Прегледај по Аутор "Gojković Cvjetković, Vesna"

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    CHROMATOGRAPHIC SEPARATION OF GLUTENIN WITH HIGH MOLECULAR WEIGHT FROM WHEAT FLOUR
    (KEY, 2020) Gojković Cvjetković, Vesna; Grujić, Radoslav; Marjanović-Balaban, Željka
    Glutenins with high molecular weight (HMW glutenins) are one of the glutenin's fractions. They play a key role in the formation of the gluten elasticity property and contribute to the formation of large glutenin polymers. The aim of this study was to investigate the effect of solvent type and column temperature on the chromatographic separation of HMW glutenins from wheat flour. For HMW glutenins extraction, 50% (v/v) ethanol, 1-propanol and isopropanol was used to which Tris-HCl (0.05 mol/L, Ph = 7.5), urea (2 mol/L) and dithioerythritol (1%) were added. The high performance liquid chromatography (HPLC) method (HPLC Agilent Technologies 1260 Infinity, Zorbax 300SB-C3 column) was used for protein separation at a column temperature 40, 45 and 50 0C. Absorbance measurements were performed at 210 nm and 280 nm. The effect of the solvents tested on the separation of HMW glutenins was shown by determining the number of observed peaks (proteins) on the chromatogram and calculating the relative concentration of HMW in the total number of glutenins from wheat flour (RC). After the extraction of glutenin proteins with 50% (v/v) ethanol, the highest number of proteins at 210 nm was observed when the column temperature was 45 0C and 50 0C (Xsr = 6.17 and RC1 = 17.76% and RC2 = 27.07%) and the lowest number at a column temperature of 40 0C (Xsr = 4, RC = 13.43%). By extraction with 50% (v/v) 1-propanol, the highest number of proteins was observed at column temperatures of 40 0C and 45 0C (Xsr = 5.17 and RC1 = 28.22% and RC2 = 31.70%, respectively) and the lowest number at 50 0C (Xsr = 4.67, RC = 34.68%) and by extraction with 50% (v/v) isopropanol the highest number of proteins was observed at a column temperature of 50 0C (Xsr = 7.17, RC = 23.61%) and the lowest number at 45 0C (Xsr = 5.83, RC = 10.67%). After the extraction of glutenin proteins with 50% (v/v) ethanol and detection at a wavelength of 280 nm, the highest number of proteins was observed at a column temperature of 45 0C (Xsr = 8.33, RC = 36.49%), and the lowest at 40 0C (Xsr = 5.50, RC = 32.57%). In the case of protein extraction with 50% (v/v) 1-propanol, the highest number of HMW glutenins was observed at 40 0C (Xsr = 7.83, RC = 61.62%) and the lowest at 50 0C (Xsr = 4.67, RC = 39.18%). When extraction was performed with 50% (v/v) isopropanol, the highest number of proteins was observed at a column temperature of 45 °C (Xsr = 7.33, RC = 21.66%), and the lowest number at a column temperature of 40 0C and 50 0C (Xsr = 7.00, RC1 = 28.64%, RC2 = 34.22%). Based on the obtained results it can be seen that the highest number of proteins was observed with 50% (v/v) ethanol (Xsr = 8.33)
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    Gliadin proteins from wheat flour the optimal determination conditions by ELISA
    (2021) Marjanović-Balaban, Željka; Gojković Cvjetković, Vesna; Grujić, Radoslav
    Introduction. The number of people with celiac disease is rapidly increasing. Gluten, is one of the most common food allergens, consists of two fractions: gliadins and glutenins. The research objective was to determine the optimal conditions for estimating gliadins by using enzyme-linked immunosorbent assay (ELISA). Study objects and methods. The experiment involved wheat flour samples (0.10, 0.20, 0.25, 0.50, and 1.0 g) suspended in different solvents (ethanol, methanol, 1-propanol, and isopropanol) of different concentrations (40, 50, 60, 70, 80, and 90% v/v). The samples were diluted with Tris buffer in ratios of 1:50, 1:100, 1:150, and 1:200. The gliadin test was performed using a Gliadin/Gluten Biotech commercial ELISA kit (Immunolab). Results and discussion. The optimal conditions for determining gliadin proteins that provided the highest gliadin concentration were: solvent – 70% v/v ethanol, extract:Tris buffer ratio – 1:50, and sample weight – 1.0 g. Conclusion. The obtained results can be of great importance to determine gliadin/gluten concentrations in food products by rapid analysis methods.
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    Separation of gliadins from wheat flour by capillary gel electrophoresis: optimal conditions
    (Kemerovo State University, 2020) Grujić, Radoslav; Gojković Cvjetković, Vesna; Marjanović-Balaban, Željka
    Introduction. Gliadin proteins are one of the gluten fractions. They are soluble in alcoholic solution and divided into four groups (α + β, γ, ω1.2, and ω5-gliadins). In this paper gliadins were extracted from wheat flour, and optimal conditions for their separation were determined. Study objects and methods. The separation was performed by capillary gel electrophoresis on Agilent apparatus, CE 7100 (a capillary with an inner diameter of 50 μm, a total length of 33 cm, and an effective length of 23.50 cm). In order to determine the optimal conditions, different solvent concentrations (50, 60, and 70% ethanol), capillary temperatures (20, 25, 30, 35, and 40°C), and electrode voltages (–14.5, –16.5, –17.5 and –18.5 kV) were applied. Migration time and relative concentration of each protein molecules within gliadin fractions in the electrophoregram were analysed using Agilent ChemStation Software. Results and discussion. The optimal conditions for gliadin separation were: solvent 70% (v/v) ethanol, capillary temperature of 25°C, and electrode voltage of –16.5 kV. Under these conditions, the total proteins were indetified as Xav = 23.50, including α + β gliadin fraction (Xav = 7.50 and relative concentration RC = 28.29%), γ-gliadins (Xav = 5.00, RC = 26.66%), ω1.2-gliadins (Xav = 4.33, RC = 14.93%), and ω5-gliadins (Xav = 6.67, RC = 30.98%). Conclusion. The results of the research can be of fundamental importance in the study of gluten proteins and the influence of technological procedures on their change and the possibility of reducing the allergic effect of gluten during processing.
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    THE EFFECT OF THE DURATION OF SEPARATION AND WAVELENGTHS OF ABSORBANCE’S MEASUREMENT ON THE EFFECTIVENESS OF ELECTROPHORETIC SEPARATION OF GLIADINS BY GCE
    (2022) Gojković Cvjetković, Vesna; Grujić, Radoslav; Marjanović-Balaban, Željka
    Gliadins are one of the gluten components which are present in most wheat products. Based on their mobility in acidic polyacrylamide gel electrophoresis (A-PAGE), gliadins are divided into α+β, γ, ω1.2, and ω5 classes. Gliadins are allergenic proteins which play a key role in celiac disease pathogenesis. Considering that the number of people with celiac disease is increasing, the aim of this study was to examine how different duration of analysis (30, 35 and 40 minutes) and wavelengths (200, 210 and 230 nm) effect the gliadin separation effectiveness by glassy carbon electrode (GCE). We examined the effect of duration of capillary gel electrophoresis (GCE) separation (30, 35 and 40 minutes) and measurement of absorbance at different wavelengths (200, 210, and 230 nm) on the effectiveness of electrophoretic separation by capillary gel electrophoresis, with the aim of faster identification and quantification of gliadin proteins. Separation of gliadin proteins by GCE was performed on an Agilent, CE 7100 apparatus. SDS-MW analysis kit, PA 800 plus (2015 Beckman Coulter, USA) was used. Based on the obtained results the optimal duration of GCE separation was 30 minutes, because the number of observed proteins is the highest and the effectiveness of electrophoretic separation is the best (Xav = 23.50). A wavelength of 200 nm proved to be the best wavelength. By measuring the absorbance at this wavelength, the total number of observed proteins is Xav=18.33. In this study a duration of 30 minutes and a wavelength of 200 nm proved to be optimal. As the duration of analysis increases, the number of proteins decreases