Browsing by Subject "hordeiini"

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  • Häkämies, Anna K. (Helsingin yliopisto, 2014)
    The literature review examined barley (Hordeum vulgare) hordein, starch granule-associated proteins and gelprotein, dough formation during mixing and starch-protein interaction in the dough. The aim of this study was to define the structure of barley dough and specify barley hordeins that form the dough structure. The purpose of the preliminary studies was to find out can ascorbic acid change barley dough structure. Mixograph was used to mix barley dough and to study dough formation. Barley dough rheology was studied using dynamic oscillation measurements. 1.5 % SDS-solution and centrifugation was used to isolate proteins from barley dough. The protein composition of the separated dough phases was analysed by SDS-PAGE. SDS sedimentation value was also determined. Ascorbic acid (120 ppm flour weight) did not change the rheological properties of the barley dough. Centrifugation separated four solid phases from barley dough; gelprotein, white layer, bran and starch. Results from the Bradford protein assay and the iodine test showed that white layer under gelprotein in the centrifuge tube contained protein and starch while gelprotein contained only proteins. SDS-PAGE showed that reduced gelprotein from barley dough consisted of D- and B-hordein but only minor amount of C-hordein. C-hordein was found, however in the dough liquid phase. The protein composition of the gelprotein was identical with the white layer. In the non-reduced gelprotein and the white layer hordeins were in polymeric form because they did not penetrate in the SDS-PAGE gel. Two studied barley doughs with the same protein content (12.0 %) differed they protein composition. In SDS-PAGE one band more B-hordeins (35 kDa) was showed in the dough which area (cm2) of the mixogramm, sedimentation value and storage modulus (G´) and loss modulus (G´´) were higher. The storage modulus (G´) of the barley dough increased during 160 min dough rest. G´ and G´´ values of the barley dough were frequency (Hz) dependent and they increased when frequency increased. Sedimentation value positively correlated with modulus (G´, G´´) and area (cm2) of the mixogramm. Based on dough rheology this study showed that barley dough structure was a weak gel. Barley hordein, especially certain B-hordeins seems to be significant in the formation of dough structure. In addition results indicated the interaction between starch and hordein in the barley dough. This study clearly showed that barley dough contained high molecular weight hordein polymers stabilized by interchain and intrachain disulfide bonds. Hordein polymer size remained unknown but the rheological properties of barley dough support the conclusion that high molecular weight gelprotein structure formed in the barley dough.
  • Perkiö, Pasi (Helsingfors universitet, 2013)
    The aim of the literature review was to examine barley’s (Hordeum vulgare) alcohol-soluble proteins – hordeins and their technological attributes. Additionally, applicability of flow field flow fractionation (FFF) separation method as well as spectrophotometric and light scattering methods for protein characterization was under investigation. The objective of the experimental research was to determine a suitable extraction method for hordeins and subsequent analysis of their molecular weight distribution, size and conformation by the use of AF4 (asymmetric flow field flow fractionation) in combination with MALS-, UV- and RI-detectors. 40 % 1 propanol combined with mild sonication treatment proved to be the most efficient method to extract hordeins from barley flour. In order to prevent deterioration of the FFF channels the solvent had to be diluted to 20 %. Same dilution was used to measure hordeins’ extinction coefficient and to calculate ?n/?c theoretically. Berry plot was found to be the most suitable fit for the data analysis. Extracted hordeins were analysed with SDS PAGE. Extracts contained monomeric C, B and ? hordeins and polymeric B, D and ? hordeins. Also, small amounts of albumins, globulins and hydrolysed proteins were present. Extracts’ fractograms had five distinctive peaks. All of the peaks’ mass fractals and polydispersity indices were above 1, which means analysed aggregates were polydisperse and shaped as complex rods. This can be explained by 1 propanol influenced protein aggregation. Some inference in light scattering was identified in the MALS detector signal. This and the use of measured extinction coefficient and calculated index of refraction caused some errors in the data. The low sample yield (19–26 %) can be explained by the hordeins’ adhering to a syringe filter and adsorbing to the surface of AF4’s ultrafiltration membrane. Also, the use of over simplifying mathematical model to calculate the results and yield could cause some errors in the results. This study showed that it is possible to study Mw, size and conformation of polymeric hordeins with AF4 combined with MALS/UV-detectors and that hordeins form big aggregates in 20 % 1 propanol. For MALS proteins should be extracted in a solvent that will not interfere with subsequent analysis and proteins net charge, which creates a challenge to find proper solvent for hordeins. Nevertheless AF4 proved to be useful and delicate tool for characterizing cereal polymeric proteins.
  • Arte, Elisa (Helsingfors universitet, 2013)
    The literature review focused on the composition of the barley hordeins and the known extraction methods. The metal-catalysed oxidation of proteins and amino acids was also reviewed. The aim of the experimental part was to develop a simple selective extraction method for the B and C hordeins and to study how metal-catalysed oxidation affects these hordeins. Hulles barley cf. Jorma was selected as test material since milling of this cultivar was simple with a Brabender Junior mill. From the milled barley, water and salt-soluble material were removed and the rest was freeze-dried. The freeze-dried sample flour was studied by gel separation, precipitation and extraction with aqueous alcohols. The aqueous alcohol extracted the C hordeins completely although there was some B hordeins present. Two-dimensional electrophoresis showed that the isoelectric point of C hordeins was between the pH 5–7. Aqueous alcohols, extraction temperatures and pH were studied for hordein extraction. None of the studied methods improved the extraction of B and C hordeins. The hordein sample used in further experiments was extracted with 55% 2-propanol at 50 ºC. The metal-catalysed oxidation of the extracted hordeins was studied by using copper or iron as a catalyst and hydrogen peroxide or ascorbic acid as an oxidant. The reactions were analysed with SDS-PAGE and SE-HPLC. The results showed that the most effective reaction was with copper and hydrogen peroxide where the B and C hordeins were degraded efficiently after 24 hours of incubation. The results from SE-HPLC showed aggregated B hordeins in the extracted sample, which were partly degraded after two hours of incubation with hydrogen peroxide and copper. The results of this study indicated that the biggest groups of hordeins, the B and C hordeins, cannot be selectively separated with extraction. The barley hordeins efficiently degraded in the metal-catalysed oxidation with hydrogen peroxide and copper.