Browsing by Subject "prolamiini"

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  • 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.
  • Rahikainen, Antti (Helsingfors universitet, 2013)
    The aim of the literature review was to research barley proteins, metal-catalyzed oxidation and subjects related to it, like antioxidants and oxidation reactions in beer. In addition ACE inhibition was looked into. The object of the experimental part was to find out if the proteins of a barley-based industrial side product can be modified by metal-catalyzed oxidation or enzymatic hydrolysis, and how these treatments affect the different proteins in the sample material. In addition, the possible ACE inhibition activity of the reaction products was determined. The sample material was a protein-rich side-product of barley starch production. Two protein fractions were extracted from the material; an alcohol soluble fraction and a reduced fraction. The modification of the proteins in the sample fractions by oxidation and hydrolysis was determined with gel electrophoresis and size exclusion chromatography. The ACE inhibitory activity of the small peptides from these reactions was determined with UV-VIS spectroscopy. Three protein groups were identified from the sample material; polymeric B hordein, monomeric B hordein and C hordein. Contrary to expectations metal-catalyzed oxidation did not break down any of the proteins in the sample; instead it aggregated the proteins into bigger units. The enzyme treatment hydrolyzed the proteins effectively. Small peptides from the enzyme hydrolysis had an ACE inhibition IC50 of 246 µg/ml, which is similar to gluten hydrolysates IC50 of 29 µg/ml. IC50 is the inhibitor concentration where 50% of enzyme activity is inhibited. Instead of breaking down the subject proteins metal-catalyzed oxidation aggregated them, and thus it could not be used to make ACE inhibitory peptides. Enzyme hydrolysis was found to be a valid method of inhibitor peptide production. The peptides produced had an ACE inhibition capacity similar to previously known ACE inhibitory food hydrolysates.
  • Väärni, Sanni (Helsingin yliopisto, 2019)
    Kaura on luontaisesti gluteeniton vilja, mutta sen kontaminoituminen gluteenia sisältävillä viljoilla alkutuotannon aikana on suuri haaste. Lisäksi gluteenipitoisuuden määrityksessä käytetään referenssinä gliadiinia, joka ei sovi gluteenipitoisuuden määritykseen kontaminantin ollessa ohra. Menetelmässä käytetty R5-vasta-aine reagoi voimakkaasti ohran C-hordeiineihin, joita on 20-30 % ohran hordeiineista. Lisäksi R5-vasta-aineen on epäilty tunnistavan kauran omia proteiineja. Työn tarkoituksena oli tutkia puhdaskauran ja -kauratuotteiden gluteenittomuuden varmistamiseen liittyviä tekijöitä. Ohrajäämien määrän vaikutusta puhdaskauran gluteenipitoisuuteen tutkittiin kontaminoimalla kauraa eri määrillä ohranjyviä. Näytteiden gluteenipitoisuudet määritettiin ELISA R5 gluteenianalyysillä PWG-gliadiini- ja C-hordeiinireferensseillä ja tuloksia vertailtiin laskennalliseen gluteenipitoisuuteen. R5-vasta-aineen reaktiivisuutta kauran aveniineihin tutkittiin ELISA R5 -gluteenianalyysillä ja elektroforeesissa erotettujen proteiinien vasta-ainevärjäyksellä. Kauramyllyn vastaanottamista puhdaskauranäytteistä löytyneiden ohrajäämien C-hordeiinien osuudet suhteessa kokonaishordeiineihin määritettiin käänteisfaasi-nestekromatografialla. Myös kauramyllyn puhdistuslaitteiden tehokkuutta seurattiin tutkimalla gluteenia sisältävien viljajäämien määriä eri puhdistusvaiheiden jälkeen. Lisäksi myllylle tehtiin näytteenottosuunnitelma lopputuotteiden gluteenipitoisuuden seuraamista varten Codex Alimentariuksen CAC/GL 50-2004 näytteenotto-ohjeiden avulla. Kontaminoiduista näytteistä gliadiinisreferenssillä saadut tulokset olivat jopa viisi kertaa suurempia kuin laskennallinen gluteenipitoisuus, kun taas C-hordeiinireferenssillä saadut tulokset olivat lähellä laskennallista arvoa. Puhtaista kauranäytteistä ELISA R5-menetelmällä määritetyt gluteenipitoisuudet olivat 3,0-5,5 mg/kg, eikä R5 tunnistanut aveniineja vasta-ainevärjäyksessä. C-hordeiinien osuus kokonaishordeiineista oli suurin Kymenlaaksosta tulleen kauran ohrajäämissä ja pienin Pohjois-Pohjanmaalta tulleen kauran ohrajäämissä. Myllyn puhdistuslaitteet puhdistivat noin 75-76 % raaka-aineessa olleista jäämistä. Niin kauan, kun gliadiini on yleisimmin käytössä oleva, sekä näin ollen myös myllyn asiakkaiden käyttämä referenssimateriaali, on myllyn ainoa vaihtoehto pyrkiä ohralla kontaminoitumattomaan lopputuotteeseen puhtaalla alku- ja lopputuotannolla, edustavalla näytteenotolla sekä tehokkaalla puhdistusprosessilla