CAPILLARY ELECTROPHORESIS OF WHEAT GLIADINS

Identification of wheat varieties by their gliadin spectrum with acidic polyacrylamide gel electrophoresis has been in use for decades. Since the early 70s electrophoresis has been one of the most frequently used analytical methods for separation and characterisation of gluten proteins. Electrophoresis separates the analytes according to the difference in their charge distribution and/or Stoke's radii. Capillary electrophoresis (CE), the miniaturised instrumental version of electrophoresis, uses similar separation principle as the traditional technique, with several advantages. These are: high electric field, thus fast separation; low sample amount (nl); low buffer consumption (5 ml/day) thus low running costs; oncolumn detection, thus quantitative analysis; use of aqueous buffers thus no environmental wastes. Due to the several advantages capillary electrophoresis is gaining popularity in a number of fields, as opposed to the standard electrophoretic techniques. In our study a capillary zone electrophoretic method has been developed to separate the gliadin fraction of wheat proteins. The effect of the buffer composition on the resolution of the separation is shown. Various wheat types have been analysed for their gliadin spectra using both the traditional and the capillary electrophoretic method. Comparing the gliadin spectra obtained by means of the two methods, capillary electrophoresis seems to be a suitable alternative to the traditional method for identification / quality control of wheat species according to their gliadin spectra

QUALITATIVE AND QUANTITATIVE DETERMINATION OF LACTOSE IN MILK AND DAIRY PRODUCTS BY CAPILLARY ELECTROPHORESIS

A capillary electrophoretic method was developed for carbohydrate analysis of various dairy products containing carbohydrates in the range of 1-10% using indirect DV detection. A thorough, yet simple sample preparation technique was developed to address the inherent problems of indirect detection (limited linear dynamic range, and lack of robustness). The effect of separation parameters (buffer, electric field strength, length of the capillary and capillary conditioning procedures) on the selectivity and efficiency of the separation were investigated. The applicability of the method is demonstrated by analysing different dairy products (cocoa milk, milk, yoghurt, sour milk, sour cream, etc.). A critical assessment of the developed analytical method is also provided

APPLICABILITY OF CAPILLARY ELECTROPHORESIS IN PROTEIN SEPARATIONS WITH REGARD TO WHEAT AND WHEAT ANALOGOUS PROTEINS

High-performance capillary electrophoresis (HPCE), among other analytical techniques (e.g. acid polyacrylamide gel electrophoresis, sodium dodecyl sulphate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography) [1]-[4] is more and more widely used also in the field of separation of cereal proteins [5]-[18]. HPCE is versatile, easy to automate, does not need toxic reagents or long analysis time, but requires only small sample size, small amount of buffer and provides high-resolution separations. So this analytical technique is particularly applicable for studying the fine structure of the composition of wheat proteins. Capillary electrophoresis has been used at our department for the determination of the fine structure of different wheat protein fractions [19]. Differences between the various wheat cultivars and changes during grain maturation process have also been studied using a home-built capillary electrophoretic system [20]. The same system has been used to investigate the electrophoretic properties of a gliadin analogous protein (BM180 - a basement membrane protein with a potential autoantigen role) [21]. The more strictly controllable nature of an automated system (especially the temperature control of the capillary) allows the use of higher voltages, so separation time decreases and resolution increases. Purchasing an automated capillary electrophoretic system we gained an opportunity to compare the two electrophoretic systems also in the field of wheat protein analyses. The aim of present work was to give an impression of the work done by capillary electrophoresis at our department

A study on the essential oil Ferulago campestris. How much does exstraction method influence the oil composition?

The essential oil of different parts of Ferulago campestris (Bess.) collected in Sicily has been extracted by microwave-assisted hydrodistillation (MAHD) and by classic hydrodistillation (HD). A comparative qualitative–quantitative study on the composition of the oils was carried out. A total of 100 compounds were identified in the oils obtained by MAHD, whereas 88 compounds characterized the HD oils. The most prominent components were, in all different parts of F. campestris and in both extraction methods, 2,4,5-trimethylbenzaldehyde and 2,4,6-trimethylbenzaldehyde isomers; the latter was not previously found. The attempt to evaluate where the oil components are located in all parts of the plant was carried out by means of a kinetic study. Then, electron microscopy observation on the different parts before and after MAHD and HD was performed

Application of Molecular Topology for the Prediction of Reaction Yields and Anti-Inflammatory Activity of Heterocyclic Amidine Derivatives

Topological-mathematical models based on multiple linear regression analyses have been built to predict the reaction yields and the anti-inflammatory activity of a set of heterocylic amidine derivatives, synthesized under environmental friendly conditions, using microwave irradiation. Two models with three variables each were selected. The models were validated by cross-validation and randomization tests. The final outcome demonstrates a good agreement between the predicted and experimental results, confirming the robustness of the method. These models also enabled the screening of virtual libraries for new amidine derivatives predicted to show higher values of reaction yields and anti-inflammatory activity

Rapid detection of Ganoderma-infected oil palms by microwave ergosterol extraction with HPLC and TLC

Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed as early detection methods for the disease. These techniques are complex, time consuming and have accuracy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This current study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70 °C and medium high power for 30 s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high performance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations.This project was supported by the Fundamental Research Grant Scheme (FRGS), administered through the Ministry of Higher Education, Malaysia (Grant No: 5524175)

Neoglycoproteins as carbohydrate antigens : synthesis, analysis, and polyclonal antibody response

The analysis and polyclonal antibody response for newly synthesized maltose-BSA conjugate neoglycoproteins is described. In this first proof of concept study, a simple carbohydrate antigen, maltose, was linked to BSA by reductive amination. An aglycone spacer was utilized to conserve the intact annular maltose structure and to promote the accessibility of the carbohydrate immunogen hapten during immunization. The neoglycoproteins were investigated by CGE and the number of conjugated maltose residues was determined by MALDI-TOF MS. The neoglycoproteins were then evaluated by immunization of BALB/c mice and the polyclonal antibody response was tested by ELISA as evidence for the presence of sugar-containing epitope-specific antibodies. Selective antibody binding was demonstrated to the synthesized neoglycoproteins with different (low and high) glycosylation degrees suggesting the possible use of this approach to generate antibodies. Moreover, the polyclonal antibody response was not inhibited by maltose or other simple carbohydrates to confirm presence of the neoglycoprotein-specific antibodies

Microbial oil produced from the fermentation of microwave-depolymerised rapeseed meal

Rapeseed meal is high in protein and carbohydrate and is a promising feedstock for microbial valorisation, however, the fibrous structure is difficult to breakdown and involves multiple chemical and enzymatic steps to release a fermentable hydrolysate. In this investigation an innovative pre-treatment of rapeseed meal was demonstrated, involving a one-step process using microwave heating and no additional chemicals or enzymes. 57% of the biomass was solubilised over just a few minutes with minimal energy input. The hydrolysate contained a mixture of monosaccharides, oligosaccharides and micronutrients. To ferment this material the oleaginous yeast Metschnikowia pulcherrima was selected and was able to metabolise the material including some of the oligosaccharides of approximately DP8 and below, producing a lipid that was highly monounsaturated. On the laboratory scale 11% lipid could be achieved from the rapeseed meal alone, with a lipid profile akin to palm oil. On the addition of glycerol to increase the C:N ratio, over 16 g/L of yeast was achieved with lipid content of 38% w/w. To demonstrate the scalability of the microbial process, the fermentation was demonstrated on depolymerised rapeseed meal with glycerol, in a 30 L pilot scale fermenter, yielding 12 g/L of yeast with 22% w/w lipid over 120 h. While the lipid production needs to be further optimized on this scale, the use of rapeseed meal as a feedstock, coupled with a one-step microwave process that does not need additional pre- and post-processing stages, is an exciting route to potential commercially viable microbial oils