starch molecules using size-exclusion chromatography. Measurement of alpha-amylase activity showed that a protein (or proteins) from semolina or pasta powder interacted with alpha-amylase, causing reduced enzymatic activity and retarding digestion of branched starch molecules with hydrodynamic radius (R-h) 100 nm. (C) 2016 Elsevier Ltd. All rights reserved

Rapid assessment of ordered molecular structure in a semi-crystalline polymer, starch, directly from solid state NMR spectra

1. Materials and Methods1.1 Materials1.2 13 C CP/MAS NMR spectroscopy1.3 Wide Angle X-Ray Diffractometry1.4 DSC1.5 Data analysi

Ultra-high performance liquid chromatography-size exclusion chromatography (UPLC-SEC) as an efficient tool for the rapid and highly informative characterisation of biopolymers

Starch has a complex molecular structure, with properties dependent on the relative chain lengths and branching structure of its constituent molecules, which varies due to variation in starch biosynthetic genes. Here we present the application of ultra-high performance size exclusion chromatography to the separation of starch chains from plant seeds. Several methods, have been used to analyse chain length distributions in starch, all with limitations in terms of analysis time, sample preparation and molecular weight range. Here we demonstrate that chain length distributions can be obtained with dramatically reduced analysis time using ultra-high performance size exclusion chromatography. The method may also show improvements in resolution of some fine structural features. Understanding links between starch fine structure and biosynthetic genes will allow bioengineering of starches with tailored properties. This technique may have application to the size separation and resolution of a range of biopolymers of value to the food, drink and pharmaceutical industries

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

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

Ten years of dynamic co-management of a multi-species reef fishery

Co-management, a governance process whereby management responsibility is shared between resource users and other collaborators, is a mainstream approach for governing social and ecological aspects of small-scale fisheries. While many assessments of co-management are available for single time periods, assessments across longer time-scales are rare–meaning the dynamic nature, and long-term outcomes, of co-management are insufficiently understood. In this study we analyse ten-years of catch and effort data from a co-managed, multi-species reef fishery in Solomon Islands. To further understand social, ecological and management dynamics we also draw on interviews with fishers and managers that had been conducted throughout the same decade. We aimed to answer (1) what are the temporal trends in fishing effort, harvesting efficiency, and catch composition within and beyond a periodically-harvested closure (i.e. a principal and preferred management tool in Pacific island reef fisheries), and, (2) what are the internal and external drivers that acted upon the fishery, and its management. Despite high fishing effort within the periodically-harvested closure, catch per unit effort remained stable throughout the ten years. Yet the taxonomic composition of catch changed substantially as species targeted early in the decade became locally depleted. These observations indicate that both the frequency of harvesting and the volumes harvested may have outpaced the turnover rates of target species. We argue that this reflects a form of hyperstability whereby declining abundance is not apparent through catch per unit effort since it is masked by a shift to alternate species. While the community sustained and adapted their management arrangements over the decade as a response to internal pressures and some signs of resource changes, some external social and ecological drivers were beyond their capabilities to govern. We argue the collaborative, knowledge exchange, and learning aspects of adaptive co-management may need even more attention to deal with this complexity, particularly as local and distal pressures on multi-species fisheries and community governance intensify

A Call to Action: A Blueprint for Academic Health Sciences in the Era of Mass Incarceration

Over 100 million Americans have criminal records, and the U.S. incarcerates seven times more citizens than most developed countries. The burden of incarceration disproportionately affects people of color and ethnic minorities, and those living in poverty. While 95% of incarcerated people return to society, recidivism rates are high with nearly 75% arrested again within five years of release. Criminal records impede access to employment and other social services such as shelter and health care. Justice-involved people have higher rates of substance, mental health, and some chronic medical disorders than the general population; furthermore, the incarcerated population is rapidly aging. Only a minority of academic health science centers are engaged in health services research, workforce training, or correctional health care. This commentary provides rationale and a blueprint for engagement of academic health science institutions to harness their capabilities to tackle one of the country\u27s most vexing public health crises

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