Structural analyses of (1->3),(1->4)-β-D-glucan of oats and barley

The structures of (1→3),(1→4)-β-D-glucans of oat bran, whole-grain oats and barley and processed foods were analysed. Various methods of hydrolysis of β-glucan, the content of insoluble fibre of whole grains of oats and barley and the solution behaviour of oat and barley β-glucans were studied. The isolated soluble β-glucans of oat bran and whole-grain oats and barley were hydrolysed with lichenase, an enzyme specific for (1→3),(1→4)-β-D-β-glucans. The amounts of oligosaccharides produced from bran were analysed with capillary electrophoresis and those from whole-grains with high-performance anion-exchange chromatography with pulse-amperometric detection. The main products were 3-O-β-cellobiosyl-D-glucose and 3-O-β-cellotriosyl-D-glucose, the oligosaccharides which have a degree of polymerisation denoted by DP3 and DP4. Small differences were detected between soluble and insoluble β-glucans and also between β-glucans of oats and barley. These differences can only be seen in the DP3:DP4 ratio which was higher for barley than for oat and also higher for insoluble than for soluble β-glucan. A greater proportion of barley β-glucan remained insoluble than of oat β-glucan. The molar masses of soluble β-glucans of oats and barley were the same as were those of insoluble β-glucans of oats and barley. To analyse the effects of cooking, baking, fermentation and drying, β-glucan was isolated from porridge, bread and fermentate and also from their starting materials. More β-glucan was released after cooking and less after baking. Drying decreased the extractability for bread and fermentate but increased it for porridge. Different hydrolysis methods of β-glucan were compared. Acid hydrolysis and the modified AOAC method gave similar results. The results of hydrolysis with lichenase gave higher recoveries than the other two. The combination of lichenase hydrolysis and high-performance anion-exchange chromatography with pulse-amperometric detection was found best for the analysis of β-glucan content. The content of insoluble fibre was higher for barley than for oats and the amount of β-glucan in the insoluble fibre fraction was higher for oats than for barley. The flow properties of both water and aqueous cuoxam solutions of oat and barley β-glucans were studied. Shear thinning was stronger for the water solutions of oat β-glucan than for barley β-glucan. In aqueous cuoxam shear thinning was not observed at the same concentration as in water but only with high concentration solutions. Then the viscosity of barley β-glucan was slightly higher than that of oat β-glucan. The oscillatory measurements showed that the crossover point of the G´ and G´´ curves was much lower for barley β-glucan than for oat β-glucan indicating a higher tendency towards solid-like behaviour for barley β-glucan than for oat β-glucan.

The effect of structure on the dilute solution properties of branched polysaccharides studied with SEC and AsFlFFF

Cereal arabinoxylans, guar galactomannans, and dextrans produced by lactic acid bacteria(LAB) are a structurally diverse group of branched polysaccharides with nutritional and industrial functions. In this thesis, the effect of the chemical structure on the dilute solution properties of these polysaccharides was investigated using size-exclusion chromatography(SEC) and asymmetric flow field-flow fractionation (AsFlFFF) with multiple-detection. The chemical structures of arabinoxylans were determined, whereas galactomannan and dextran structures were studied in previous investigations. Characterization of arabinoxylans revealed differences in the chemical structures of cereal arabinoxylans. Although arabinoxylans from wheat, rye, and barley fiber contained similar amounts of arabinose side units, the substitution pattern of arabinoxylans from different cereals varied. Arabinoxylans from barley husks and commercial low-viscosity wheat arabinoxylan contained a lower number of arabinose side units. Structurally different dextrans were obtained from different LAB. The structural effects on the solution properties could be studied in detail by modifying pure wheat and rye arabinoxylans and guar galactomannan with specific enzymes. The solution characterization of arabinoxylans, enzymatically modified galactomannans, and dextrans revealed the presence of aggregates in aqueous polysaccharide solutions. In the case of arabinoxylans and dextrans, the comparison of molar mass data from aqueous and organic SEC analyses was essential in confirming aggregation, which could not be observed only from the peak or molar mass distribution shapes obtained with aqueous SEC. The AsFlFFF analyses gave further evidence of aggregation. Comparison of molar mass and intrinsic viscosity data of unmodified and partially debranched guar galactomannan, on the other hand, revealed the aggregation of native galactomannan. The arabinoxylan and galactomannan samples with low or enzymatically extensively decreased side unit content behaved similarly in aqueous solution: lower molar mass samples stayed in solution but formed large aggregates, whereas the water solubility of the higher-molar-mass samples decreased significantly. Due to the restricted solubility of galactomannans in organic solvents, only aqueous galactomannan solutions were studied. The SEC and AsFlFFF results differed for the wheat arabinoxylan and dextran samples. Column matrix effects and possible differences in the separation parameters are discussed, and a problem related to the non-established relationship between the separation parameters of the two separation techniques is highlighted. This thesis shows that complementary approaches in the solution characterization of chemically heterogeneous polysaccharides are needed to comprehensively investigate macromolecular behavior in solution. These results may also be valuable when characterizing other branched polysaccharides.

Characterization of hydrophobin proteins at interfaces and in solutions using x rays

Hydrophobins are a group of particularly surface active proteins. The surface activity is demonstrated in the ready adsorption of hydrophobins to hydrophobic/hydrophilic interfaces such as the air/water interface. Adsorbed hydrophobins self-assemble into ordered films, lower the surface tension of water, and stabilize air bubbles and foams. Hydrophobin proteins originate from filamentous fungi. In the fungi the adsorbed hydrophobin films enable the growth of fungal aerial structures, form protective coatings and mediate the attachment of fungi to solid surfaces. This thesis focuses on hydrophobins HFBI, HFBII, and HFBIII from a rot fungus Trichoderma reesei. The self-assembled hydrophobin films were studied both at the air/water interface and on a solid substrate. In particular, using grazing-incidence x-ray diffraction and reflectivity, it was possible to characterize the hydrophobin films directly at the air/water interface. The in situ experiments yielded information on the arrangement of the protein molecules in the films. All the T. reesei hydrophobins were shown to self-assemble into highly crystalline, hexagonally ordered rafts. The thicknesses of these two-dimensional protein crystals were below 30 Å. Similar films were also obtained on silicon substrates. The adsorption of the proteins is likely to be driven by the hydrophobic effect, but the self-assembly into ordered films involves also specific protein-protein interactions. The protein-protein interactions lead to differences in the arrangement of the molecules in the HFBI, HFBII, and HFBIII protein films, as seen in the grazing-incidence x-ray diffraction data. The protein-protein interactions were further probed in solution using small-angle x-ray scattering. Both HFBI and HFBII were shown to form mainly tetramers in aqueous solution. By modifying the solution conditions and thereby the interactions, it was shown that the association was due to the hydrophobic effect. The stable tetrameric assemblies could tolerate heating and changes in pH. The stability of the structure facilitates the persistence of these secreted proteins in the soil.

Non-enzymatic Degradation of (1→3)(1→4)-β-D-glucan in Aqueous Processing of Oats

Cereal water-soluble β-glucan [(1→3)(1→4)-β-D-glucan] has well-evidenced health benefits and it contributes to the texture properties of foods. These functions are characteristically dependent on the excellent viscosity forming ability of this cell wall polysaccharide. The viscosity is affected by the molar mass, solubility and conformation of β-glucan molecule, which are further known to be altered during food processing. This study focused on demonstrating the degradation of β-glucan in water solutions following the addition of ascorbic acid, during heat treatments or high pressure homogenisation. Furthermore, the motivation of this study was in the non-enzymatic degradation mechanisms, particularly in oxidative cleavage via hydroxyl radicals. The addition of ascorbic acid at food-related concentrations (2-50 mM), autoclaving (120°C) treatments, and high pressure homogenisation (300-1000 bar) considerably cleaved the β-glucan chains, determined as a steep decrease in the viscosity of β-glucan solutions and decrease in the molar mass of β-glucan. The cleavage was more intense in a solution of native β-glucan with co-extracted compounds than in a solution of highly purified β-glucan. Despite the clear and immediate process-related degradation, β-glucan was less sensitive to these treatments compared to other water-soluble polysaccharides previously reported in the literature. In particular, the highly purified β-glucan was relatively resistant to the autoclaving treatments without the addition of ferrous ions. The formation of highly oxidative free radicals was detected at the elevated temperatures, and the formation was considerably accelerated by added ferrous ions. Also ascorbic acid pronounced the formation of these oxidative radicals, and oxygen was simultaneously consumed by ascorbic acid addition and by heating the β-glucan solutions. These results demonstrated the occurrence of oxidative reactions, most likely the metal catalysed Fenton-like reactions, in the β-glucan solutions during these processes. Furthermore, oxidized functional groups (carbonyls) were formed along the β-glucan chain by the treatments, including high pressure homogenisation, evidencing the oxidation of β-glucan by these treatments. The degradative forces acting on the particles in the high pressure homogenisation are generally considered to be the mechanical shear, but as shown here, carbohydrates are also easily degraded during the process, and oxidation may have a role in the modification of polysaccharides by this technique. In the present study, oat β-glucan was demonstrated to be susceptible to degradation during aqueous processing by non-enzymatic degradation mechanisms. Oxidation was for the first time shown to be a highly relevant degradation mechanism of β-glucan in food processing.

A computational study of Compton profiles of water - methanol solutions

The molecular level structure of mixtures of water and alcohols is very complicated and has been under intense research in the recent past. Both experimental and computational methods have been used in the studies. One method for studying the intra- and intermolecular bindings in the mixtures is the use of the so called difference Compton profiles, which are a way to obtain information about changes in the electron wave functions. In the process of Compton scattering a photon scatters inelastically from an electron. The Compton profile that is obtained from the electron wave functions is directly proportional to the probability of photon scattering at a given energy to a given solid angle. In this work we develop a method to compute Compton profiles numerically for mixtures of liquids. In order to obtain the electronic wave functions necessary to calculate the Compton profiles we need some statistical information about atomic coordinates. Acquiring this using ab-initio molecular dynamics is beyond our computational capabilities and therefore we use classical molecular dynamics to model the movement of atoms in the mixture. We discuss the validity of the chosen method in view of the results obtained from the simulations. There are some difficulties in using classical molecular dynamics for the quantum mechanical calculations, but these can possibly be overcome by parameter tuning. According to the calculations clear differences can be seen in the Compton profiles of different mixtures. This prediction needs to be tested in experiments in order to find out whether the approximations made are valid.

Syntheses and Self-Assembling Characteristics of Amphiphilic Star Diblock Copolymers

Uusien polymeeripohjaisten teknologioiden ja materiaalien myötä räätälöityjen polymeerien tarve on kasvanut. Viime vuosituhannen lopussa kehitetyt kontrolloidut polymerointimenetelmät ovat avanneet uusia mahdollisuuksia paitsi monimutkaisten polymeerien synteesiin, myös itsejärjestyvyyteen perustuvien funktionaalisten nanorakenteiden suunnitteluun ja valmistukseen. Nämä voivat jäljitellä luonnossa esiintyviä rakenteita, joita muodostavat esimerkiksi lipidit ja proteiinit. Itsejärjestyvät molekyylit ovat usein amfifiilisiä eli ne koostuvat hydrofiilisistä ja hydrofobisista osista ja polymeereissä nämä osat voivat olla omina lohkoinaan, jolloin puhutaan amfifiilisistä lohko- tai blokkikopolymeereistä. Riippuen järjestyneiden rakenteiden koostumuksesta ja muodosta, amfifiilisiä blokkikopolymeerejä on tutkittu tai jo käytetty nanoteknologiassa, elastomeereissä, voiteluaineissa, pinta-aktiivisina aineina, lääkkeenannostelussa, maaleissa, sekä elektroniikka-, kosmetiikka- ja elintarviketeollisuudessa. Tavallisimmin käytetyt amfifiiliset blokkikopolymeerit ovat olleet lineaarisia, mutta viime aikoina tutkimus on suuntautunut kohti monimutkaisempia rakenteita. Tällaisia ovat esimerkiksi tähtipolymeerit. Tähtimäisissä polymeereissä miselleille tyypillinen ydin-kuori-rakenne säilyy hyvin alhaisissakin polymeerikonsentraatioissa, koska polymeeriketjut ovat kiinni toisissaan yhdessä pisteessä. Siten ne ovat erityisen kiinnostavia tutkimuskohteita erilaisten hydrofobisten orgaanisten yhdisteiden sitomiseksi ja vapauttamiseksi. Tässä työssä on tarkasteltu amfifiilisten tähtipolymeerien itsejärjestymistä vesiliuoksissa sekä kokeellisesti ja tietokonesimulaatioin. Työ koostuu kahdesta osasta: tähtipolymeerien synteesistä makrosyklisillä initiaattoreilla ja amfifiilisten tähtimäisten blokkikopolymeerien ominaisuuksien tutkimisesta.

Organized Nanostructures of Thermoresponsive Poly(N-isopropylacrylamide) Block Copolymers Obtained Through Controlled RAFT Polymerization

Kontrolloidut radikaalipolymerointimenetelmät, kuten RAFT-polymerointi, ovat moderni tapa valmistaa polymeerejä säädellysti. RAFT-polymeroinnilla polymeerien ketjunpituutta, moolimassajakaumaa, mikrorakennetta (taktisuus, järjestys), koostumusta ja funktionaalisuutta kyetään hallitsemaan. Siten menetelmällä voidaan valmistaa uudenlaisia polymeeriarkkitektuureja, kuten blokki- ja tähtipolymeerejä, sekä hybridimateriaaleja ja biokonjugaatteja. Polymeeristen rakennuspalikoiden itsejärjestyminen, missä huolellisesti syntetisoidut polymeerit järjestyvät halutulla tavalla nanoskaalassa, on suosittu tutkimuskohde materiaalitieteessä. On huomattava, että blokkipolymeerien itsejärjestyminen on vielä suhteellisen nuori tutkimusaihe. Tämän hetkiset polymeeriset nanomateriaalit ovat suhteellisen yksinkertaisia luonnon luomuksiin verrattuina, tarjoten jatkuvasti uusia mahdollisuuksia seuraavan sukupolven polymeereille. Tässä työssä RAFT-polymeroinnilla syntetisoitiin amfifiilisiä di- ja triblokkikopolymeerejä sekä tutkittiin niiden järjestymistä nanorakenteiksi. Kaikissa blokkikopolymeereissä käytettiin lämpöherkkää poly(N-isopropyyliakryyliamidia). Siten polymeerit ja tutkitut materiaalit reagoivat lämpötilanmuutokseen ympäristössä eli ovat ns. ympäristöherkkiä. Työssä tutkittiin taktisuuden kontrollointia N-isopropyyliakryyliamidin RAFT-polymeroinnissa. Polymeerin taktisuutta sekä ketjunpituutta ja blokkijärjestystä säätämällä voitiin hallita polymeerin itsejärjestymistä vesiliuoksessa. Amfifiiliset polymeerit järjestyivät laimeissa vesiliuoksissa erilaisiksi misellirakenteiksi, muodostaen ns. mikrosäiliöitä. Tällaisilla polymeereillä odotetaan olevan sovelluksia esim. lääkeainevapautuksessa. Amfifiilejä käytetään myös esimerkiksi apuaineina pinnoitteissa ja kosmetiikassa. Kiinteässä tilassa tutkitut triblokkikopolymeerit muodostivat teoreettisesti ennustettuja morfologioita. Lämpöherkän materiaalin hydrogeelit toimivat suodatinmembraanina nanokokoluokassa. RAFT-polymeroinnilla syntetisoituja polymeereja voidaan sellaisenaan käyttää kultananopartikkeleiden päällystämiseen. Kultananopartikkelit ovat erittäin kiinostavia mm. niiden stabiilisuuden ja ainutlaatuisten pintaominaisuuksien vuoksi. Kun amfifiilisiä polymeerejä kiinnitettiin kultapartikkelin pinnalle, sen liuos- ja optisia ominaisuuksia voitiin säädellä pH:n ja lämpötilan avulla. Tällaisilla kultananopartikkeleilla on sovelluksia mm. diagnostiikassa, sensoreina ja solukuvauksessa.