Physicochemical characterisation of kafirins extracted from sorghum grain and dried distillers grain with solubles related to their biomaterial functionality.

Kafirin, the hydrophobic prolamin storage protein in sorghum grain is enriched when the grain is used for bioethanol production to give dried distillers grain with solubles (DGGS) as a by-product. There is great interest in DDGS kafirin as a new source for biomaterials. There is however a lack of fundamental understanding of how the physicochemical properties of DDGS kafirin having been exposed to the high temperature conditions during ethanol production, compare to kafirin made directly from the grain. An understanding of these properties is required to catalyse the utilisation of DDGS kafirin for biomaterial applications. The aim of this study was to extract kafirin directly from sorghum grain and from DDGS derived from the same grain and, then perform a comparative investigation of the physicochemical properties of these kafirins in terms of: polypeptide profile by sodium-dodecyl sulphate polyacrylamide gel electrophoresis; secondary structure by Fourier transform infra-red spectroscopy and x-ray diffraction, self-assembly behaviour by small-angle x-ray scattering, surface morphology by scanning electron microscopy and surface chemical properties by energy dispersive x-ray spectroscopy. DDGS kafirin was found to have very similar polypeptide profile as grain kafirin but contained altered secondary structure with increased levels of ß-sheets. The structure morphology showed surface fractals and surface elemental composition suggesting enhanced reactivity with possibility to endow interfacial wettability. These properties of DDGS kafirin may provide it with unique functionality and thus open up opportunities for it to be used as a novel food grade biomaterial.

Lupin seed hydrolysate promotes G-protein-coupled receptor, intracellular Ca2+ and enhanced glycolytic metabolism-mediated insulin secretion from BRIN-BD11 pancreatic beta cells.

Lupin seed proteins have been reported to exhibit hypoglycaemic effects in animals and humans following oral administration, however little is known about its mechanism of action. This study investigated the signalling pathway(s) responsible for the insulinotropic effect of the hydrolysate obtained from lupin (Lupinus angustifolius L.) seed extracts utilizing BRIN-BD11 ß-cells. The extract was treated with digestive enzymes to give a hydrolysate rich in biomolecules ≤7 kDa. Cells exhibited hydrolysate induced dose-dependent stimulation of insulin secretion and enhanced intracellular Ca2+ and glucose metabolism. The stimulatory effect of the hydrolysate was potentiated by depolarizing concentrations of KCl and was blocked by inhibitors of the ATP sensitive K+ channel, Gαq protein, phospholipase C (PLC) and protein kinase C (PKC). These findings reveal a novel mechanism for lupin hydrolysate stimulated insulin secretion via Gαq mediated signal transduction (Gαq/PLC/PKC) in the ß-cells. Thus, lupin hydrolysates may have potential for nutraceutical treatment in type 2 diabetes.

Reverse phase HPLC method for detection and quantification of lupin seed γ-conglutin.

A simple, selective and accurate reverse phase HPLC method was developed for detection and quantitation of γ-conglutin from lupin seed extract. A linear gradient of water and acetonitrile containing trifluoroacetic acid (TFA) on a reverse phase column (Agilent Zorbax 300SB C-18), with a flow rate of 0.8ml/min was able to produce a sharp and symmetric peak of γ-conglutin with a retention time at 29.16min. The identity of γ-conglutin in the peak was confirmed by mass spectrometry (MS/MS identification) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The data obtained from MS/MS analysis was matched against the specified database to obtain the exact match for the protein of interest. The proposed method was validated in terms of specificity, linearity, sensitivity, precision, recovery and accuracy. The analytical parameters revealed that the validated method was capable of selectively performing a good chromatographic separation of γ-conglutin from the lupin seed extract with no interference of the matrix. The detection and quantitation limit of γ-conglutin were found to be 2.68µg/ml and 8.12µg/ml respectively. The accuracy (precision and recovery) analysis of the method was conducted under repeatable conditions on different days. Intra-day and inter-day precision values less than 0.5% and recovery greater than 97% indicated high precision and accuracy of the method for analysis of γ-conglutin. The method validation findings were reproducible and can be successfully applied for routine analysis of γ-conglutin from lupin seed extract.

Kafirin adsorption on ion-exchange resins: isotherm and kinetic studies.

Kafirin is a natural, hydrophobic and celiac safe prolamin protein obtained from sorghum seeds. Today kafirin is found to be useful in designing delayed delivery systems and coatings of pharmaceuticals and nutraceuticals where its purity is important and this can be obtained by adsorptive chromatography. This study is the first scientific insight into the isotherm and kinetic studies of kafirin adsorption on anion- and cation-exchange resins for practical applications in preparative scale chromatography. Adsorption isotherms of kafirin were determined for five anion- and two cation-exchange resins in batch systems. Isotherm parameters such as maximum binding capacity and dissociation constant were determined from Langmuir isotherm, and adsorptive capacity and affinity constant from Freundlich isotherm. Langmuir isotherm was found to fit the adsorption equilibrium data well. Batch uptake kinetics for kafirin adsorption on these resins was also carried out and critical parameters including the diffusion coefficient, film mass transfer coefficient, and Biot number for film-pore diffusion model were calculated. Both the isotherm and the kinetic parameters were considered for selection of appropriate resin for kafirin purification. UNOsphere Q (78.26 mg/ml) and Toyopearl SP-650M (57.4 mg/ml) were found to offer better kafirin binding capacities and interaction strength with excellent uptake kinetics under moderate operating conditions. With these adsorbents, film diffusion resistance was found to be major governing factor for adsorption (Bi<10 and δ<1). Based on designer objective function, UNOsphere Q was found be best adsorbent for binding of kafirin. The data presented is valuable for designing large scale preparative adsorptive chromatographic kafirin purification systems.