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Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Lignocellulosic material is composed of three major macromolecule components i.e., cellulose, hemicelluloses and lignin. These components are chemically associated and directly linked to each other through covalent bonding which is scientifically denoted as lignin-carbohydrate complexes (LCCs) and their interaction is fundamentally important as to understand wood formation and reactivity during chemical and biological processing e.g. pulping and enzymatic hydrolysis. The association of lignin with polysaccharides (covalent linkages) has been surrounded by contradictions and controversy in several wood chemistry studies. These linkages exist in lignocellulosic materials from wood to herbaceous plants. In woody plants, they consist of ester and ether linkages through sugar hydroxyl to α-carbonyl of phenyl-propane unit on lignin. However, in herbaceous plants ferulic and p-coumaric acids are esterified to hemicelluloses and lignin respectively. In recent studies, the existence of the bonds has been shown by applying indirect analysis strategies which resulted to low yields and contaminations. The general aim of the current study was to isolate and fractionate LCCs from raw lignocellulosic materials (E. grandis and sugarcane bagasse) and corresponding processed materials (chemical pulps and water-insoluble residues (WIS)) in order to determine the chemical structure of the residual lignin associated with polysaccharides and how they affected industrial processing. The objective of the study is to compile a document that when the development of pulping and bio-ethanol bio-refinery will greatly depends on the detailed wood chemistry on how the components interact with each before and after hemicelluloses pre-extraction prior to pulping and steam explosion pre-treatment prior to enzymatic hydrolysis. The current study was focusing on understanding the effect LCCs isolated from two different industrial processing methods, i.e. pulping and enzymatic hydrolysis (EH). There were two lignocelluloses feedstocks used for pulping, i.e. Eucalyptus grandis and sugarcane bagasse whereas sugarcane bagasse was the only feedstock used for enzymatic hydrolysis. Hemicelluloses pre-extracted (mild alkali or dilute acid and autohydrolysis for sugarcane bagasse) pulps of Kraft or soda AQ from E. grandis and sugarcane bagasse were used to understand the effect of xylan pre-extraction prior to pulping on lignin-carbohydrate complexes has not been reported to the best knowledge of the primary author. Also prior to EH the material was subjected to two different treatment methods, i.e. steam explosion and ionic liquid fractionation in varying conditions. The study illustrated the types of extracted and fractionated LCCs from hemicelluloses pre-extracted pulps and WIS in comparison to the non-extracted pulps and reports from the literature. Lignin-carbohydrate complexes (LCCs) were isolated and fractionated by an inorganic method which yielded reasonable quantification quantities and no contamination and low yields for the hardwood compared to reports of using an enzymatic method. To the best knowledge of the authors, no work has been done on WIS material. The lignocelluloses were subjected to ball milling which was followed by a sequence of inorganic solvents swelling and dissolution into 2 fractions i.e. glucan-lignin and xylan-lignin-glucan. Characterisation of the isolated LCCs was made using a variety of analytical tools such as FTIR-PCA, HPLC, GPC and GC-MS. LCCs were evident when FTIR and HPLC studies were conducted. Residual lignin isolated from the lignocelluloses was assumed to be chemically bonded to carbohydrates and mostly to xylan. Approximately 60% and 30% of the lignin was linked to xylan while for the second and first fractions respectively. It is reported that lignin associated with xylan is more resistant and reduce the delignification process than when linked to glucan that is easily hydrolysable. With the FTIR and GPC analyses of LCC fractions, it was evident that the ester bonds of LCCs were destroyed through pre-extraction and pre-treatment, where this resulted to more cellulose being more accessible to alkaline pulping and enzymatic hydrolysis respectively. The linkages were either partially broken down or completely destroyed leading to significant changes of chemical structures. The polydispersity of the LCCs assisted in determining the structure of lignin, either existing as monolignols on the surfaces of fibres or a as complex two or three-dimensional structure that is linked to carbohydrates as the Mw increased or decreased. In general, these findings may have an important implication for the overall efficiency on bio-refinery. The molecular weights (Mw) of the extracted LCCs were measured by gel permeation chromatography. From the chromatograms, it was observed that the materials that were subjected to pre-processing prior to further processing, the Mw shifted to lower Mws regions. It was found that LCCs isolated from mild alkali pre-extracted pulps had high lignin syringyl to guaiacyl lignin contents than LCCs isolated from dilute acid pre-extracted pulps. High syringyl/guaiacyl ratio (S/G ratio) was an indication of low lignin content as a result of processing which will result to high product yields after downstream processing. The 5 average S/G ratio for the pulps from E. grandis and sugarcane bagasse was ranging between 1.1 to 19.01 and 1.4 to 18.16 respectively, while for the WIS-material generated from ionic liquid fractionated and steam exploded materials ranged from 3.29 to 9.27 and 3.5 to 13.3 respectively. The S/G ratios of the LCCs extracted from E. grandis and sugarcane bagasse pulps ranged from 0.42 to 2.39 and 0.041 to 0.31 was respectively while for the LCCs extracted from water-insoluble-solids (WIS) material generated from steam exploded material was from 4.87 to 10.40. The determination of S/G ratio is recommended for the LCC extraction and characterisation study as an evaluation of residual lignin in processed materials such as pulps and WIS. The obtained saccharifications were low, possibly due to the severity of the steam explosion pre-treatment and ionic liquid fractionation conditions which resulted on high accumulation of acetic acid and increased in cellulose crystallinity respectively. From quantitative analysis of the LCCs perspective it could be concluded that free lignin was present in mild alkali pre-extracted pulps than for the dilute acid pre-extracted pulps. / AFRIKAANSE OPSOMMING: Cellulose materiaal is saamgestel uit drie groot makromolekule komponente naamlik, sellulose, hemisellulose en lignien. Hierdie komponente is chemies verwante en direk met mekaar verbind deur kovalente binding wat wetenskaplik aangedui as lignien-koolhidraat komplekse (LCCs) en hul interaksie is fundamenteel belangrik as hout vorming en reaktiwiteit tydens chemiese en biologiese verwerking bv om te verstaan verpulping en ensiematiese hidrolise. Die vereniging van lignien met polisakkariede (kovalente verbindings) is omring deur teenstrydighede en omstredenheid in verskeie hout chemie studies. Hierdie skakeling bestaan in cellulose materiaal uit hout te kruidagtige plante. In houtagtige plante, hulle bestaan uit ester en eter bindings deur suiker hidroksiel te α-karboniel van feniel-propaan eenheid op lignien. Maar in kruidagtige plante ferulic en p-coumaric sure veresterd te hemisellulose en lignien onderskeidelik. In onlangse studies, het die bestaan van die bande is getoon deur die toepassing van indirekte analise strategieë wat gelei tot lae opbrengste en kontaminasie. Die algemene doel van die huidige studie was om te isoleer en fraksioneer LCCs van rou cellulose materiaal (E. grandis en suikerriet bagasse) en die ooreenstemmende verwerkte materiaal (chemiese pulp en water-oplosbare residue (WIS)) ten einde die chemiese struktuur van die te bepaal oorblywende lignien wat verband hou met polisakkariede en hoe hulle geaffekteerde industriële verwerking. Die doel van die studie is 'n dokument op te stel dat wanneer die ontwikkeling van verpulping en bio-etanol bio-raffinadery sal grootliks afhang van die gedetailleerde hout chemie oor hoe om die komponente met mekaar voor en na hemisellulose pre-onttrekking voor verpulping en stoom ontploffing pre-behandeling voor ensiematiese hidrolise. Die huidige studie was die fokus op die begrip van die effek LCCs geïsoleerd van twee verskillende industriële verwerking, maw verpulping en ensiematiese hidrolise (EH). Daar was twee lignocelluloses voerstowwe gebruik vir verpulping, dws Eucalyptus grandis en suikerriet bagasse terwyl suikerriet bagasse was die enigste grondstof gebruik vir ensiematiese hidrolise. Hemisellulose pre-onttrek (ligte alkali of verdunde suur en autohydrolysis vir suikerriet bagasse) pulp van Kraft of soda AQ van E. grandis en suikerriet bagasse is gebruik om die effek van Xylan pre-onttrekking te voor verstaan verpulping op lignien-koolhidraat komplekse het nie aan die berig is beste kennis van die primêre outeur. Ook voor EH die materiaal is onderworpe aan twee verskillende behandeling metodes, naamlik stoom ontploffing en ioniese vloeistof fraksionering in wisselende toestande. Die studie geïllustreer die tipes onttrek en gefractioneerd LCCs van hemisellulose pre-onttrek pulp en WIS in vergelyking met die nie-onttrek pulp en verslae van die literatuur. Lignien-koolhidraat komplekse (LCCs) is geïsoleer en gefraksioneer deur 'n anorganiese metode wat redelike kwantifisering hoeveelhede en geen besoedeling en lae opbrengste opgelewer vir die hardehout vergelyking met verslae van die gebruik van 'n ensiematiese metode. Na die beste kennis van die skrywers, het geen werk op WIS materiaal gedoen. Die lignocelluloses is onderworpe aan die bal maal wat gevolg is deur 'n reeks van anorganiese oplosmiddels swelling en ontbinding in 2 breuke dws glucan-lignien en Xylan-lignien-glucan. Karakterisering van die geïsoleerde LCCs is gemaak met behulp van 'n verskeidenheid van analitiese gereedskap soos FTIR-PCA, HPLC, GPC en GC-MS. LCCs was duidelik wanneer FTIR en HPLC studies is uitgevoer. Residuele lignien geïsoleerd van die lignocelluloses is aanvaar moet word chemies gebind aan koolhidrate en meestal te xylan. Ongeveer 60% en 30% van die lignien is gekoppel aan xylan terwyl dit vir die tweede en eerste breuke onderskeidelik. Dit is gerapporteer dat lignien wat verband hou met Xylan is meer bestand en die delignification proses as wanneer gekoppel aan glucane wat maklik hidroliseerbare verminder. Met die FTIR en GPC ontledings van LCC breuke, was dit duidelik dat die ester bande van LCCs is deur pre-ontginning en pre-behandeling, waar dit gelei tot meer sellulose om meer toeganklik te alkaliese verpulping en ensiematiese hidrolise onderskeidelik vernietig. Die skakeling is óf gedeeltelik afgebreek of heeltemal vernietig lei tot beduidende veranderinge van chemiese strukture. Die polydispersity van die LCCs bygestaan in die bepaling van die struktuur van lignien, hetsy bestaande as monolignols op die oppervlak van die vesel of 'n as komplekse twee of drie-dimensionele struktuur wat gekoppel is aan koolhidrate as die Mw vermeerder of verminder. In die algemeen, kan hierdie bevindinge het 'n belangrike implikasie vir die algehele doeltreffendheid op bio-raffinadery. Die molekulêre gewigte (Mw) die onttrek LCCs gemeet deur gelpermeasie- chromatografie. Van die chromatograms, was dit opgemerk dat die materiaal wat blootgestel is aan die pre-verwerking voor verdere verwerking, die Mw verskuif MWS streke te verlaag. Daar is gevind dat LCCs geïsoleerd van ligte alkali pre-onttrek pulp het hoë lignien syringyl lignien inhoud as LCCs geïsoleerd van verdunde suur vooraf onttrek pulp te guaiacyl. Hoë syringyl / guaiacyl verhouding (S/G-verhouding) was 'n aanduiding van 'n lae lignien inhoud as 'n resultaat van verwerking wat sal lei tot 'n hoë produk opbrengste ná stroomaf verwerking. Die gemiddelde S/G-verhouding vir die pulp van E. grandis en suikerriet bagasse was wat wissel tussen 1,1-19,01 en 1,4-18,16 onderskeidelik, terwyl dit vir die WIS-materiaal gegenereer uit ioniese vloeistof gefraksioneer en stoom ontplof materiaal het gewissel 3,29-9,27 en 3.5 13,3 onderskeidelik. Die S/G verhoudings van die LCCs onttrek uit E. grandis en suikerriet bagasse pulp gewissel 0,42-2,39 en ,041-,31 was onderskeidelik terwyl dit vir die LCCs onttrek uit water-oplosbare-vastestowwe (WIS) materiaal gegenereer uit stoom ontplof materiaal was van 4,87-10,40. Die bepaling van S/G-verhouding word aanbeveel vir die LCC ontginning en karakterisering studie as 'n evaluering van die oorblywende lignien in verwerkte materiaal soos pulp en WIS. Die verkry saccharifications was laag, moontlik as gevolg van die erns van die stoom ontploffing pre-behandeling en ioniese vloeistof fraksionering voorwaardes wat gelei op 'n hoë opeenhoping van asynsuur en vermeerder in sellulose kristalliniteit.

Thesis (MScAgric) -- Stellenbosch University, 2003. / ENGLISH ABSTRACT: Cellulose is by far the most abundant carbohydrate available from plant biomass. These biopolymers are therefore an important renewable source of food, fuels and chemicals. Cellulose is embedded in a matrix of hemicellulose, lignin and pectin and is composed of repeating glucose units linked by p-1,4-glycosidic bonds. The individual molecules are held together by hydrogen bonds, forming largely crystalline fibres. The hemicellulose, which is a low molecular weight heteropolysaccharide, coats and binds the cellulose microfibrils, preventing the cellulose from becoming too crystalline. Three predominant types of hemicelluloses are recognised, namely 1,3- and 1,4-p-D-galactans, 1,4-p-D-mannans and 1,4-p-D-xylans, which are named according to the sugar type that forms the polymer backbone. Pectic substances contain rhamnogalacturonan backbones in which 1,4-linked a-D-galacturonan chains are interrupted at intervals with a-L-rhamnopyranosyl residues carrying neutral side chains. Two groups of enzymes, cellulases and pectinases, are required for the microbial utilisation of crystalline cellulose and pectin. Cellulases are multicomponent complexes that are often composed of endoglucanases, exoglucanases and cellobiases. Cellobiose is the major end product of concerted endoglucanase and exoglucanase activity. Cellobiose is then hydrolysed to glucose by p-glucosidases. The enzymatic breakdown of pectic polymers occurs by the deesterifying action of the saponifying enzymes, pectinesterase, releasing the methyl groups of the pectin molecule, and by hydrolase or lyase action of the depolymerases (pectin lyase, pectate lyase and polygalacturonase), splitting the a- 1.4-glycosidic linkages in the polygalacturonate chain. The yeast Saccharomyces cerevisiae has been used extensively in the alcoholic beverage industry for fermentations of wine, beer and other alcoholic beverages for many years. However, it is unable to produce extracellular depolymerising enzymes that can efficiently degrade polysaccharides, which are the main cause of clarification and filtration problems. Enzyme preparations have been used in the alcoholic beverage industries to degrade haze-forming polysaccharides, thereby improving the filterability and quality of products such as beer and wine. An alternative would be to develop S. cerevisiae strains that produce extracellular polysaccharidases, enabling the yeast to degrade polysaccharides without the addition of commercial enzyme preparations. These strains can also be very useful in improving the quality of wine, as well as cutting the costs of the winemaking process. The objective of this study was to investigate the effects of two transformed S. cerevisiae strains on different wine grape varieties. The following genes have been cloned and characterised previously: the Aspergillus niger endo-p-xylanase gene (xynC), the Butyrivibrio fibrisolvens endo-|3- 1.4-glucanase gene (endl), the Erwinia chrysanthemi pectate lyase gene (pelE) and the Erwinia carotovora polygalacturonase gene (p e h l). The yeast alcohol dehydrogenase I gene promoter (ADH1p), the alcohol dehydrogenase II gene terminator (ADH2j), the tryptophan synthase gene terminator (TRP5r) and the yeast mating-type pheromone a-factor secretion signal sequence (MFcrfs) were used to compile the following gene constructs: ADH1 p-MFa1 s-end1-TRP5r (designated END1), A DH1 p-xyn C-A DH2T (designated XYN4), ADH1 p-MFa1 s-peh1 -TRP5t (designated PEH1) and ADH1 p-MFa1 s-pelE-TRP5r (designated PELE). Two yeast integrating plasmids were constructed, one containing the END1 and XYN4 gene cassettes and the other containing the PEH1-PELE cassette. These two plasmids were then integrated into the URA3 locus of two separate industrial wine yeast strains of S. cerevisiae. To facilitate selection of the industrial yeast transformants in the absence of auxotrophic markers, the integrating plasmid containing the END1 and XYN4 gene cassettes was issued with the dominant selectable Geneticin G418-resistance {G f) marker. The integrating plasmid harbouring the PEH1-PELE gene cassette was issued with the dominant selectable sulphumetronmethyl resistance (SMR1) marker. The introduction of these plasmids into commercial wine yeast strains directed the synthesis of END1, XYN4, PELE and PEFI1 transcripts and the production of extracellular biologically active endo-P-1,4- glucanase, endo-(3-xylanase, pectate lyase and polygalacturonase. These recombinant yeasts were capable of extracting more colour from grape skins of certain varieties, as well as leading to more freeflow wine as a result of the more effective degradation of glucans, xylans and pectins in the skins. They also led to decreased turbidity in the wine, making it more filterable. Future work will entail further investigation of the effects of these recombinant yeasts on different white and red wine grape varieties. Another objective of this study was to screen non-Saccharomyces wine yeasts for the production of extracellular hydrolytic enzymes. The reason for this part of the thesis was to determine the types of extracellular hydrolytic enzymes that are produced and to determine which genera produce which kinds of extracellular enzymes. A total of 237 yeast isolates, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and Kluyveromyces, were screened for the production of extracellular pectinases, proteases, (3-glucanases, lichenases, p-glucosidases, cellulases, xylanases, amylases and sulphite reductase activity. These yeasts were all isolated from grapes and clarified grape juice to ensure that they were yeasts found in must during the initial stages of fermentation. This information can be used to pave the way to pinpoint the specific effects in wine of these enzymes produced by the so-called wild yeasts associated with grape must. This information can also be used to transform Saccharomyces wine yeasts with some of the genes from these non-Saccharomyces yeasts for the production of extracellular hydrolytic enzymes. However, future research will have to be done to determine the extent of the activity of these enzymes in wine fermentations and to obtain better knowledge of the physiological and metabolical features of non-Saccharomyces yeasts. / AFRIKAANSE OPSOMMING: Sellulose is verreweg die volopste koolhidraat in plantbiomassa. Hierdie biopolimere is dus ‘n baie belangrike hernubare bron van voedsel, brandstof en chemikaliee. Sellulose is in 'n matriks van hemisellulose, lignien en pektien gebed en is uit herhaalde glukose eenhede, wat deur middel van (3-1,4-glukosidiese bindings geheg is, saamgestel. Die individuele molekules word deur waterstofbindings aan mekaar geheg, wat aanleiding gee tot die vorming van kristallyne vesels. Die hemisellulose, wat 'n lae molekulere gewig heteropolisakkaried is, bedek en bind die sellulose vesels en verhoed daarmee die vorming van vesels wat te kristallyn is. Drie predominante tipes hemisellulose word herken en sluit 1,3- en 1,4-p-D-galaktane, 1,4-p-D-mannane en 1,4-p-D-xylane in, wat vernoem word volgens die suikereenhede wat die polimeerruggraat vorm. Pektiene bestaan uit 'n rhamnogalakturonaanruggraat waarin 1,4-gekoppelde a-D-galakturonaankettings periodiek met a-L-rhamnopiranosiel residue, bevattende neutrale sykettings, onderbreek word. Twee groepe ensieme, nl. pektinase en sellulase, word deur mikrobes vir die benutting van kristallyne pektinase en sellulase vereis. Sellulase is multikomponent komplekse wat dikwels uit endoglukanase, ekso-glukanase en sellobiase saamgestel is. Sellobiose is die hoof eindproduk van die saamgestelde aktiwiteit tussen endoglukanase en ekso-glukanase en word verder gehidroliseer tot glukose deur |3-glukosidases. Die ensimatiese afbraak van pektien polimere vind deur die de-esterifiserings aksie van die versepings ensiem, pektienesterase, plaas. Dit lei tot die vrystelling van die metielgroepe van die pektienmolekuul. Deur die hidrolase of liase aksie van die depolimerase (pektien liase, pektaatliase en poligalakturonase), split die a-1,4-glukosidiese verbindings in die poligalakturonaatketting. Die gis Saccharomyces cerevisiae word al vir jare ekstensief in die alkoholbedryf vir die fermentasie van verskeie produkte, veral druiwe, gebruik. S. cerevisiae besit egter nie die vermoe om ekstrasellulere depolimiserende ensieme wat vir die effektiewe degradasie van polisakkariede verantwoordelik is, te produseer nie, wat die hoof oorsaak van die verhelderings- en filtreringsprobleme in onder andere wyn en bier is. Dit veroorsaak ook dat S. cerevisiae nie oor die vermoe beskik om waasvormende polisakkariede in wyn te degradeer nie. Tans word ensiempreparate in die alkoholiese bedryf vir die degradasie van die probleem polisakkariede gebruik. Sodoende word die filtreerbaarheid en kwaliteit van wyn en bier verbeter. ‘n Goeie alternatief is die ontwikkeling van S. cerevisiae-rasse wat oor die vermoe beskik om ekstrasellulere polisakkarase te produseer en dus polisakkariede self sonder die byvoeging van eksterne kommersiele ensiempreparate te degradeer. Hierdie rasse sal baie voordelig wees vir die verbetering van wynkwaliteit, sowel as vir die vermindering van die kostes verbonde aan die wynmaakproses. Die objektief van hierdie studie is dus om die uitwerking van twee getransformeerde S. cerevisiae rasse, wat ekstrasellulere polisakkarases produseer, op verskillende wyndruifvarieteite na te vors. Die volgende gene is reeds voorheen gekloneer en gekarakteriseer: die endo-pxylanase- geen (xynC) van Aspergillus niger, die endo-p-1,4-glukanase-geen (endl) van Butyrivibrio fibrisolvens, die pektaatliase-geen (pe/E) van Erwinia chrysanthemi en die poligalakturonase-geen (p e h l) van Erwinia carotovora. Die alkoholdehidrogenase-geenpromotor (ADH1P), die alkoholdehidrogenase IIgeentermineerder (ADH2T), die gistriptofaansintase geen se termineerder (TRP5t) en die sekresiesein van die gisferomoon a-faktor (MFa1s) is gebruik om die volgende geenkonstrukte saam te stel: ADH1 p-MFa1 s-end1 -TRP5t (toekend as END1), ADH1 p-xynC-ADH2T (bekend as XYN4), ADH1 p-MFa1 s-peh1-TRP5T fbekend as PEH1), and ADH1 p-MFa1 s-pelE-TRP5T (bekend as PELE). Twee gisintegrerings plasmiede is gekonstrueer, een wat die END1- en XYN4- geenkassette bevat en die ander wat die PEH1-PELE-kasset besit. Hierdie twee plasmiede is daarna in twee aparte industriele wyngisrasse van S. cerevisiae by die URA3 lokus geintegreer. Vir die seleksie van die industriele wyngistransformante in die afwesigheid van ouksotrofiese merkers, is die dominante selekteerbare Geneticin G418 weerstandbiedende (G f) merker in die END1- en XYA/4-geenkassetbevattende plasmied geintegreer. Die dominante selekteerbare sulfumetronmetielweerstandbiedende (SMR1) merker is in die integreringsplasmied, wat die PEH1- PELE-geenkasset bevat, geintegreer vir seleksie. Transformasie van hierdie plasmiede in kommersiele wyngisrasse het tot die direkte sintese van die END1-, XYN4-, PELE- en PEH1-transkripte aanleiding gegee, sowel as tot die produksie van die biologies aktiewe ekstrasellulere endo-P-1,4-glukanase, endo-P-xylanase, pektaatliase en poligalaturonase. Tydens die wynmaakproses het bogenoemde rekombinante giste aanleiding gegee tot verhoogde kleurekstraksie uit die druifdoppe van sekere varieteite, asook tot verhoogde vryvloei wyn. Dit is verkry deur die effektiewe degradasie van die glukane, xilane en pektiene in die doppe. Die rekombinante giste het ook verlaagde turbiditeit in die wyn tot gevolg gehad, wat die wyne makliker filtreerbaar maak. Hierdie werk was net die eerste stap. In die toekoms sal verdere navorsing gedoen moet word om die presiese effekte van hierdie rekombinante giste op verskillende rooi en wit druifvarieteite te bepaal. ‘n Ander fokus van hierdie tesis was om nie-Saccharomyces wyngiste vir die produksie van ekstrasellulere hidrolitiese ensieme te selekteer. Die rede hiervoor is om te bepaal watter tipes ekstrasellulere hidrolitiese ensieme geproduseer word, asook watter ensieme deur watter genera geproduseer word, ‘n Totaal van 237 gisisolate wat tot die generas Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora en Kluyveromyces behoort, is vir die produksie van ekstrasellulere pektinase, protease, p-glukanase, lichenase, p-glukosidase, sellulase, xilanase, amilase en sulfiet reduktase-aktiwiteit getoets. Hierdie giste is almal vanaf druiwe en druiwesap geVsoleer om te verseker dat dit wel giste is wat gedurende die beginfases van fermentasie in die mos teenwoordig is. Hierdie inligting kan nou verder gebruik word om die spesifieke effekte wat hierdie ensieme, wat deur die sogenaamde wilde giste geproduseer word, tydens die beginfases van fermentasies op die mos het, te bepaal. Hierdie inligting kan ook in die toekoms gebruik word om Saccharomyces-wyngiste met gene van die ri\e-Saccharomycesgiste te transformeer om ekstrasellulere hidrolitiese ensieme vir die degradasie van die problematiese polisakkariede in wyn te produseer. Daar sal egter in die toekoms baie navorsing gedoen moet word om die omvang van hierdie ensiemaktiwiteite in wynfermentasies te bepaal, asook om meer kennis te bekom oor die fisiologiese en metaboliese samestelling van nie-Saccfraromyces wyngiste.

PAE, CAPES / Neste estudo, o amido de mandioca foi reticulado com o trimetafosfato de sódio (TMFS). Nesta matriz, foi incorporado o fármaco diclofenaco de sódio, onde se avaliou sua cinética de liberação, com o objetivo de verificar a possibilidade de empregar as micropartículas de amido de mandioca reticulado com TMFS na liberação controlada de fármacos. Empregou-se um delineamento experimental onde testou-se duas concentrações de amido (-1 = 5%; 1= 10%), duas concentrações de reticulante (-1 = 15%; 1 = 30%) e dois tempos (-1 = 2; 1 = 8 horas), além de quatro repetições para o ponto central que correspondeu a concentração de amido 7,5%, reticulante 22,5% e tempo de 5 horas, em um total de doze experimentos. Verificou-se que o único fator que influenciou significativamente a um nível de confiança de 90% na incorporação do diclofenaco de sódio foi à concentração do amido, sendo a incorporação maior quando a concentração de amido se aproxima de 10%. Selecionou-se a amostra que incorporou a maior massa de diclofenaco de sódio de cada grupo com concentração diferente de amido (A5R15t2; A10R30t2 e A7,5R22,5t5 4) para a caracterização por espectroscopia de infravermelho, análise térmica, densidade de reticulação, microscopia eletrônica de varredura e também para o estudo cinético de liberação. Verificou-se que a amostra que apresentou a maior reticulação (A5R15t2) também incorporou mais diclofenaco de sódio, a amostra A7,5R22,5t5 apresentou a maior quantidade de grupamentos fosfato não ligados (reticulação não efetiva) e apresentou a menor quantidade de diclofenaco de sódio incorporado. As amostras reticuladas apresentaram uma resistência térmica maior em comparação ao amido puro, as amostras reticuladas e com diclofenaco de sódio incorporado apresentaram resistência térmica superior ao amido puro, porém inferior às amostras de amido reticulado. Os modelos cinéticos que melhor representaram a liberação do fármaco foram: primeira ordem, Peppas e Higuchi, para o modelo de Peppas calculou-se o coeficiente de difusão (n) do sistema, obtendo-se valores de 0,14; 0,26 e 0,12 para as amostras A5R15t2DS; A7,5R22,5t5DS e A10R30t2DS respectivamente, estes valores são diferentes dos referenciados na literatura, atribui-se a variação aos diferentes tamanhos de partícula obtidos e difícil solubilidade do diclofenaco de sódio, características confirmadas pela microscopia eletrônica de varredura. / In this study, the cassava starch was crosslinked with sodium trimetaphosphate (TMFS). In this matrix was incorporated the sodium diclofenac drug, where it was evaluated its release kinetics, with the objective of verifying the possibility of employing the cassava starch microparticules crosslinked with TMFS in controlled release of drugs. It was used an experimental design which tested two concentrations of starch (-1 = 5%; 1 = 10%), two concentrations of crosslinkers (-1 = 15%; 1 = 30%) and two periods of time (-1= 2; 1 = 8 hours) besides, four repetitions for the main point which corresponds to concentration of starch 7,5%, crosslinkers 22,5% and time of 5 hours, a total of twelve experiments. It was found that the only factor that significantly influenced in a confidence level of 90% in incorporation of sodium diclofenac was the concentration of starch, the incorporation is greater when the starch concentration approaches 10%. It was selected the sample which incorporated the greatest amount of sodium diclofenac in each group with a different concentration of starch (A5R15t2; A10R30t2 and A7,5R22,5t5 4) for characterization by infrared spectroscopy, thermal analysis, cross-linking density, scanning electronic microscopy and also for the kinetic study of the release. It was verified that the sample with the highest crosslinking (A5R15t2) also incorporated more sodium diclofenac, the A7,5R22,5t5 sample showed the highest amount of phosphate groups not linked (no effective crosslinking) and had the lowest amount of sodium diclofenac incorporated. The crosslinked samples presented a higher thermal resistance compared to pure starch, crosslinked samples with the sodium diclofenac had higher thermal stability than pure starch, but lower than the samples crosslinked starch. The kinetic models that best represented the release of the drug were: first-order, Peppas and Higuchi, for Peppas model it was calculated the diffusion coefficient (n) of the system, obtaining values of 0,14; 0,26 and 0,12 for samples A5R15t2DS; A7,5R22,5t5DS and A10R30t2DS respectively, these values are different from those reported in the literature, the variation is attributed to the different particle sizes obtained and difficult solubility of sodium diclofenac, aspects confirmed by scanning electronic microscopy.

La faible stabilité thermique et les problèmes d’agrégations irréversibles limitent l’utilisation de nanocomposites polymères à renfort cellulosique. Dans ce contexte, des CNC thermiquement stable et fortement dispersés ont été préparés par des procédés verts, basés sur des méthodes en milieu aqueux, telle que l'adsorption physique et la modification de surface. Ces deux types de CNC extrudables ont été utilisés comme renfort dans des polymères réputés hydrophobes. Les composites biosourcés à matrice polymère ainsi réalisés sont caractérisés par une amélioration du module visqueux, de la résistance à la traction et du module d’Young. On constate également sur les images de microscopie électronique à balayage, qu’à la différence des observations réalisées avec les CNC non traités, qu’aucun micro-agrégat cellulosique n’est observé dans la matrice polymère. Ces deux méthodes, développées en milieu aqueux, apparaissent ainsi comme des solutions industriellement viables. / The low thermal stability and irreversible agglomeration issues are limiting to process the polymer nanocomposites using CNC as the reinforcing phase. In this context, thermally stable and highly dispersed CNC were prepared by green process (Aqueous based methods) like physical adsorption and surface modification. These two different Extrudable CNC was reinforced in to hydrophobic polymers. Ensuing polymer nanocomposites had a positive impact on the storage modulus, tensile strength, Young’s modulus. Importantly, no evidence of micro aggregates in the matrix was observed in the scanning electron microscopy images contrary to non-treated CNC. Both the surface modification and adsorption are the water based methods and is an industrially viable solution. Also, it can be applicable at industrial level.

CNPq / A lasiodiplodana é uma β-glucana exocelular que apresenta funcionalidades biológicas como atividade antioxidante, antiproliferativa, hipocolesterolêmica, atividade protetora contra danos no DNA induzidos por doxorubicina e atividade hipoglicêmica. A derivatização química de macromoléculas polissacarídicas tem sido considerada como um mecanismo potencializador de bioatividade. Neste cotexto, o presente trabalho propôs a derivatização da lasiodiplodana por acetilação. Anidrido acético foi utilizado como agente derivatizante e piridina como catalisador e meio reacional. Os derivados obtidos foram avaliados quanto a hidrossolubilidade, grau de substituição (DS), potencial antioxidante, e caracterizados por espectroscopia no infravermelho (FT-IR), análise termogravimétrica, calorimetria diferencial de varredura, difratometria de raios X e microscopia eletrônica de varredura. Derivados acetilados com diferentes graus de substituição (1,26; 1,03; 0,66 e 0,48) foram obtidos, havendo correlação entre a concentração do agente derivatizante e o DS. A análise de espectroscopia FT-IR confirmou a inserção de grupos acetil nas biomacromoléculas derivatizadas (LAS-AC) através de bandas especificas referentes a grupo carbonila (C=O) e aumento na vibração C-O. Análise por MEV indicou que a lasiodiplodana nativa apresenta estrutura morfológica na forma de filme fino, com aspecto translucido e dobras ao longo de sua extensão. A derivatização levou a mudanças morfológicas no polímero, incluindo aspectos de espessura, translucidez e aglomeração do filme. A análise térmica indicou que a amostra nativa e o derivado com DS 0,48 apresentaram três estágios de perda de massa. O primeiro estágio ocorreu até 125 °C (perda de água) e houve dois eventos consecutivos de perda de massa (200 °C - 400 °C) atribuídos a degradação da molécula. As amostras com DS 1,26; 1,03 e 0,66 apresentaram quatro estágios de perda de massa. O primeiro estagio ocorreu até 130 °C (perda de água), na sequencia dois eventos consecutivos de perda de massa (200°C – 392 °C) atribuídos a degradação do biopolímero foram verificados. O quarto estágio ocorreu entre 381 °C e 532 °C (decomposição final) com picos exotérmicos entre 472 °C e 491 °C. Os padrões da difração de raios X demonstraram que a lasiodiplodana nativa e acetilada apresentam estrutura amorfa com regiões semicristalina. A derivatização não contribuiu para a solubilidade da macromolécula mas potencializou sua capacidade antioxidante. A acetilação da lasiodiplodana permitiu a obtenção de uma nova biomacromolécula com maior potencial antioxidante do que a molécula nativa e com propriedades tecnológicas aplicáveis em diversos setores industriais. / Lasiodiplodan is an exocellular β-glucan with biological functionalities such as antioxidant, antiproliferative, hypocholesterolemic, protective activity against DNA damage induced by doxorubicin and hypoglycemic activity. Chemical derivatization of polysaccharide macromolecules has been considered as a potentiating mechanism for bioactivity. In this context, this work proposes the derivatization of lasiodiplodan by acetylation. Acetic anhydride was used as derivatizing agent and pyridine as catalyst and reaction medium. The derivatives obtained were evaluated by its water solubility, degree of substitution (DS), antioxidant potential, and characterized by infrared spectroscopy (FT-IR), thermal analysis, differential scanning calorimetry, X-ray diffraction and scanning electron microscopy. Acetylated derivatives with different degrees of substitution (1.26; 1.03; 0.66 and 0.48) were obtained, and there was correlation between the concentration of derivatizing agent and DS. FT-IR spectroscopy analysis confirmed the insertion of acetyl groups into derivatized macromolecules (LAS-AC) through of specific bands concerning to carbonyl group (C = O) and increase in C-O vibration. SEM analysis indicated that native lasiodiplodan presents morphological structure in the form of thin films with translucent appearance and folds along its length. Derivatization led to morphological changes in the polymer, including aspects thickness, translucency and agglomeration. Thermal analysis indicated the native sample and derivative with DS 0.48 presented three weight loss stages. The first stage occurred until 125 ° C (loss of water) and there were two consecutive events of weight loss (200 ° C - 400 ° C) attributed to molecule degradation. Samples with DS 1.26; 1.03 and 0.66 demonstrated four weight loss stages. The first stage occurred until 130 ° C (loss of water), following by two consecutive events of weight loss (200 ° C - 392 ° C) attributed to degradation of the biopolymer. The fourth stage was between 381 ° C and 532 ° C (final decomposition) with exothermic peaks between 472 ° C and 491 ° C. X-ray diffraction patterns showed that native and acetylated lasiodiplodan have amorphous structure with semicrystalline regions. Derivatization did not contribute to increased solubility of the macromolecule, but potentiated its antioxidant capacity. Acetylation of lasiodiplodan allowed to obtaining a new macromolecule with higher antioxidant potential than the native molecule and with technological properties applicable in various industrial sectors.

CNPq / A lasiodiplodana é uma β-glucana exocelular que apresenta funcionalidades biológicas como atividade antioxidante, antiproliferativa, hipocolesterolêmica, atividade protetora contra danos no DNA induzidos por doxorubicina e atividade hipoglicêmica. A derivatização química de macromoléculas polissacarídicas tem sido considerada como um mecanismo potencializador de bioatividade. Neste cotexto, o presente trabalho propôs a derivatização da lasiodiplodana por acetilação. Anidrido acético foi utilizado como agente derivatizante e piridina como catalisador e meio reacional. Os derivados obtidos foram avaliados quanto a hidrossolubilidade, grau de substituição (DS), potencial antioxidante, e caracterizados por espectroscopia no infravermelho (FT-IR), análise termogravimétrica, calorimetria diferencial de varredura, difratometria de raios X e microscopia eletrônica de varredura. Derivados acetilados com diferentes graus de substituição (1,26; 1,03; 0,66 e 0,48) foram obtidos, havendo correlação entre a concentração do agente derivatizante e o DS. A análise de espectroscopia FT-IR confirmou a inserção de grupos acetil nas biomacromoléculas derivatizadas (LAS-AC) através de bandas especificas referentes a grupo carbonila (C=O) e aumento na vibração C-O. Análise por MEV indicou que a lasiodiplodana nativa apresenta estrutura morfológica na forma de filme fino, com aspecto translucido e dobras ao longo de sua extensão. A derivatização levou a mudanças morfológicas no polímero, incluindo aspectos de espessura, translucidez e aglomeração do filme. A análise térmica indicou que a amostra nativa e o derivado com DS 0,48 apresentaram três estágios de perda de massa. O primeiro estágio ocorreu até 125 °C (perda de água) e houve dois eventos consecutivos de perda de massa (200 °C - 400 °C) atribuídos a degradação da molécula. As amostras com DS 1,26; 1,03 e 0,66 apresentaram quatro estágios de perda de massa. O primeiro estagio ocorreu até 130 °C (perda de água), na sequencia dois eventos consecutivos de perda de massa (200°C – 392 °C) atribuídos a degradação do biopolímero foram verificados. O quarto estágio ocorreu entre 381 °C e 532 °C (decomposição final) com picos exotérmicos entre 472 °C e 491 °C. Os padrões da difração de raios X demonstraram que a lasiodiplodana nativa e acetilada apresentam estrutura amorfa com regiões semicristalina. A derivatização não contribuiu para a solubilidade da macromolécula mas potencializou sua capacidade antioxidante. A acetilação da lasiodiplodana permitiu a obtenção de uma nova biomacromolécula com maior potencial antioxidante do que a molécula nativa e com propriedades tecnológicas aplicáveis em diversos setores industriais. / Lasiodiplodan is an exocellular β-glucan with biological functionalities such as antioxidant, antiproliferative, hypocholesterolemic, protective activity against DNA damage induced by doxorubicin and hypoglycemic activity. Chemical derivatization of polysaccharide macromolecules has been considered as a potentiating mechanism for bioactivity. In this context, this work proposes the derivatization of lasiodiplodan by acetylation. Acetic anhydride was used as derivatizing agent and pyridine as catalyst and reaction medium. The derivatives obtained were evaluated by its water solubility, degree of substitution (DS), antioxidant potential, and characterized by infrared spectroscopy (FT-IR), thermal analysis, differential scanning calorimetry, X-ray diffraction and scanning electron microscopy. Acetylated derivatives with different degrees of substitution (1.26; 1.03; 0.66 and 0.48) were obtained, and there was correlation between the concentration of derivatizing agent and DS. FT-IR spectroscopy analysis confirmed the insertion of acetyl groups into derivatized macromolecules (LAS-AC) through of specific bands concerning to carbonyl group (C = O) and increase in C-O vibration. SEM analysis indicated that native lasiodiplodan presents morphological structure in the form of thin films with translucent appearance and folds along its length. Derivatization led to morphological changes in the polymer, including aspects thickness, translucency and agglomeration. Thermal analysis indicated the native sample and derivative with DS 0.48 presented three weight loss stages. The first stage occurred until 125 ° C (loss of water) and there were two consecutive events of weight loss (200 ° C - 400 ° C) attributed to molecule degradation. Samples with DS 1.26; 1.03 and 0.66 demonstrated four weight loss stages. The first stage occurred until 130 ° C (loss of water), following by two consecutive events of weight loss (200 ° C - 392 ° C) attributed to degradation of the biopolymer. The fourth stage was between 381 ° C and 532 ° C (final decomposition) with exothermic peaks between 472 ° C and 491 ° C. X-ray diffraction patterns showed that native and acetylated lasiodiplodan have amorphous structure with semicrystalline regions. Derivatization did not contribute to increased solubility of the macromolecule, but potentiated its antioxidant capacity. Acetylation of lasiodiplodan allowed to obtaining a new macromolecule with higher antioxidant potential than the native molecule and with technological properties applicable in various industrial sectors.

nÃo hà / PolissacarÃdeos sulfatados de algas marinhas sÃo polÃmeros heterogÃneos que representam biomolÃculas de interesse comercial, principalmente nas indÃstrias alimentÃcia e farmacÃutica. O presente trabalho teve como finalidade avaliar os efeitos dos polissacarÃdeos sulfatados totais (PST) da alga marinha verde Caulerpa mexicana em modelos clÃssicos de nocicepÃÃo e inflamaÃÃo aguda em animais. Foram utilizados camundongos Swiss machos (18-25 g) e ratos Wistar machos (120-260 g). O rendimento de PST obtidos da extraÃÃo por digestÃo enzimÃtica foi de 1,7%. Na anÃlise por espectroscopia na regiÃo do infravermelho foram caracterizados como polissacarÃdeos contendo, principalmente, galactose sulfatada e Ãcido urÃnico. Os PST da alga C. mexicana foram capazes de reduzir (p<0,05) o nÃmero de contorÃÃes abdominais induzidas por Ãcido acÃtico (1%) em 31,8; 74,5 e 88,9% nas doses de 5, 10 e 20 (mg/kg; i.v.), respectivamente. No teste da formalina, os PST inibiram (p<0,05) o tempo de lambedura da pata somente na segunda fase do teste (88,6 e 98,5% para as doses 10 e 20 mg/kg; i.v., respectivamente). No teste da placa quente, os PST nÃo demonstraram efeito antinociceptivo no decorrer dos 90 minutos do teste. Desta forma, os resultados sugerem que os PST exerceram um efeito antinociceptivo de aÃÃo perifÃrica. Em adiÃÃo, os PST (5, 10 e 20 mg/kg) apresentaram efeito anti-inflamatÃrio quando administrados por via s.c. em ratos Wistar no modelo de edema de pata induzido por carragenana, dextrana e histamina. Para analisar o envolvimento da via heme oxigenase (HO) no efeito anti-inflamatÃrio dos PST (20 mg/kg; s.c.), os animais foram prÃ-tratados por via s.c. com um inibidor de HO especÃfico (zinco protopofirina IX). Os resultados obtidos demonstraram que os PST foram capazes de reduzir o edema de pata induzido por carragenana na terceira hora, cujo resultado foi comprovado pela quantificaÃÃo da mieloperoxidase. AlÃm disso, os PST reduziram o edema induzido por dextrana ou histamina em modelos de edema de pata nos primeiros 30 minutos apÃs a aplicaÃÃo dos agentes flogÃsticos. O efeito anti-inflamatÃrio dos PST no edema de pata induzido por carragenana nÃo foi observado apÃs inibiÃÃo prÃvia por zinco protopofirina IX. Portanto, sugerimos que o efeito anti-inflamatÃrio dos PST da alga C. mexicana pode estar relacionado com a inibiÃÃo da liberaÃÃo de histamina, reduÃÃo da migraÃÃo celular e ativaÃÃo da via da HO. Para avaliar os efeitos sistÃmicos dos PST, estes foram administrados por via i.v. (20 mg/kg) em camundongos em dose Ãnica e as alteraÃÃes sistÃmicas avaliadas durante 48 h. Os resultados obtidos demonstraram que os PST nÃo causaram mortalidade e nem alteraÃÃes macroscÃpicas significativas dos ÃrgÃos e dos parÃmetros bioquÃmicos avaliados, sendo, portanto, considerados seguros na dose testada. Em resumo, os PST da alga C. mexicana apresentaram efeitos antinociceptivos e anti-inflamatÃrios significativos, tornando-se uma importante ferramenta biotecnolÃgica para estudos posteriores. / Sulfated polysaccharides from seaweed are heterogeneous polymers representing biomolecules of commercial interest, especially in food and pharmaceutical industries. The aim this study was to evaluate the effects of total sulfated polysaccharides (TSP) of the green seaweed Caulerpa mexicana in classical models of nociception and acute inflammation in animals. Swiss male mice (18-25 g) and male Wistar rats (120-260 g) were used. The yield of the extraction of TSP obtained by enzymatic digestion was 1.7%. In analysis by Fourier transformed infrared were characterized as polysaccharides containing mainly sulfated galactose and uronic acid. The TSP of the alga C. mexicana were able to decrease (p <0.05) the number of writhes induced by acetic acid (1%) on 31.8, 74.5 and 88.9% at doses of 5, 10 and 20 mg/kg; i.v., respectively. In the formalin test, the TSP inhibited (p <0.05) the licking time of the paw only on the second of the test phase (88.6 and 98.5% for the doses 10 and 20 mg/kg; i.v., respectively). In the hot plate test, the TSP did not show antinociceptive effect in the course of 90 minutes of the test. Thus, the results suggest that the TSP presented analgesic effect by a peripheral action. In addition, TSP (5, 10 and 20 mg/kg) showed anti-inflammatory effect when administered s.c. in rats on paw edema models induced by carrageenan, dextran or histamine. To analyze the involvement of heme oxygenase (HO) pathway in the anti-inflammatory effect of TSP (20 mg/kg, sc), the animals were pretreated (s.c.) with a specific HO inhibitor (zinc protoporphirin IX). The results showed that the TSP were able to reduce the paw edema induced by carrageenan in the third hour, which was confirmed by myeloperoxidase quantification. In addition, the TSP reduced the edema induced by dextran or histamine in paw edema models in the first thirty minutes after application the flogistic agents. The anti-inflammatory effect of TSP on paw edema induced by carrageenan was not observed after prior inhibition by zinc protoporphyrin IX. Therefore, we suggest that the anti-inflammatory effect of TSP of the alga C. mexicana algae may be related to inhibition of the histamine release, reduction of cell migration and activation of the HO pathway. To evaluate the systemic effects of TSP, they were administered (i.v.) in mice (20 mg/kg) in a single-dose and evaluated the systemic changes for 48 h. The results showed that the TSP did not cause mortality or significant alterations on organs and on biochemical parameters being considered safe in the tested dose. In summary, the TSP from the algae C. mexicana have showed significant antinociceptive and anti-inflammatory effects, becoming an important biotechnological tool for further studies.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / PolissacarÃdeos ocorrem em grandes quantidades nas sementes como componentes da parede celular ou como reserva. Dentre estes Ãltimos, incluem-se as xiloglucanas cotiledonÃrias e as galactomananas endospÃrmicas. As xiloglucanas apresentam uma cadeia principal de &#946;-D-(1&#8594;4)-glucana ramificada com ligaÃÃes &#945;-(1&#8594;6) por resÃduos D-xilopiranosÃdeos ou &#946;-D-galactopiranosÃdeo-(1&#8594;2)-D-xilopiranosÃdeos, enquanto as galactomananas endospÃrmicas consistem em cadeias polimÃricas de resÃduos &#946;-D-manopiranosil (1&#8594;4) ligados, substituÃdos em O-6 por unidades de &#945;-D-galactopiranosil. O objetivo deste trabalho foi analisar a interaÃÃo de xiloglucanas e galactomananas com lectinas galactose-ligantes e, assim, sugerir o uso de tais polissacarÃdeos como alternativa barata e eficaz na preparaÃÃo de matrizes cromatogrÃficas para o isolamento e determinaÃÃo da especificidade anomÃrica de novas lectinas. Dessa forma, as interaÃÃes das lectinas das sementes de Artocarpus integrifolia (frutalina), Artocarpus incisa (jacalina), Ricinus communis (ricina) e Arachis hypogaea (PNA) com matrizes de xiloglucanas de sementes de Copaifera langsdorffii, Mucuna sloanei e Hymenaea courbaril (MC, MMu, MJ, respectivamente) e de galactomananas de Mimosa scabrella, Stryphnodendron barbatiman, Adenanthera pavonina e Dimorphandra mollis (MM, MS, MA; MD, respectivamente) foram analisadas. As galactomananas apresentaram melhor capacidade de retenÃÃo da jacalina (MA â 0,92 mg ; MM â 1,48 mg ; MD â0,88 mg ; MS â 0,83 mg) e da frutalina (MA â 0,99 mg ; MM â 1,09 mg ; MD â 0,94 mg ; MS â 0,85 mg), lectinas que possuem especificidade por &#945;-D-galactose. Vale destacar que a galactomanana de M. scabrella apresentou melhor capacidade de retenÃÃo da lectinas testadas. Por outro lado a ricina, capaz de ligar-se aos dois anÃmeros, mas que se liga preferencialmente ao anÃmero &#946;, teve maior massa retida nas colunas de xiloglucana (MMu â 2,17 mg ;MJ â 1,30 mg; MC â 2,83 mg). Houve diferenÃa nos perfis de retenÃÃo da PNA, que tambÃm se liga aos anÃmeros &#945; e &#946; da galactose, sendo que a melhor retenÃÃo foi na coluna contendo matriz de M. sloanei (0,12 mg). As colunas foram todas saturadas com extrato bruto a partir das farinhas das sementes, para que se utilizasse a capacidade mÃxima de retenÃÃo de cada matriz. Atividade hemaglutinante foi detectada em ambos os picos PI e PII. Para a ricina, atividade tÃxica foi realizada e detectada para todos os PII obtidos. Por meio de SDS-PAGE, a pureza de cada uma das lectinas foi confirmada. Diante dos resultados expostos, pode-se sugerir o uso de xiloglucanas e galactomananas para o isolamento, purificaÃÃo e determinaÃÃo da especificidade anomÃrica de lectinas galactose-ligantes. / Polysaccharides are found in large quantity in seeds and they represent the main compounds of cell wall or reservoir. Among reservoir compounds, it included cotyledonary xyloglucans and endospermic galactomannans. The xyloglucans are made of a main chain of &#946;-D-(1&#8594;4)-glucan with &#945;-(1&#8594;6) ramifications of D-xylopyranoside or &#946;-D-galactopyranoside-(1&#8594;2)-D-xylopyranoside residues. Endospermic galactomannans are polimeric chains of &#946;-D-mannopyranosil (1&#8594;4) and replaceabled in O-6 for units of &#945;-D-galactopyranosil. The aim of this work is investigate the interaction of xyloglucans and galactomannans with galactose bounding lectins and show the possibility of the usage of these polysaccharides as cheap and useful chromatographic matrices for isolation and determination of anomeric specificity of galactose bounding lectins. The interactions of lectins from seeds of Artocarpus integrifolia (frutalin), Artocarpus incisa (jacalin), Ricinus communis (ricin) e Arachis hypogaea (PNA) were performed with coluns of xyloglucans of seeds from Copaifera langsdorffii, Mucuna sloanei and Hymenaea courbaril (MC, MMu, MJ, respectively) and galactomannans from Mimosa scabrella, Stryphnodendron barbatiman, Adenanthera pavonina and Dimorphandra mollis (MM, MS, MA; MD, respectively). The galactomannans showed the best colun interaction capacity for the jacalin (MA â 0,92 mg ; MM â 1,48 mg ; MD â0,88 mg ; MS â 0,83 mg) and frutalin (MA â 0,99 mg ; MM â 1,09 mg ; MD - 0,94 mg ; MS - 0,85 mg) lectins. Remarkably the M. scabrella galactomannan showed the best colun interaction among all lectins analysed. On the other hand, ricin was better hold in coluns made of xyloglucan (MMu â 2,17 mg ;MJ â 1,30 mg; MC â 2,83 mg). For PNA lectin, differences were detected in colun interaction capacity. The best colun interaction was with the M. sloanei matrix (0,12 mg) for PNA lectin. All coluns were fill with sample extract of flour from seeds and hemagglutination assays was performed with PI and PII. In these assays, hemagglutination activity was detected in both PI and PII from the coluns. For ricin, toxic activity was made and it was detected for all obtained chromatographic samples. With SDS-PAGE it was possible confirmed the purification of the studied lectins. The bands in polyacrilamid gel were the same for the lectins purified. In conclusion, it can be suggested the usage of xyloglucans and galactomannans for isolation, purification and determination of anomeric specificity of galactose-bounding lectins.

Orientadores: Jaime Aparecido Cury, Hyun Koo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-10T06:35:17Z (GMT). No. of bitstreams: 1 Aires_CarolinaPatricia_D.pdf: 3028727 bytes, checksum: 2801d7baaa2bd663ff7b8748629b5d05 (MD5) Previous issue date: 2008 / Resumo: Sacarose é o mais cariogênico dos carboidratos da dieta e há evidências que sua cariogenicidade aumenta quando associada ao amido. Isso seria explicado pelas mudanças na matriz do biofilme formado, pois de acordo com estudo in vitro um ¿novo¿ polissacarídeo extracelular (EPS) é sintetizado pela glicosiltransferase (GTF) B de S. mutans a partir de sacarose e hidrolisados do amido. Entretanto, isso precisa ser confirmado em biofilmes, pois o efeito desse ¿novo¿ polissacarídeo pode não ser evidenciado quando da ação simultânea das diferentes GTFs de S. mutans e de outras bactérias. Em acréscimo, a maior cariogenicidade da associação amido-sacarose é conhecida para o esmalte, mas não para dentina. Na realidade, não há evidência nem mesmo se amido isoladamente seria cariogênico para dentina e cárie radicular é uma preocupação atual considerando o declínio de cárie, maior retenção de dentes e o aumento da expectativa de vida das populações. Assim, o objetivo da presente tese foi estudar a influência da associação amido-sacarose em biofilmes in vitro e in situ. In vitro, biofilmes de S. mutans foram formados sobre discos de hidroxiapatita na presença de amido, sacarose ou associações. In situ, a cariogenicidade da associação amido-sacarose foi testada através de um estudo cruzado e em cada fase blocos de dentina radicular foram submetidos extraoralmente a tratamentos com soluções de amido, sacarose ou suas combinações. Os biofilmes formados in vitro e in situ foram analisados quanto às suas composições bioquímicas e microbiológicas, enquanto que cárie dentinária foi avaliada somente in situ. O biofilme de S. mutans formado in vitro na presença da associação amido-sacarose mostrou composição distinta em EPS em relação àquele formado na presença dos açúcares isolados, porém o mesmo não foi evidenciado in situ. Quanto à expressão dos genes gtfBCD, resultados preliminares promissores foram encontrados mas aparentemente distintos para as condições in vitro e in situ. Amido se mostrou levemente cariogênico para dentina em comparação com sacarose, mas a associação amido-sacarose não foi mais cariogênica que sacarose isolada. Conclui-se que embora haja evidências que a associação amido-sacarose possa aumentar a cariogenicidade do biofilme formado, isso não foi confirmado in situ para dentina radicular, pois a associação não se mostrou mais cariogênica que sacarose isolada / Abstract: Sucrose is the most cariogenic of the dietary carbohydrates but there is evidence that its cariogenicity is enhanced when used in combination with starch. This could be explained by changes in biofilm matrix, since an in vitro study showed that a novel extracellular polysaccharide (EPS) is synthesized by glicosiltransferase (GTF) B of S. mutans in presence of sucrose and starch hydrolysates. However, this should be confirmed in biofilms since the effect of this novel polysaccharide could not be evidenced when different GTFs from S. mutans and from other bacteria are synthesized simultaneously. In addition, the cariogenicity of starch-sucrose combination is well established for enamel but not for dentine. Also, evidence is still lacking on the cariogenicity of starch for root dentine, and root caries is a current concern considering the decline of caries, higher tooth retention and the increase of the life expectancy for populations. Thus, the aim of this study was to evaluate the influence of starch, sucrose and their combinations in in vitro and in situ biofilms. In vitro, S. mutans biofilms were formed on hydroxyapatite discs in presence of starch, sucrose or their combination. In situ, the cariogenicity of starch-sucrose association was tested by a crossover study and during each phase root dentine slabs were submitted extraorally to solutions of starch, sucrose or starch-sucrose association. In vitro and in situ biofilms were analyzed for biochemical and microbiological composition, and root dentine caries was evaluated in situ. In vitro biofilms formed in presence of starch-sucrose association showed distinct EPS composition in relation to those formed in the presence of isolated sugars, but the same trend was not evidenced in situ. In relation to gtfBCD expression, promising preliminary results were found, but they were distinct for in vitro and in situ studies. Starch was moderately cariogenic for dentine in comparison to sucrose but starch-sucrose association was not more cariogenic than sucrose alone. In conclusion, although starch-sucrose association could enhance the cariogenicity of the biofilm formed, this trend was not confirmed in situ for root dentine since the association was not more cariogenic than sucrose alone / Doutorado / Cariologia / Doutor em Odontologia