20.500.12556/RUL-108846 Histokemijsko določanje ß-glukanov v zrnih ječmena Histochemical determination β-glucans in barley grains Ječmen je eno od najstarejših žit na svetu in spada v botanično družino trav (Poaceae). Glavna gradbena komponenta notranjega dela celičnih sten škrobnega endosperma so (1→3),(1→4)-β-D-β-glukani. To so linearni polisaharidi, ki spadajo v skupino prehranskih vlaknin in so pomemben del človekove prehrane. Glavni nalogi β-glukanov sta stabilizacija rastlinske celične stene ter mobilizacija nastale energije pri njihovi hidrolizi. Namena našega raziskovalnega dela sta bila histokemijska lokalizacija β-glukanov v zrnu ječmena ter ovrednotenje intenzitete fluorescence pri štirih vrstah ječmena kot osnova za semi-kvantitativno določanje vsebnosti beta glukanov. Kot izhodni material smo vzeli po nekaj zrn treh različnih sort ječmena z že znanimi vsebnostmi β-glukanov ter nekaj zrn sorte ječmena z neznano vsebnostjo β-glukanov. Vzorce smo s tehniko zamrzovanja in odmrzovanja (angl. freeze-thaw) pripravili za rezanje in barvanje. Pri barvanju smo uporabili dve fluorescentni barvili, kalkofluor beli (Sigma-Aldrich) in fluorestentni osvetljevalec 28 (Sigma-Aldrich), ki se vežeta na β-1,3 in β-1,4 polisaharide. Ugotovili smo, da se β-glukani nahajajo znotraj zrna ječmena, natančneje v škrobnem endospermu in alevronski plasti. Na slikah pobarvanih rezin vzorcev smo pomerili intenziteto fluorescence s programom ImageJ. S pomočjo povprečnih vrednosti intenzitete fluorescence smo izdelali umeritveno krivuljo, s pomočjo katere smo določili vsebnost β-glukanov v zrnu ječmena z neznano vsebnostjo β-glukanov. Zaključimo lahko, da je histokemijska metoda primerna za semi-kvantitativno določanje količine β-glukanov v zrnih ječmena, pri čemer jih lahko razdelimo v razrede z manjšo, srednjo in veliko vsebnostjo β-glukanov. Barley is one of the oldest cultivated cereals in the world and is classified as a botanical member of the grass family (Poaceae). The primary structural components in the cell wall interiors of the starchy endosperm are (1→3), (1→4)-β-D-β-glucans. They are linear polysaccharides that belong to the group of dietary fibres and are a crucial part of human nutrition. β-glucan’s primary functions are the stabilisation of a plant’s cell wall and the mobilisation of energy produced by its hydrolysis. Our research project’s goals were the histochemical localisation of β-glucans within the grain of barley and the evaluation of fluorescence intensity in four different barley types for the basis of quantitative determination of the content of β-glucans. For source material, we gathered a few grains from three different types of barley with known concentrations of β-glucans, and a few grains of a type of barley with an unknown concentration of β-glucans. The specimens were prepared for cutting and dyeing with the freeze-thaw technique. Dyeing was achieved with two fluorescent dyes; Calcofluor White (Sigma-Aldrich) and the Fluorescent Brightener 28 (Sigma-Aldrich), which bind to the β-1,3 and β-1,4 polysaccharides. We determined that the β-glucans are present within the grain of barley, specifically within the starchy endosperm and the aleurone layer. We measured the intensity of fluorescence on the images of dyed slice samples with the imageJ programme. With the help of the average values of fluorescence intensity, we created a measuring curve, which we used to determine the content of β-glucans in the grain of barley with an unknown content of β-glucans. We can conclude that the histochemical method is appropriate for semi-quantitative assessment of the β-glucan levels in grains of barley, which can then be classified into grades of fewer, medium, and higher β-glucan content. ječmen barley true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2019-07-30 05:33:49 2019-08-20 12:35:24 2025-04-17 04:53:48 0000-00-00 00:00:00 2019 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 12534089 diploma_PDF_Urska_Keber_2019.pdf diploma_PDF_Urska_Keber_2019.pdf 1 6F2DB57D7B86CE01F2351EDDF61265FF f0c9090f6357eba55e00ad632dcf2e40278e9a2b8390f5953169abee01c78db2 022841b2-1ab3-11f0-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=206796 Pedagoška fakulteta 0 0 0