Isolation and Biophysical Characterisation of Bioactive Polysaccharides from Cucurbita Moschata (Butternut Squash).

Cucurbits are plants that have been used frequently as functional foods. This study includes the extraction, isolation, and characterisation of the mesocarp polysaccharide of Cucurbita moschata. The polysaccharide component was purified by gel filtration into three fractions (NJBTF1, NJBTF2, and NJBTF3) of different molecular weights. Characterisation includes the hydrodynamic properties, identification of monosaccharide composition, and bioactivity. Sedimentation velocity also indicated the presence of small amounts of additional discrete higher molecular weight components even after fractionation. Sedimentation equilibrium revealed respective weight average molecular weights of 90, 31, and 19 kDa, with the higher fractions (NJBTF1 and NJBTF2) indicating a tendency to self-associate. Based on the limited amount of data (combinations of 3 sets of viscosity and sedimentation data corresponding to the 3 fractions), HYDFIT indicates an extended, semi-flexible coil conformation. Of all the fractions obtained, NJBTF1 showed the highest bioactivity. All fractions contained galacturonic acid and variable amounts of neutral sugars. To probe further, the extent of glycosidic linkages in NJBTF1 was estimated using gas chromatography-mass spectrometry (GCMS), yielding a high galacturonic acid content (for pectin polysaccharide) and the presence of fructans-the first evidence of fructans (levan) in the mesocarp. Our understanding of the size and structural flexibility together with the high bioactivity suggests that the polysaccharide obtained from C. moschata has the potential to be developed into a therapeutic agent.

Characterisation of insulin analogues therapeutically available to patients.

The structure and function of clinical dosage insulin and its analogues were assessed. This included 'native insulins' (human recombinant, bovine, porcine), 'fast-acting analogues' (aspart, glulisine, lispro) and 'slow-acting analogues' (glargine, detemir, degludec). Analytical ultracentrifugation, both sedimentation velocity and equilibrium experiments, were employed to yield distributions of both molar mass and sedimentation coefficient of all nine insulins. Size exclusion chromatography, coupled to multi-angle light scattering, was also used to explore the function of these analogues. On ultracentrifugation analysis, the insulins under investigation were found to be in numerous conformational states, however the majority of insulins were present in a primarily hexameric conformation. This was true for all native insulins and two fast-acting analogues. However, glargine was present as a dimer, detemir was a multi-hexameric system, degludec was a dodecamer (di-hexamer) and glulisine was present as a dimer-hexamer-dihexamer system. However, size-exclusion chromatography showed that the two hexameric fast-acting analogues (aspart and lispro) dissociated into monomers and dimers due to the lack of zinc in the mobile phase. This comprehensive study is the first time all nine insulins have been characterised in this way, the first time that insulin detemir have been studied using analytical ultracentrifugation and the first time that insulins aspart and glulisine have been studied using sedimentation equilibrium. The structure and function of these clinically administered insulins is of critical importance and this research adds novel data to an otherwise complex functional physiological protein.

Application of recent advances in hydrodynamic methods for characterising mucins in solution.

Mucins are the primary macromolecular component of mucus--nature's natural lubricant--although they are poorly characterised heterogeneous substances. Recent advances in hydrodynamic methodology now offer the opportunity for gaining a better understanding of their solution properties. In this study a combination of such methods was used to provide increased understanding of a preparation of porcine intestinal mucin (PIM), MUC2 mucin, in terms of both heterogeneity and quantification of conformational flexibility. The new sedimentation equilibrium algorithm SEDFIT-MSTAR is applied to yield a weight average (over the whole distribution) molar mass of 7.1 × 10(6) g mol(-1), in complete agreement with size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), which yielded a value of 7.2 × 10(6) g mol(-1). Sedimentation velocity profiles show mucin to be very polydisperse, with a broad molar mass distribution obtained using the Extended Fujita algorithm, consistent with the elution profiles from SEC-MALS. On-line differential pressure viscometry coupled to the SEC-MALS was used to obtain the intrinsic viscosity [η] as a function of molar mass. These data combined with sedimentation coefficient data into the global conformation algorithm HYDFIT show that PIM has a flexible linear structure, with persistence length L p ~10 nm and mass per unit length, M L ~2380 g mol(-1) nm(-1), consistent with a Wales-van Holde ratio of ~1.2 obtained from the concentration dependence of the sedimentation coefficient.

Ultracentrifuge Methods for the Analysis of Polysaccharides, Glycoconjugates, and Lignins.

Although like proteins, polysaccharides are synthesized by enzymes, unlike proteins there is no template. This means that they are polydisperse, do not generally have compact folded structures, and are often very large with greater nonideality behavior in solution. This chapter considers the relevant analytical ultracentrifuge methodology available for characterizing these and related carbohydrate-based systems and information this methodology supplies, in terms of sizes, shapes, and interactions using a comprehensive range of examples, including glycoconjugates and lignins. The relevance and potential of recent software developments such as SEDFIT-MSTAR, the Extended Fujita algorithm, and HYDFIT are considered.

Recent advances in the analysis of macromolecular interactions using the matrix-free method of sedimentation in the analytical ultracentrifuge.

Sedimentation in the analytical ultracentrifuge is a matrix free solution technique with no immobilisation, columns, or membranes required and can be used to study self-association and complex or "hetero"-interactions, stoichiometry, reversibility and interaction strength of a wide variety of macromolecular types and across a very large dynamic range (dissociation constants from 10-12 M to 10-1 M). We extend an earlier review specifically highlighting advances in sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge applied to protein interactions and mucoadhesion and to review recent applications in protein self-association (tetanus toxoid, agrin), protein-like carbohydrate association (aminocelluloses), carbohydrate-protein interactions (polysaccharide-gliadin), nucleic-acid protein (G-duplexes), nucleic acid-carbohydrate (DNA-chitosan) and finally carbohydrate-carbohydrate (xanthan-chitosan and a ternary polysaccharide complex) interactions.

Hydrodynamic characterisation of chitosan and its interaction with two polyanions: DNA and xanthan.

Chitosan, a soluble polycationic derivative of insoluble chitin, has been widely considered for use in the food, cosmetic and pharmaceutical industries. Commercial ("C") and in-house laboratory ("L") prepared chitosan samples extracted from crustaceous shells with different molecular weight and degrees of acetylation (25% and 15%) were compared with regards to (i) weight-average molecular weight (Mw); (ii) sedimentation coefficient (s(o)(20,w)) distribution, and (iii) intrinsic viscosity ([η]). These parameters were estimated using a combination of analytical ultracentrifugation (AUC), size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALS) and differential pressure viscometry. Polydisperse distributions were seen from sedimentation coefficient distributions and elution profiles from SEC-MALS. Mw values obtained for each sample by sedimentation equilibrium measurements were in excellent agreement with those obtained from SEC-MALS. Mark-Houwink-Kuhn-Sakurada (MHKS) and Wales van Holde analyses of the data all suggest a semi-flexible conformation. The principle of co-sedimentation was then used to monitor the interactions of the two different molecular weights of L chitosans with two polyanions, DNA and xanthan (another double helical high molecular weight molecule). Interactions were clearly observed and then quantified from the changes in the sedimentation coefficient distribution of the mixture compared to unmixed controls using sedimentation velocity. The interactions appeared to show a strong dependence on molecular weight. The relevance of this for DNA condensation applications is indicated.

An integrated approach: advances in the use of Clostridium for biofuel.

Almost 90% of our energy comes from fossil fuels, which are both limited and polluting, hence the need to find alternative sources. Biofuels can provide a sustainable and renewable source of energy for the future. Recent significant advances in genetic engineering and fermentation technology have made microbial bio-based production of chemicals from renewable resources more viable. Clostridium species are considered as promising micro-organisms for the production of a wide range of chemicals for industrial use. However, a number of scientific challenges still need to be overcome to facilitate an economically viable production system. These include the use of cheap non-food-based substrates, a better understanding of the metabolic processes involved, improvement of strains through genetic engineering and innovation in process technology. This paper reviews recent developments in these areas, advancing the use of Clostridium within an industrial context especially for the production of biofuels.

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 D-chiro-inositol (DCI), possess hypoglycemic properties and could assist in maintaining glycemic control.

Can dietary fibre help provide safer food products for sufferers of gluten intolerance? A well-established biophysical probe may help towards providing an answer.

Gluten intolerance is a condition which affects an increasing percentage of the world's population and for which the only current treatment is a restrictive gluten free diet. However could the inclusion of a particular polysaccharide, or blends of different types, help with the provision of 'safer' foods for those individuals who suffer from this condition? We review the current knowledge on the prevalence, clinical symptoms and treatment of gluten intolerance, and the use and properties of the allergens responsible. We consider the potential for dietary fibre polysaccharides to sequester peptides that are responsible for activation of the disease in susceptible individuals, and consider the potential of co-sedimentation in the analytical ultracentrifuge as a molecular probe for finding interactions strong enough to be considered as useful.

Application and use of Inulin as a tool for therapeutic drug delivery.

Inulin is a polysaccharide with an extensive range of therapeutic uses such as a vehicle in drug delivery vehicle, as a diagnostic/analytical tool or as a dietary fibre with additional health benefits. In the main, much research has focussed on inulin as a drug delivery carrier for colon-targeted drug delivery. The justification for this is its potential to survive the stomach's acidic environment. This unique stability and strength is utilized in many ways to deliver drugs safely to colon, where they can be easily absorbed through the gut epithelium into the blood. Inulin based hydrodynamic research will be useful to discover the potential of inulin.

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

Extended Fujita approach to the molecular weight distribution of polysaccharides and other polymeric systems.

In 1962 H. Fujita (H. Fujita, Mathematical Theory of Sedimentation Analysis, Academic Press, New York, 1962) examined the possibility of transforming a quasi-continuous distribution g(s) of sedimentation coefficient s into a distribution f(M) of molecular weight M for linear polymers using the relation f(M)=g(s)·(ds/dM) and showed that this could be done if information about the relation between s and M is available from other sources. Fujita provided the transformation based on the scaling relation s=κ(s)M(0.5), where κ(s) is taken as a constant for that particular polymer and the exponent 0.5 essentially corresponds to a randomly coiled polymer under ideal conditions. This method has been successfully applied to mucus glycoproteins (S.E. Harding, Adv. Carbohyd. Chem. Biochem. 47 (1989) 345-381). We now describe an extension of the method to general conformation types via the scaling relation s=κM(b), where b=0.4-0.5 for a coil, ∼0.15-0.2 for a rod and ∼0.67 for a sphere. We give examples of distributions f(M) versus M obtained for polysaccharides from SEDFIT derived least squares g(s) versus s profiles (P. Schuck, Biophys. J. 78 (2000) 1606-1619) and the analytical derivative for ds/dM performed with Microcal ORIGIN. We also describe a more direct route from a direct numerical solution of the integral equation describing the molecular weight distribution problem. Both routes give identical distributions although the latter offers the advantage of being incorporated completely within SEDFIT. The method currently assumes that solutions behave ideally: sedimentation velocity has the major advantage over sedimentation equilibrium in that concentrations less than 0.2mg/ml can be employed, and for many systems non-ideality effects can be reasonably ignored. For large, non-globular polymer systems, diffusive contributions are also likely to be small.

Structure and heterogeneity of gliadin: a hydrodynamic evaluation.

A study of the heterogeneity and conformation in solution [in 70% (v/v) aq. ethanol] of gliadin proteins from wheat was undertaken based upon sedimentation velocity in the analytical ultracentrifuge, analysis of the distribution coefficients and ellipsoidal axial ratios assuming quasi-rigid particles, allowing for a range of plausible time-averaged hydration values. All classical fractions (alpha, gamma, omega(slow), omega(fast)) show three clearly resolved components. Based on the weight-average sedimentation coefficient for each fraction and a weight-average molecular weight from sedimentation equilibrium and/or cDNA sequence analysis, all the proteins are extended molecules with axial ratios ranging from ~10 to 30 with alpha appearing the most extended and gamma the least.

Application of food safety management systems (ISO 22000/HACCP) in the Turkish poultry industry: a comparison based on enterprise size.

The objectives of this study were to determine the extent of food safety management systems (ISO 22000/HACCP) implementation in the Turkish poultry industry. A survey was conducted with 25 major poultry meat producers, which account for close to 90% of national production, and a comparison was made between the procedures of small-to-medium enterprises (SMEs) and large firms (LFs). The survey revealed that there is a high level of application of ISO 22000 (72%), which is seen to aid the export market. LFs were shown to adopt more stringent schemes and make better use of governmental support services than SMEs. LFs were also more aware of, and able to deal with, risks from a greater range of contaminants.

The effects of heat treatment on technological properties in Red-bud maple (Acer trautvetteri Medw.) wood.

Heat treatment is often used to improve the dimensional stability of wood. In this study, the effects of heat treatment on technological properties of Red-bud maple (Acer trautvetteri Medw.) wood were examined. Samples obtained from Düzce Forest Enterprises, Turkey, were subjected to heat treatment at varying temperatures (120 degrees C, 150 degrees C and 180 degrees C) and for varying durations (2h, 6h and 10h). The technological properties of heat-treated wood samples and control samples were tested. Compression strength parallel to grain, bending strength, modulus of elasticity in bending, janka-hardness, impact bending strength, and tension strength perpendicular to grain were determined. The results showed that technological strength values decreased with increasing treatment temperature and treatment times. Red-bud maple wood could be utilized by using proper heat treatment techniques with minimal losses in strength values in areas where working, and stability such as in window frames, are important factors.