Guy A. Channell

Extraction, isolation and characterisation of oil bodies from pumpkin seeds for therapeutic use

Pumpkin, a member of the Cucurbitaceae family has been used frequently as functional medicines for therapeutic use. Several phytochemicals such as polysaccharides, phenolic glycosides, 13-hydroxy-9Z, 11E-octadecatrienoic acid from the leaves of pumpkin, proteins from germinated seeds, have been isolated. Here the influence of pH, ionic strength, and temperature on the properties and stability of oil bodies from pumpkin (Cucurbita) were determined with a view to patterning oil body size and structure for future therapeutic intervention. Oil bodies from pumpkin seeds were extracted, isolated, characterised using optical microscopy, zeta potential and particle size distribution obtained. During microscopic analysis, the oil bodies were more intact and in an integrated form at the time of extraction but were ruptured with time. Water extracted oil bodies were spherical for all four layers where cream had larger oil bodies then upper curd. Lower curd and supernatant had considerably smaller size with lower curd densely packed and seemed to be rich in oil bodies than any of the four layers. At pH 3, in the absence of salt, the zeta potential is approximately +30 mV, but as the salt concentration increases, the ζ potential rises at 10 mM but then decreases over the salt range. This trend continues for the upper curd, lower curd and the supernatant and the degree of the reduction (mV) in zeta potential is of the order cream<upper curd<lower curd<supernatant. At pH 7.4, physiological pH, the changes in salt concentration from 10 to 100 mM reduce the zeta potential significantly across all layers such that increased salt concentrations induce negative potentials. Increasing the salt concentrations still further, however, does not make the ζ potential more negative. However, at pH 9 the zeta potential falls from 0 to -50 mV as the salt concentration increases with the largest reduction shown with 100 mM salt. Particle size distribution at increasing pH salt concentration shows the average size distribution of pumpkin seed oil bodies at an increasing pH (3, 7.4 and 9) and salt concentration (0, 10, 50 and 100 mM) across all four layers. The lowest average size distributions are seen at pH 7.4 across all four layers especially within the cream and upper curd layers. At pH 3 and 9, the highest average size distributions are seen in the lower curd and cream layers. Oil bodies can be extracted, isolated and from pumpkins using an aqueous extraction method and may prove to be a useful new source of lipids for application in patterning therapeutics for clinical use.

The Hypoglycemic Effect of Pumpkin Seeds, Trigonelline (TRG), Nicotinic Acid (NA), and D-Chiro-inositol (DCI) in Controlling Glycemic Levels in Diabetes Mellitus

In the contemporary society, diabetes mellitus is considered as a common, growing, serious, costly, and potentially preventable public health problem. It is forecasted that in 2030, the number of people with diabetes will go up from 117 million in 2000 to 366 million in 2030. The prevalence of diabetes will place a huge burden on health and financial structures of countries, and these will impact on individuals, as well as families and nations. Polysaccharides, para-aminobenzoic acid, fixed oils, sterol, proteins, and peptides are biologically active ingredients, which are found in pumpkins. The chemicals within pumpkins such as the fruit pulp, oil from ungerminated seeds, and protein from germinated seeds have hypoglycemic properties. Preliminary investigation showed that pumpkin seeds, and the macromolecules, therein, such as Trigonelline (TRG), Nicotinic acid (NA), and dchiro-inositol (DCI), possess hypoglycemic properties and could assist in maintaining glycemic control.

Hydrodynamics of the VanA-type VanS histidine kinase: an extended solution conformation and first evidence for interactions with vancomycin

VanA-type resistance to glycopeptide antibiotics in clinical enterococci is regulated by the VanSARA two-component signal transduction system. The nature of the molecular ligand that is recognised by the VanSA sensory component has not hitherto been identified. Here we employ purified, intact and active VanSA membrane protein (henceforth referred to as VanS) in analytical ultracentrifugation experiments to study VanS oligomeric state and conformation in the absence and presence of vancomycin. A combination of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge (SEDFIT, SEDFIT-MSTAR and MULTISIG analysis) showed that VanS in the absence of the ligand is almost entirely monomeric (molar mass M = 45.7 kDa) in dilute aqueous solution with a trace amount of high molar mass material (M ~ 200 kDa). The sedimentation coefficient s suggests the monomer adopts an extended conformation in aqueous solution with an equivalent aspect ratio of ~(12 ± 2). In the presence of vancomycin over a 33% increase in the sedimentation coefficient is observed with the appearance of additional higher s components, demonstrating an interaction, an observation consistent with our circular dichroism measurements. The two possible causes of this increase in s – either a ligand induced dimerization and/or compaction of the monomer are considered.

Identification and monitoring of intermediates and products in the acrylamide pathway using online analysis.

Acrylamide formation under controlled processing conditions was studied in a starch matrix by analyzing volatile compounds in the gas phase using online mass spectrometry. Compounds were identified using mass spectral analysis, authentic standards, and the labeling patterns from isotopically labeled asparagine and sugars. Acrylamide, 3-aminopropanamide, methylpyrazine, 3-oxopropanamide, and aminopropan-2-one were assigned to the ions at m/ z 72, 89, 95, 88, and 74, respectively. Ion m/ z 60 was proposed as the transamination product of glyoxal, but labeling experiments did not support this assignment. Temporal formation of acrylamide and related compounds was studied in 51 samples containing asparagine and selected sugars or carbonyls. Data from the experiments were analyzed to investigate correlations between the amounts of acrylamide, intermediates, and pyrazines formed. A strong correlation between 3-aminopropanamide and acrylamide was found in all samples, whereas other correlations were reactant specific. Preliminary multiway analysis of the data identified temporal similarities in the ion profiles and showed that dynamic monitoring can follow the production and utilization of intermediates leading to acrylamide.

A simple cell-alignment protocol for sedimentation velocity analytical ultracentrifugation to complement mechanical and optical alignment procedures

In establishing the sources of data variability within sedimentation velocity analysis in the analytical ultracentrifuge and their relative importance, recent studies have demonstrated that alignment of the sample cells to the centre of rotation is the most significant contributing factor to overall variability, particularly for the characterisation of low levels of protein aggregation. Accurate mechanical and optical alignment tools have been recently designed. In this study, we (1) confirm the effect of misalignment observed by others on the estimated amounts of bovine serum albumin (BSA) monomer and dimer, and the sedimentation coefficient value for the BSA dimer; and (2) demonstrate the high performance of a mechanical alignment tool and the usefulness of a simple and complementary enhanced manual alignment protocol which should be useful for situations where these tools are not available.

Thermal flavour generation: insights from mass spectrometric studies

Abstract Fundamental studies of the reaction between glucose and L-asparagine demonstrated effects of moisture and of temperature on the generation of Strecker degradation products, exemplified here by acrylamide. Increasing the humidity of the reaction environment significantly enhanced acrylamide production. Irrespective of humidity, acrylamide was not observed until the reaction temperature was ramped from 100 to 830°C or above. Studies using a more complex food system (wheat bread) have also demonstrated that humidifying the environment during cooking can change the resulting concentrations of key flavour compounds such as pyrazines and Strecker aldehydes. The generation of acrylamide and of 2,5-dimethylpyrazine in a hydrated potato flake system were followed at 180 and 200°C using an on-line gas-phase MS-MS technique. Each of these compounds can be formed subsequent to Strecker-type reactions between amino acids and a common pool of carbonylic intermediates. Production of the pyrazine preceded that of acrylamide, perhaps, indicating that it began at a lower temperature.

On line monitoring of acrylamide formation.

A system to monitor the formation of acrylamide in model systems and from real food products under controlled conditions of temperature, time and moisture content has been developed. By humidifying the gas that flows through the sample, some control over moisture content can be affected. Results are presented to show the validity and reproducibility of the technique and its ability to deliver quantitative data. The effects of different processing conditions on acrylamide formation and on the development of color, due to the Maillard reaction, are evaluated.

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.