Dissertations / Theses on the topic 'Polysaccharide oxidation'

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Gelatina à uma proteÃna obtida por desnaturaÃÃo do colÃgeno que tem gerado grande interesse na preparaÃÃo de materiais para uso biomÃdico devido Ãs propriedades como, biocompatibilidade e bioadesividade. No entanto, devido a solubilidade de microesferas de gelatina em meio aquoso, a interaÃÃo com outros polÃmeros e reticulaÃÃo tem sido proposta para melhorar suas propriedades fÃsicoquÃmicas. A goma do cajueiro foi modificada por reaÃÃo de sulfataÃÃo (GCS) e oxidaÃÃo (GCX), e os derivados foram utilizados no desenvolvimento de microesferas por interaÃÃo com gelatina. Os derivados da goma foram caracterizados por espectroscopia na regiÃo do infravermelho, ressonÃncia magnÃtica nuclear, anÃlise elementar, cromatografia de permeaÃÃo em gel, viscosidade intrÃnseca e anÃlise termogravimÃtrica. As reaÃÃes foram eficientes na modificaÃÃo, resultando grau de substituiÃÃo de 0,02 a 0,88 para a goma sulfatada, e relaÃÃo de 10:3 e 10:4 de unidades glicosÃdicas/unidades oxidadas. AnÃlises de RMN 13C DEPT indicam sulfataÃÃo em C-6 da galactose. Os espectros de RMN 1H das GCXÂs mostraram sinal de aldeÃdo em 8,3 ppm. As microesferas foram preparadas pelo mÃtodo de emulsÃo Ãleo/Ãgua e caracterizadas por MEV, RMN 1H e IV. Essas tÃcnicas mostraram mostraram a presenÃa dos dois polÃmeros nas esferas. Microesferas de GEGC reticuladas com genipina por 24 e 72 h (RGEGC) foram caracterizadas quanto a capacidade de intumescimento. O aumento da concentraÃÃo de GC nas esferas de RGEGC e o tempo de reticulaÃÃo (24 e 72h) diminuem a capacidade de intumescimento. Para as esferas de GECX, o aumento da oxidaÃÃo da goma (GCX) de 10:3 para 10:4 diminui em atà 12,3% a capacidade de intumescimento, o que indica maior grau de reticulaÃÃo, via formaÃÃo da base de Schiff, entre gelatina e GCX (10:4). As microesferas de GEGCS mostraram maior capacidade de intumescimento em relaÃÃo a todos os sistemas estudados neste trabalho.
Gelatin is a protein obtained from denaturation of collagen that has been used in the preparation of biomedical materials due to its biocompatibility and bioadhesive properties. However due to gelatin solubility in aqueous solution, the interaction of gelatin with other polymers and also its cross-linking have been carried out in order to improve the gelatin physico-chemical properties. Cashew gum was modified by sulfation (CGS) and oxidation (CGX) reactions. The derivatives were characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy, elemental analysis and gel permeation chromatography (GPC). The degree of substitution for sulfation reaction range from 0.02 to 0.88 and the oxidation reaction produced two samples of 10:3 and 10:4 (number of anhydrogalactose units per number of oxidized units). The 13C DEPT NMR experiment shows that the sulfation occurs at C-6 of galactose. 1H-NMR spectra of CGX samples shows aldehyde proton at 8.3 ppm. Cashew gum and its derivatives were used on the preparation of microsphere with gelatin by oil/water emulsion method. Microspheres were characterized by IR, NMR and thermogravimetric analysis (TGA). That method shows modifications that indicates the presence of both polymers on the microspheres. Microspheres with cashew gum unmodified (GECG) and crosslinked with genipin for 24 and 72 h (RGECG) were characterized in terms of the swelling behavior. The increase of CG content and crosslinking time decrease the swelling behavior in water of RGECG sample. For microspheres of gelatin and oxidated cashew gum (GECGX), the increase of oxidation from 10:3 to 10:4 decrease the swelling ratio in 12.3% indicating an increase in crosslinking density by Schiff base formation between gelatin and CGX sample (10:4). Microsphere with sulfated cashew gum (GECGS) shows the highest swelling ratio among all systems investigated.

Galactomannans constitute one of the major families of natural polysaccharides. They can be obtained from the endosperm of the seed of some leguminous plants and are widely employed in a number of industrial fields as rheology modifiers. Each galactomannan has a unique mannose-to-galactose ratio (M/G), which strongly influences its physico-chemical properties, including solubility and viscosity. Usually, polysaccharides are characterized by nuclear magnetic resonance (NMR), whereas mass spectrometry (MS) has not seen a wide application in this field. In this thesis, a quantification method for sugar diastereomers in galactomannans was developed, exploiting the typical fragmentation pathways of mannose and galactose in tandem MS analyses. The tandem MS spectra of commercially available galactose and mannose display an accumulation of the same fragments for both monosaccharides, but significant differences in the intensities of the product ions. Mixtures with known concentrations of mannose and galactose have been analysed employing the same procedure in order to evaluate the applicability of this method for quantification aims. The relative intensities over the entire spectrum can be deconvoluted as a linear combination of the intensities of pure mannose and galactose spectra, leading to a reliable and reproducible quantification of these monosaccharides. After validation, the method has been applied to the quantification of chemical hydrolysis products of galactomannans from different leguminous plants, like guar (Cyamopsis tetragonolobus), sesbania (Sesbania bispinosa) and tara (Caesalpina spinosa), to evaluate specific susceptibility to different chemical hydrolysis conditions. Galactomannans are commonly employed not only in their native, but also in their chemically and/or biochemically modified form. In this thesis, gums from guar, sesbania and fenugreek (Trigonella foenum-graecum L.) have been oxidised by a laccase/TEMPO-mediated system. Chemo-enzymatic oxidation of galactomannans in aqueous solution caused a viscosity increase up to fifteen-fold, generating structured and stable hydrogels. Upon lyophilisation of these hydrogels, water-insoluble aerogels were generated, capable of uptaking aqueous solutions several times their own weight. The aerogels have been characterized by scanning electron microscopy, calorimetry and X-ray diffraction. Analyses by stability assays, electrospray ionisation MS, NMR and Fourier-transform infrared spectroscopy demonstrate that the chemo-enzymatic treatment leads to the formation of carbonyl and carboxyl groups from primary hydroxyls on the polymers and subsequent establishment of hemiacetal and ester bonds, cross-linking the gels. Fenugreek-based materials emerged to be more structured and stable compared to guar and sesbania aerogels, which is likely due to the higher amount of oxidable galactose units present in fenugreek gum (i.e., fenugreek has a M/G of 1, whereas guar and sesbania have a M/G between 1.3 and 1.6) and, therefore, to more extensive cross-linking of the resulting elastic gel. Active principles have been absorbed into guar, sesbania and fenugreek aerogels by incubating the materials in aqueous solutions of different actives (polymyxin B, nisin, enzymes). The aerogels were rinsed, blotted on filter paper and re-lyophilised, and the release of the active principles was tested in appropriate media. The release of polymyxin B was evaluated against six Gram-negative bacterial strains, whereas the release of nisin was tested against two Gram-positive bacterial strains. Protease and lipase release was evaluated in solution by monitoring the increase in protein concentration and enzymatic activity. The analyses performed during this project suggest that these aerogels represent versatile bio-compatible delivery systems based on renewable raw materials and could be employed for biomedical and industrial applications.

L'etude des complexes du titre a ete envisagee selon deux approches: a) la premiere correspond a la synthese de composes mixtes modeles, avec differentes aglycones mono ou dimeriques, correspondant a des liaisons glycosidiques et a des liaisons ether benzylique. La sensibilite de ces liaisons vis-a-vis de l'oxydation a ete testee. B) la seconde est une etude biochimique. On prepare des polymeres a base d'alcool coniferylique a l'aide d'un extrait brut enzymatique en presence d'hydrates de carbone (polysaccharides ou nucleotides-oses) pour obtenir in vitro les complexes recherches

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Algas marinhas do filo Rhodophyta sÃo fontes naturais de polissacarÃdeos sulfatados que sÃo amplamente utilizados na indÃstria alimentÃcia e na indÃstria farmacÃutica. O presente trabalho teve como finalidade obter os polissacarÃdeos sulfatados totais da alga marinha vermelha Gracilaria caudata (PSG) por extraÃÃo enzimÃtica, determinar a sua estrutura quÃmica e testar o seu potencial antioxidante. As anÃlises quÃmicas revelaram a presenÃa de 85% de aÃÃcares totais e 1% de proteÃnas contaminantes no extrato obtido. AtravÃs de espectrometria de emissÃo Ãptica com plasma (ICP-OES), os PSG apresentaram 0,9% de Ãtomos de enxofre e um grau de sulfataÃÃo de 0,14%. A massa molar mÃdia dos PSG foi determinada por cromatografia em permeaÃÃo em gel (GPC) e mostrou ser da ordem de 116,51 kDa. Os polissacarÃdeos sulfatados totais foram submetidos a testes de caracterizaÃÃo estrutural atravÃs da anÃlise por espectroscopia de infravermelho com transformada de Fourier e ressonÃncia magnÃtica nuclear (RMN) de prÃton (1H) e carbono (13C), identificando os PSG como galactana do tipo agarana. A atividade antioxidante in vitro dos PSG foi avaliada atravÃs de testes, tais como, ensaios de eliminaÃÃo do radical DPPH, quelaÃÃo do Ãon ferroso e capacidade antioxidante total. Os resultados indicaram que tais polissacarÃdeos possuem capacidade de sequestrar radicais livres de maneira significativa e concentraÃÃo-dependente. A atividade antioxidante in vivo dos PSG foi avaliada em modelo de estresse oxidativo induzido pelo 2,2â-azobis-2-amidinopropano (AAPH) em ratos, com posterior dosagem de marcadores do sistema antioxidante enzimÃtico, como catalase (CAT) e superÃxido desmutase (SOD), alÃm da quantificaÃÃo de marcadores de dano oxidativo, como nitrito e tiol. O resultado demonstrou uma melhora no desequilÃbrio redox pelo aumento da atividade da CAT e aumento da atividade da SOD, com melhor resposta na dose de 3 mg/kg. Devido a estes resultados, os polissacarÃdeos sulfatados obtidos a partir da alga marinha Gracilaria caudata mostram potencial de virem a ser utilizados na indÃstria alimentÃcia e farmacÃutica.
Red algae are natural sources of sulfated polysaccharides, which are widely used in the food and pharmaceutical industries. This study aims to obtain the total sulfated polysaccharides from the red seaweed Gracilaria caudata (PSG) through enzymatic extraction, determine their chemical structure and their antioxidant potential. Chemical analysis revealed that the obtained extract is comprised of 85% total sugars and 1% of contaminating proteins. Through Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES), the PSG showed a percentage of 0.9% sulfur atoms and a degree of sulfation of 0.14%. The average molar mass of PSG was determined through gel permeation chromatography (GPC) and was determined as 116.51 kDa. The total sulfated olysaccharides were subjected to structural characterization tests through infrared Fourier transform spectroscopy and C13 and H1 Nuclear Magnetic Resonance (NMR) analysis, identifying the PSG as galactan from the agaran type. The in vitro antioxidant activity of PSG was determined using tests such as elimination of DPPH radical, chelation of ferrous ion and total antioxidant capacity. The results indicated that such polysaccharides have the capacity to scavenge free radicals significantly and in a concentration-dependent maner. The in vivo antioxidant activity of PSG was valuated in an oxidative stress model induced by 2,2'-azobis- -amidinopropane (AAPH) in rats, with subsequent dosage of antioxidant enzyme system markers, such as catalase (CAT) and superoxide dismutase (SOD), and the quantitation of oxidative damage markers such as nitrite and thiol. The results showed an improvement in the redox imbalance through increased CAT activity and increased SOD activity with the best response found at a dose of 3 mg / kg. Because of these results, the sulfated polysaccharide obtained from seaweed Gracilaria caudata shows potential for their being used in the food and pharmaceutical industry.

Des solutions d'acides carboxyliques contenant principalement de l'acide formique sont obtenues par oxydation photo-Fenton des polysaccharides. Une irradiation dans le visible de 60 W sous des conditions douces suffit à fournir des résultats reproductibles. Les produits d'oxydation issus d'amidon de pomme de terre et d'amidon de blé ont montré des capacités de complexation comparables aux solutions commerciales d'acide gluconique et glucuronique.Un système d'oxydation innovant, comprenant des sels d'halogénures et du DMSO, convertit quantitativement le HMF en DFF. Cette méthode permet aussi la transformation one-pot du fructose en DFF avec de bons rendements. Selon l'étude mécanistique, le bromo-HMF serait un intermédiaire réactionnel
Solutions of carboxylic acids, containing mainly formic acid, are produced under photo-Fenton conditions. Visible irradiation with a 60 W spot is sufficient to provide reproducible results under mild conditions. The oxidation products of potato starch and wheat starch have shown Ca sequestering properties similar to those of gluconic and glucuronic acids.Using halide salts and DMSO, an innovating method has been elaborated for the selective oxidation of HMF to DFF with quantitative yields. The one-pot transformation of fructose to DFF occurs with fair yields. Based on the mechanistic study, Br-HMF would be the reaction intermediate

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第7891号
農博第1049号
新制||農||778(附属図書館)
学位論文||H11||N3254(農学部図書室)
UT51-99-G485
京都大学大学院農学研究科食品工学専攻
(主査)教授 松野 隆一, 教授 池田 篤治, 教授 北畠 直文
学位規則第4条第1項該当

The improved synthesis of two methyl glycosides, viz methyl 4-O-methyl-α-D-glucopyranoside and methyl 4-O-(α-D-glucopyranosyl)-α-D-glucopyranoside (methyl α-maltoside) from readily available starting materials is described for use as model compounds for oxidative studies of the industrially important polysaccharide amylose. Attempts to acetylate methyl-α-D-glucoside regioselectivity proved fruitless, whilst regioselective pivaloylation was more successful, albeit in yields of poor synthetic utility. In a variation to this approach, the glucoside, is synthesized via selective tri-O-benzoylation of methyl-α-D-glucoside with the regiospecificity of the reaction being a function of the relative strengths of intramolecular hydrogen bound rings, which are governed by the conditions under which the reaction is performed. In the second step, methylation of the free hydroxy group can only be achieved under pressure with silver oxide as catalyst. The target compound is obtained in the final step by conventional cleavage of the ester functions. Initial attempts to synthesise methyl-α-maltoside were convergent, being conducted with variants of the Koenigs-Knorr methodology and using reactions of previously prepared haloglucosides with variously protected 4-hydroxymethyl-α-D-glucosides. No combination of catalysts could be found to activate either of the substrates, or in an alternative strategy, mediate the reaction between a halomaltoside and an alkoxy nucleophile. The target maltoside is formed eventually via the selective anomeric deprotection of per-O-acetylated maltose, followed by stereospecific methylation in a manner similar to that described previously. It is found that in this penultimate step an intramolecular silver alkoxy - esteric salt inhibits mutarotation, thus allowing the predominant formation of the α-anomer. Further deacetylation gives rise to methyl-α-maltoside in 63% yield and in only three steps.

Doutoramento em Bioquímica
Os polissacarídeos são os componentes maioritários dos grãos de café verde e torrado e da bebida de café. Os mais abundantes são as galactomananas, seguindo-se as arabinogalactanas. Durante o processo de torra, as galactomananas e arabinogalactanas sofrem modificações estruturais, as quais estão longe de estar completamente elucidadas devido à sua diversidade e à complexidade estrutural dos compostos formados. Durante o processo de torra, as galactomananas e arabinogalactanas reagem com proteínas, ácidos clorogénicos e sacarose, originando compostos castanhos de alto peso molecular contendo nitrogénio, designados de melanoidinas. As melanoidinas do café apresentam diversas atividades biológicas e efeitos benéficos para a saúde. No entanto, a sua estrutura exata e os mecanismos envolvidos na sua formação permanecem desconhecidos, bem como a relação estrutura-atividade biológica. A utilização de sistemas modelo e a análise por espectrometria de massa permitem obter uma visão global e, simultaneamente, detalhada das modificações estruturais nos polissacarídeos do café promovidas pela torra, contribuindo para a elucidação das estruturas e mecanismos de formação das melanoidinas. Com base nesta tese, oligossacarídeos estruturalmente relacionados com a cadeia principal das galactomananas, (β1→4)-Dmanotriose (Man3), e as cadeias laterais das arabinogalactanas, (α1→5)-Larabinotriose (Ara3), isoladamente ou em misturas com ácido 5-Ocafeoilquínico (5-CQA), o ácido clorogénico mais abundante nos grãos de café verde, e péptidos compostos por tirosina e leucina, usados como modelos das proteínas, foram sujeitos a tratamento térmico a seco, mimetizando o processo de torra. A oxidação induzida por radicais hidroxilo (HO•) foi também estudada, uma vez que estes radicais parecem estar envolvidos na modificação dos polissacarídeos durante a torra. A identificação das modificações estruturais induzidas por tratamento térmico e oxidativo dos compostos modelo foi feita por estratégias analíticas baseadas principalmente em espectrometria de massa, mas também em cromatografia líquida. A cromatografia de gás foi usada na análise de açúcares neutros e ligações glicosídicas. Para validar as conclusões obtidas com os compostos modelo, foram também analisadas amostras de polissacarídeos do café obtidas a partir de resíduo de café e café instantâneo. Os resultados obtidos a partir dos oligossacarídeos modelo quando submetidos a tratamento térmico (seco), assim como à oxidação induzida por HO• (em solução), indicam a ocorrência de despolimerização, o que está de acordo com estudos anteriores que reportam a despolimerização das galactomananas e arabinogalactanas do café durante a torra. Foram ainda identificados outros compostos resultantes da quebra do anel de açúcares formados durante o tratamento térmico e oxidativo da Ara3. Por outro lado, o tratamento térmico a seco dos oligossacarídeos modelo (individualmente ou quando misturados) promoveu a formação de oligossacarídeos com um maior grau de polimerização, e também polissacarídeos com novos tipos de ligações glicosídicas, evidenciando a ocorrência de polimerização através reações de transglicosilação não enzimática induzidas por tratamento térmico a seco. As reações de transglicosilação induzidas por tratamento térmico a seco podem ocorrer entre resíduos de açúcares provenientes da mesma origem, mas também de origens diferentes com formação de estruturas híbridas, contendo arabinose e manose como observado nos casos dos compostos modelo usados. Os resultados obtidos a partir de amostras do resíduo de café e de café instantâneo sugerem a presença de polissacarídeos híbridos nestas amostras de café processado, corroborando a ocorrência de transglicosilação durante o processo de torra. Além disso, o estudo de misturas contendo diferentes proporções de cada oligossacarídeo modelo, mimetizando regiões do grão de café com composição distinta em polissacarídeos, sujeitos a diferentes períodos de tratamento térmico, permitiu inferir que diferentes estruturas híbridas e não híbridas podem ser formadas a partir das arabinogalactanas e galactomananas, dependendo da sua distribuição nas paredes celulares do grão e das condições de torra. Estes resultados podem explicar a heterogeneidade de estruturas de melanoidinas formadas durante a torra do café. Os resultados obtidos a partir de misturas modelo contendo um oligossacarídeo (Ara3 ou Man3) e 5-CQA sujeitas a tratamento térmico a seco, assim como de amostras provenientes do resíduo de café, mostraram a formação de compostos híbridos compostos por moléculas de CQA ligadas covalentemente a um número variável de resíduos de açúcar. Além disso, os resultados obtidos a partir da mistura contendo Man3 e 5-CQA mostraram que o CQA atua como catalisador das reações de transglicosilação. Por outro lado, nas misturas modelo contendo um péptido, mesmo contendo também 5-CQA e sujeitas ao mesmo tratamento, observou-se uma diminuição na extensão das reações transglicosilação. Este resultado pode explicar a baixa extensão das reações de transglicosilação não enzimáticas durante a torra nas regiões do grão de café mais ricas em proteínas, apesar dos polissacarídeos serem os componentes maioritários dos grãos de café. A diminuição das reações de transglicosilação na presença de péptidos/proteínas pode dever-se ao facto de os resíduos de açúcares redutores reagirem preferencialmente com os grupos amina de péptidos/proteínas por reação de Maillard, diminuindo o número de resíduos de açúcares redutores disponíveis para as reações de transglicosilação. Além dos compostos já descritos, uma diversidade de outros compostos foram formados a partir dos sistemas modelo, nomeadamente derivados de desidratação formados durante o tratamento térmico a seco. Em conclusão, a tipificação das modificações estruturais promovidas pela torra nos polissacarídeos do café abre o caminho para a compreensão dos mecanismos de formação das melanoidinas e da relação estrutura-atividade destes compostos.
Polysaccharides are the major components of green and roasted coffee beans, and coffee brew. The most abundant ones are galactomannans, followed by arabinogalactans. During the roasting process, galactomannans and arabinogalactans undergo structural modifications that are far to be completely elucidated due to their diversity and complexity of the compounds formed. During the roasting process, galactomannans and arabinogalactans react with proteins, chlorogenic acids, and sucrose, originating high molecular weight brown compounds containing nitrogen, known as melanoidins. Several biological activities and beneficial health effects have been attributed to coffee melanoidins. However, their exact structures and the mechanisms involved in their formation remain unknown, as well as the structure-biological activity relationship. The use of model systems and mass spectrometry analysis allow to obtain an overall view and, simultaneously, detailed, of the structural modifications in coffee polysaccharides promoted by roasting, contributing to the elucidation of the structures and formation mechanisms of melanoidins. Based on this thesis, oligosaccharides structurally related to the backbone of galactomannans, (β1→4)-D-mannotriose, and the side chains of arabinogalactans, (α1→5)-Larabinotriose, alone or in mixtures with 5-O-caffeoylquinic acid, the most abundant chlorogenic acid in green coffee beans, and dipeptides composed by tyrosine and leucine, used as models of proteins, were submitted to dry thermal treatments, mimicking the coffee roasting process. The oxidation induced by hydroxyl radicals (HO•) was also studied, since these radicals seem to be involved in the modification of the polysaccharides during roasting. The identification of the structural modifications induced by thermal and oxidative treatment of the model compounds was performed mostly by mass spectrometry-based analytical strategies, but also using liquid chromatography. Gas chromatography was used in the analysis of neutral sugars and glycosidic linkages. To validate the conclusions achieved with the model compounds, coffee polysaccharide samples obtained from spent coffee grounds and instant coffee were also analysed. The results obtained from the model oligosaccharides when submitted to thermal treatment (dry) or oxidation induced by HO• (in solution) indicate the occurrence of depolymerization, which is in line with previous studies reporting the depolymerization of coffee galactomannans and arabinogalactans during roasting. Compounds resulting from sugar ring cleavage were also formed during thermal treatment and oxidative treatment of Ara3. On the other hand, the dry thermal treatment of the model oligosaccharides (alone or when mixed) promoted the formation of oligosaccharides with a higher degree of polymerization, and also polysaccharides with new type of glycosidic linkages, evidencing the occurrence of polymerization via non-enzymatic transglycosylation reactions induced by dry thermal treatment. The transglycosylation reactions induced by dry thermal treatment can occur between sugar residues from the same origin, but also of different origins, with formation of hybrid structures, containing arabinose and mannose in the case of the model compounds used. The results obtained from spent coffee grounds and instant coffee samples suggest the presence of hybrid polysaccharides in these processed coffee samples, corroborating the occurrence of transglycosylation during the roasting process. Furthermore, the study of mixtures containing different proportions of each model oligosaccharide, mimicking coffee bean regions with distinct polysaccharide composition, subjected to different periods of thermal treatment, allowed to infer that different hybrid and non-hybrid structures may be formed from arabinogalactans and galactomannans, depending on their distribution in the bean cell walls and on roasting conditions. These results may explain the heterogeneity of melanoidins structures formed during coffee roasting. The results obtained from model mixtures containing an oligosaccharide (Ara3 or Man3) and 5-CQA and subjected to dry thermal treatment, as well as samples derived from spent coffee grounds, showed the formation of hybrid compounds composed by CQA molecules covalently linked to a variable number of sugar residues. Moreover, the results obtained from the mixture containing Man3 and 5-CQA showed that CQA acts as catalyst of transglycosylation reactions. On the other hand, in the model mixtures containing a peptide, even if containing 5-CQA and subjected to the same treatment, it was observed a decrease in the extent of transglycosylation reactions. This outcome can explain the low extent of non-enzymatic transglycosylation reactions during roasting in coffee bean regions enriched in proteins, although polysaccharides are the major components of the coffee beans. The decrease of transglycosylation reactions in the presence of peptides/proteins can be related with the preferential reactivity of reducing residues with the amino groups of peptides/proteins by Maillard reaction, decreasing the number of reducing residues available to be directly involved in the transglycosylation reactions. In addition to the compounds already described, a diversity of other compounds were formed from model systems, namely dehydrated derivatives formed during dry thermal treatment. In conclusion, the identification of the structural modifications in coffee polysaccharides promoted by roasting pave the way to the understanding of the mechanisms of formation of melanoidins and structure-activity relationship of these compounds.

Pullulan is a non-ionic water-soluble polysaccharide which is produced from starch by the yeast-like fungus Aureobasidium pullulans. Pullulan is known for its non-toxicity and biocompatibility. Most pullulan modifications are intended to reduce its water solubility or to introduce charged or reactive groups for functionality. Polysaccharides that have been hydrophobically modified and contain carboxyl groups are commonly used in drug delivery systems because of their ability to provide pH-controlled drug release. We demonstrated in this dissertation the regioselective synthesis of a range of 6-carboxypullulan ethers that are promising anionic derivatives for drug delivery applications. These compounds have also shown impressive surfactant properties. Another class of pullulan derivatives was synthesized by regioselective introduction of amine and amide groups to the pullulan backbone. These chemical groups are known to play a fundamental role in the biological activity of important polysaccharides, such as chitin and chitosan, therefore, the pullulan derivatives synthesized herein, which are structural isomers of those polymers, possess great potential for biomedical applications. Clarithromycin (CLA) is an aminomacrolide antibiotic whose physical properties are fascinating and challenging. It has very poor solubility at neutral intestinal pH, but much higher solubility under acidic conditions. Therefore, CLA dissolves better in the stomach than in the small intestine; but CLA is also quite labile towards acid-catalyzed degradation. We report herein a study on amorphous solid dispersion (ASD) of CLA with promising carboxyl-containing cellulose derivatives, both as macro and nanoparticles. This approach was intended to improve CLA solubility in neutral media, to protect it from acid degradation, and thereby increase its uptake from the small intestine and ultimately its bioavailability. We have also prepared ASDs of selected anti-HIV drugs, ritonavir (RTV), efavirenz (EFV) and etravirine (ETR) with the cellulosic derivative carboxymethyl cellulose acetate butyrate (CMCAB). This polymer was efficient in stabilizing RTV and EFV in their amorphous form in the solid phase and all ASDs provided significant enhancement of drug solution concentration.
Ph. D.

Les travaux de thèse présentés dans ce manuscrit portent essentiellement sur la préparation, le criblage et l’utilisation des catalyseurs hétérogènes à base de polysaccharides. L’objectif principal du projet de thèse a plus particulièrement consisté à évaluer deux polysaccharides : les alginates et le chitosane en tant que supports renouvelables pour la catalyse hétérogène. Ces deux types de polysaccharides ont des structures et des propriétés physico-chimiques très différentes : les alginates sont connus pour être de bons complexants de métaux di ou trivalents de par la présence des fonctions carboxylates dans leur matrice, le chitosane résulte quant à lui de l’assemblage d’unités N-glucosamine pouvant être facilement modifiées chimiquement.Dans un premier temps, nos travaux ont essentiellement portés sur des catalyseurs bimétalliques Mn+ Pd supportés sur alginate dont nous avons évalué l’activité catalytique dans les réactions de couplage C—C de Mizoroki-Heck, Sonogashira et Suzuki-Miyaura. D’une manière générale, seuls les catalyseurs à base de nanoparticules de palladium ont montré une réactivité intéressante pour la catalyse de la réaction de Suzuki-Miyaura. Par la suite, nous avons également étudié les réactions d’oxydations d’alcools catalysées par du palladium (II) complexé à l’alginate. Cette étude nous a permis d’identifier deux catalyseurs actifs vis-à-vis de l’oxydation d’alcools allyliques et benzyliques.Dans un deuxième temps, nous avons développé de nouveaux ligands de type NHC en vue de les greffer sur la matrice chitosane : un ligand NHC pour les réactions de métathèse d’oléfines, et plusieurs ligands NHC de type pincer CNC pour les réactions de couplage C—C dans l’eau. Bien que les performances catalytiques des systèmes hétérogènes correspondant soient limitées, ces travaux ont conduit à l’élaboration de nouveaux ligands amphiphiles construits autour d’un noyau pyrazine porteurs de quatre ligands carbéniques. Après complexation de métaux tels que le palladium ou l’or, ces systèmes conduisent à des nanocatalyseurs ayant des performances catalytiques intéressantes. Enfin, dans un troisième temps, nous avons développé une nouvelle réaction de cyanation décarboxylante pallado-catalysée permettant de transformer en une étape des acides carboxyliques aromatiques en benzonitriles correspondants. Outre son intérêt synthétique, cette réaction présente un grand intérêt pour le marquage isotopique
This work describes the preparation, screening and use of heterogeneous catalysts based on polysaccharides. The main goal of our project was to evaluate two polysaccharides: alginates and chitosan as renewable supports for heterogeneous catalysis.Alginates are known to form gels with most di- and multivalent cations due to the presence of the carboxylate functions of their matrix. And chitosan is an attractive polysaccharide for application in catalysis owing to the presence of readily functionalizable amino group and its insolubility in organic solvents.First, our work focused on evaluating the catalytic activity of bimetallic Mn+-Pd catalysts supported on alginate in C—C coupling reactions. Among them, one system demonstrated remarkable catalytic properties for the Suzuki-Miyaura coupling. Then, the oxidation of alcohols catalyzed by Alginate-Mn+-Pd2+ catalyst was investigated. Two catalysts demonstrated good activity for oxidation of benzylic and allylic alcohol.In a second time, we developed new NHC ligands in order to anchor them on chitosan: two new NHC ligands for olefin metathesis and several NHC pincer CNC ligands for C—C coupling reactions in water. A palladium complex obtained with one our new ligand bearing long alkyl chains showed good activity in the Suzuki-Miyaura coupling in pure water.Finally, a new palladium (II) catalyzed decarboxylative cyanation reaction was investigated. This methodology is the first example of direct conversion of aryl carboxylic acid into the corresponding aryl nitrile. This reaction is well adapted to labeled compound synthesis

La première partie de ce travail est réalisé dans le but de préparer des produits pouvant jouer un rôle très important de l’imagerie cellulaire des lipopolysaccharides sur la surface des cellules bactériennes, tout en utilisant des méthodes fiables telle que la ‘’chimie click’’. Dans un premier temps, nous avons synthétisé une première génération d’outils fluorescents à base de rhodamine B et fluorescéine modifiée par une fonction oxyde de nitrile. Dans un deuxième temps nous avons cherché les meilleures conditions d’applications de la chimie click 3+2 sans cuivre, entre la fonction oxyde de nitrile et le motif saccharidique complémentaire portant une fonction vinylique. Finalement, nous avons appliquée, avec succès, la méthode click avec cuivre sur plusieurs types de bactéries portant sur leur membrane cellulaire des lipopolysaccharides ayant un motif ‘’Kdo’’ fonctionnalisé par un groupement azoture déjà synthétiser au sein de notre équipe, et une sonde fluorescente porteuse d’une fonction alcyne. Une nouvelle génération d’outils de marquage saccharidique est en cours de finalisation dans le but d’optimiser les conditions finales, tel que le dérivé de ‘’Kdo’’ fonctionnalisé cette fois-ci par des dérivées cycliques, bicycliques et aromatiques porteurs d’une fonction alcyne ou alcène, pour réaliser les derniers essais de marquage in vitro. Le stress oxydant est lié au vieillissement des cellules et à de nombreuses maladies: cardiovasculaire, cancer, diabète, Alzheimer… Il est dû à un déséquilibre entre le système oxydant / antioxydant au niveau des cellules, et se caractérise par la présence principalement de substance radicalaires réactives oxygénées. Dans le but d'identifier le taux de substances réactives oxygénées dans les cellules, le travail dans la deuxième partie de la thèse reposait sur la synthèse multi étape d'une molécule fluorescente dérivée de la coumarine. Le composé ciblé est le peroxyde d'oxygène, connu sous le nom d'eau oxygénée. Possédant un ester vinyl-boronique, notre molécule sera oxydée et réarrangée en aldéhyde conduisant à la condensation intramoléculaire avec le groupement aminé adjacent pour former un cycle indolique, libérant ainsi de la fluorescence. La recherche des conditions optimales de la dernière étape de la voie synthétique sont toujours en cours d’optimisation. Dans le futur, on cherchera les conditions optimales de la dernière étape de la synthèse de la sonde spécifique au peroxyde d’oxygène. Cette molécule est d'une importance remarquable comme sonde du stress oxydant. En réussissant cette étape, on pourra avoir en main une bibliothèque de ‘’KDO’’ fonctionnalisé afin d’avoir des résultats satisfaisants in vivo
The first part of this work is done in order to prepare products that play a very important role in cellular imaging lipopolisaccharides on the surface of bacterial cells, while using reliable methods such as '' click chemistry ''. Initially, we synthesized the first generation of tools based fluorescent rhodamine B and fluorescein -modified nitrile oxide function. In a second step we sought the best possible applications of click chemistry 3+2 copper free, between the nitrile oxide function and the additional saccharide unit with a vinyl function. Finally, we applied successfully, the click method with copper on several types of bacteria on their cell membrane lipopolysaccharides having a pattern Kdo functionalized azide group already synthesized within our team, and a probe carrying a fluorescent alkyne. A new generation of tools saccharide marking is being finalized in order to optimize the final terms, such as derivative functionalized Kdo this time by cyclic, bicyclic and aromatic derivatives holders of an alkene or alkyne function to perform final testing of in vitro labeling. Oxidative stress is linked to cell aging and many diseases: cardiovascular disease, cancer, diabetes, Alzheimer's ... It is due to an imbalance between oxidant system/antioxidant in cells, and is characterized mainly by the presence of radical substance reactive oxygen. In order to identify the rate of reactive oxygen substances in cells, work in the second part of the thesis based on multi- step synthesis of a fluorescent molecule derived from coumarin. The target compound is oxygen peroxide, known as the name of hydrogen peroxide. Having a vinyl boronic ester, this molecule will be oxidized and rearranged aldehyde leading to intramolecular condensation with the adjacent amino group to form an indolique ring , thereby releasing fluorescence. The search for optimal conditions for the last step of the synthetic pathway is still being optimized. In the future, the optimal conditions for the last step of the synthesis of specific probe oxygen peroxide are sought. This molecule is remarkable importance as a probe of oxidative stress. By passing this step, we will have on hand a library of KDO functionalized to have satisfactory results in vivo

Dans le contexte actuel, il devient nécessaire de rendre les alternatives au pétrole, tel que le bioéthanol 2G, disponibles à grande échelle. Cependant, l’étape d’hydrolyse par les enzymes de Trichoderma reesei reste un verrou à un procédé économiquement stable et rentable. Ces travaux de thèse, s'intègrent dans le cadre du projet Futurol et ont pour objectifs d'identifier et de caractériser de nouvelles enzymes fongiques pour améliorer l'hydrolyse de la biomasse lignocellulosique. A partir des données protéomiques disponibles pour Podospora anserina et Fusarium verticillioides, une douzaine d'enzymes candidates ont été identifiées dans leurs sécrétomes. Ce travail de thèse s'est plus particulièrement focalisé sur les AA9s « Lytic Polysaccharide Monooxygenases » (LPMOs) de P. anserina. Parmi les LPMOs étudiées, PaLPMO9A, PaLPMO9E et PaLPMO9H, qui possèdent un CBM1, sont les plus actives sur la cellulose. La détermination de la régiosélectivité d'action a mis en évidence que PaLPMO9A et PaLPMO9H clivent la cellulose en position C1 et C4 alors que la PaLPMO9E génère uniquement des produits oxydés en C1. La PaLPMO9H est la plus versatile puisqu’elle est active sur les cello-oligosaccharides solubles et sur les polysaccharides hémicellulosiques liés en β-(1,4) (i.e., xyloglucane, glucomannane). La supplémentation du cocktail de T. reesei avec PaLPMO9E ou PaLPMO9H a permis de doubler les rendements d'hydrolyse du miscanthus prétraité. Les travaux réalisés au cours de cette thèse ont permis de démontrer l'importance de ces enzymes oxydatives dans les phénomènes de déconstruction de la lignocellulose chez les champignons filamenteux
In the current context, it becomes essential to make alternative to oil, such as the 2G bioethanol, available at large scale. However, the hydrolysis step by Trichoderma reesei enzymes remains the major bottleneck for an economically sustainable process. The present work is part of the Futurol project, and aims at identifying and characterizing new fungal enzymes to improve the hydrolysis of lignocellulosic biomass. From the proteomic data available for Podospora anserina and Fusarium verticillioides, a dozen of interesting enzymes were identified in their secretomes. This work focuses, mainly, on the AA9s « Lytic Polysaccharide Monooxygenases » (LPMOs) from P. anserina. Among all the LPMOs studied, PaLPMO9A, PaLPMO9E and PaLPMO9H that harbored a CBM1 were the most active on cellulose. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan). The hydrolysis yield of the pretreated miscanthus was significantly improved up to 2 fold, when the PaLPMO9E, or PaLPMO9H were supplemented to the T. reesei cocktail. This work demonstrated the importance of these oxidative enzymes for lignocellulose deconstruction by fungi. These biocatalysts open new prospects to improve the enzymatic conversion of plant biomass for 2G bioethanol production

Avec 800 000 morts par année, les maladies diarrhéiques sont la seconde cause de mortalité chez les enfants de moins de cinq ans. La shigellose, causée par des bactéries Gram négatif appelées Shigella, est l’une des quatre grandes maladies entériques touchant cette population. L’infection naturelle protège contre la réinfection et la composante polysaccharidique du lipopolysaccharide bactérien est la principale cible de l’immunité humorale. Chez S. sonnei, espèce prévalente dans les pays en développement et développés, ce polysaccharide spécifique, à caractère zwitterionique, a pour unité répétitive un disaccharide composé de deux hexosamines rares : l’acide 2-acétamido-2-désoxy-L-altruronique (A) et le 2-acétamido-4-amino-2,4,6-tridésoxy-D-galactose (B, aussi appelé AAT) associés par des liens glycosidiques 1,2-trans (I). ->4-a-L-AltpNAcA-(1->3)-b-D-FucpNAc4N-(1-> (I). Ces travaux s’intègrent dans un programme visant le développement d’un vaccin issu de sucres de synthèse à couverture large contre les infections par Shigella. Le premier objectif de la stratégie développée contre les infections par S. sonnei est l’identification des épitopes saccharidiques, cibles des anticorps protecteurs. Dans ce but, nous avons entrepris la synthèse d’une diversité de fragments du polysaccharide d’intérêt. Des synthèses multi-grammes de précurseurs orthogonalement protégés des monosaccharides A et B ont été mises au point afin d’accéder aux intermédiaires donneurs et accepteurs impliqués dans les étapes de glycosylation. En particulier, deux voies originales d’accès au précurseur B ont été développées. D’autre part, l’optimisation des conditions de glycosylation et d’oxydation a conduit à un bloc disaccharidique AB compatible avec la synthèse d’oligosaccharides d’ordres supérieurs. Les synthons mono- et disaccharidiques identifiés ont été validés à travers l’obtention de quatre disaccharides portant ou non des modifications de la répartition des charges, de deux trisaccharides ainsi que d’un tétrasaccharide
800,000 children die each year of diarrhoeal diseases, making it the second cause of death among children under five. Shigellosis, caused by a Gram negative bacterium, Shigella, is one of the four major forms of diarrhoeal diseases in this population. Natural infection protects against reinfection and the humoral response is primarily directed against the specific polysaccharide moiety of the bacterial lipopolysaccharide. S. sonnei, the prevalent species in developed and transitional countries, displays a zwitterionic polysaccharide, whose disaccharide repeating unit is made of two rare aminosugars: a 2-acetamido-2-deoxy-L-altruronic acid (A) and a 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (B, AAT) 1,2-trans linked to one another (I). ->4-a-L-AltpNAcA-(1->3)-b-D-FucpNAc4N-(1-> (I). This work is part of the program aimed at the development of a synthetic carbohydrate-based broad coverage vaccine against Shigella infections. In order to define the protective epitopes located on the O-specific polysaccharide of S. sonnei, we tackled the synthesis of fragments thereof. First, multigram-scale syntheses of orthogonally protected precursors to residues A and B were undertaken to access donor and acceptor intermediates in the glycosylation reactions. In particular, two original routes to precursors of residue B were developed. Careful optimisation of the glycosylation and oxidation reaction conditions gave the disaccharide building block AB equipped for the synthesis of chain extension at both ends. Selected mono- and disaccharide building blocks were validated by the synthesis of four disaccharides, bearing modification of the charge pattern or not, two trisaccharides and a tetrasaccharide

碩士
國立臺灣師範大學
生命科學研究所
102
Most age-related neurodegenerative diseases are characterized by accumulation of aberrant protein aggregates/inclusions in the affected brain regions. Several lines of evidence suggest that increased oxidative stress may involve in the pathogenesis of neurodegenerative diseases. Studies have shown that polysaccharides from several algal species have antioxidant activity. This study investigated the antioxidant activities of polysaccharide extracts from brown seaweed (fucoidan, a sulfated polysaccharide), Nostoc sphaeroides (a nitrogen-fixing cyanobacterium) and Chlorella sorokiniana (a green alga) and their application in polyQ-mediated spinocerebellar ataxias (SCA). Firstly, the polysaccharides from these algae were examined for their 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion radicals scavenging potential. The polysaccharides of these algae exhibited an effective scavenging capability on superoxide anion radical. Then their antioxidative activity was evaluated by measuring cellular production of reactive oxidative species (ROS) using CellRox green fluorescent probe on a flow cytometry. Pretreatment with polysaccharides from these algae protected both human embryonic kidney HEK-293 and neuroblastoma SH-SY5Y cells against the H2O2-induced cell death and reduced ROS production. Lastly, human SCA type 3 full length cDNAs with normal (ATXN3/Q14) and expanded (ATXN3/Q75) polyQ were constructed and transiently expressed in HEK-293T cells. Pre-treatment with algae polysaccharides significantly reduced ROS production in ATXN3/Q14~75 transfected cells. However, only fucoidan effectively enhanced hemeoxygenase (decycling) 1 (HMOX1) expression in ATXN3/Q75 expressing cells.

During the extraction of apple juice an insoluble residue, called apple pomace, is generated. Apple pomace represents the fruit cell wall material, identified as a source of polysaccharides and polyphenols. However, apple pomace is mostly discarded by the industries. In the one hand, this occurs because some polysaccharides and polyphenols still correspond to molecules of unknown applications and structures resulting from oxidation phenomena that occur during juice extraction. On the other hand, the industries lack drying methodologies capable of meeting their needs for valorization of apple pomace compounds. The adoption of different extraction and fractionation methodologies gives an overview of the properties and structures of the compounds that can be available from apple pomace after the implementation of a drying process to preserve the byproduct. Based on this hypothesis, apple pomace was subjected to extractions with water, methanol and acetone:water (60:40; v:v), without or with urea, and microwave superheated water extractions to unravel the existence of phenolic structures other than those naturally occurring in the fresh fruit. Additionally, for identification of chemically modified carbohydrates due to polyphenol oxidation, hot water extractions were performed followed by dialysis (12-14 kDa) and ethanol precipitation. Extraction with hot water, methanol and acetone followed by analysis by liquid chromatography allowed to observe the presence of flavan-3-ols, flavonols, dihydrochalcones and hydroxycinamic acids, totaling 5 g/kg of dry apple pomace. Oxidation products of dihydrochalcones and hydroxycinnamic acids were also detected in apple pomace. Alkaline fusion analysis of water-insoluble material demonstrated the occurrence of non-extractable oxidized procyanidins, whose prevalence was 4-fold higher than in native apple polyphenols. Extraction with microwave superheated water followed by ethanol precipitation of the obtained extracts showed that procyanidins were covalently linked to the pectic polysaccharides, xyloglucans and cellulose present in the water-insoluble material and could account for up to 40% of the polyphenols that can be obtained from apple pomace. Solid phase extraction of the hot water-soluble material using C18 cartridges showed that, along with polyphenols, polysaccharides were also extracted, some of them exhibiting a hydrophobic behavior at pH 7 and/or pH 3. Dialysis followed by ethanol precipitation, alongside with glycosidic linkages analysis by gas chromatography, nuclear magnetic ressonance, size-exclusion chromatography, and alkaline fusion, led to the conclusion that polyphenols covalently linked to polysaccharides are responsible for their hydrophobicity, constituting xyloglucan-polyphenols-pectic polysaccharide and arabinan-polyphenol complexes. To understand the formation of polysaccharide-polyphenol complexes established by covalent bonds, the interactions of arabinans with 5-O-caffeoylquinic acid and phloridzin were studied. Through their diffusion along dialysis membranes in the presence or absence of polysaccharide, linear arabinans showed 10-fold and 2-fold greater retention of 5-O-caffeoylquinic acid and phloridzin than branched arabinans suggesting that a higher degree of branching limits polysaccharides interaction with polyphenols. The same trend was observed for the interaction of arabinans with procyanidins by isothermal titration calorimetry, where linear arabinans presented higher affinity constants (Ka 540 M-1) than branched arabinans (Ka 391 M-1). This approach also demonstrated that the occurrence of covalently linked polyphenols to arabinans restricts further interactions with polyphenols (Ka 85 M-1). The analysis of the material that precipitated after interaction between arabinans and procyanidins showed that procyanidins of higher degree of polymerization and less branched polysaccharides tended to form insoluble aggregates. In order to ensure the stability of apple pomace for further extraction of compounds, this byproduct was dried via microwave hydrodiffusion and gravity (MHG) technology using delivery powers of 300-900 W. MHG drying proved to be a three phases process: heating, drying and burning point phase. This methodology presented an estimated 80% energy efficiency, as well as drying times (1-2.6 h) and water flows (5.1-13.9 mL/min) up to 4-fold higher than those verified by hot air drying (3.6-9.9 he 1-3.5 mL/min). Using MHG drying, it was also possible to obtain extracts composed by polyphenols and polysaccharides. The addition of ethanol during the drying process can increase the obtained extract mass. The dried apple pomace was stable for at least 2 years, after which was possible to obtain extracts rich in polyphenols and polysaccharides. In conclusion, the adoption of different extraction and fractionation procedures allowed a better understanding of the structures and chemical properties of the compounds present in apple pomace, especially those derived from oxidation reactions of polyphenols. This, together with the efficient and rapid drying verified for MHG technology, opens new research perspectives and applications that should culminate in the possible mitigation of apple pomace as an agro-industrial residue.
Durante a extração do sumo de maçã é gerado um resíduo insolúvel denominado bagaço de maçã. O bagaço de maçã representa o material da parede celular do fruto, identificado como fonte de polissacarídeos e polifenóis. No entanto, o bagaço de maçã é maioritariamente descartado pelas indústrias. Por um lado, pelo facto de alguns polissacarídeos e compostos fenólicos corresponderem a moléculas de aplicações e estruturas desconhecidas, resultantes de fenómenos de oxidação que ocorrem durante a extração de sumo. Por outro, as indústrias carecem de metodologias de secagem capazes de corresponder às suas necessidades para valorização dos compostos do bagaço de maçã. A adoção de diferentes metodologias de extração e fracionamento permitem obter uma visão global das propriedades e estruturas dos compostos passíveis de serem recolhidos do bagaço após implementação de um processo de secagem para preservação do subproduto. Com base nesta hipótese, o bagaço de maçã foi submetido a extrações com água, metanol e acetona:água (60:40; v:v), sem ou com ureia, e água superaquecida por micro-ondas com o objetivo de desvendar a existência de outras estruturas fenólicas além daquelas que ocorrem naturalmente no fruto. Adicionalmente, para identificação de carbohidratos quimicamente modificados como consequência da oxidação de polifenóis, foram realizadas extrações com água quente seguidas de fracionamento por diálise (12-14 kDa) e precipitação em etanol. A extração com água quente, metanol e acetona seguida de análise por cromatografia líquida permitiu identificar a presença de flavan-3-óis, flavonóis, di-hidrocalconas e ácidos hidroxicinâmico, totalizando 5 g/kg de bagaço seco. Também foram detectados produtos de oxidação de di-hidrocalconas e ácidos hidroxicinâmicos no bagaço de maçã. A análise por fusão alcalina do material insolúvel em água demonstrou a ocorrência de procianidinas oxidadas não-extractáveis, cuja prevalência foi 4 vezes superior aos compostos fenólicos nativos da maçã. A extração com água superaquecida por micro-ondas seguida de precipitação em etanol dos extratos obtidos mostrou que estas se encontravam covalentemente ligadas aos polissacarídeos pécticos, xiloglucanas e celulose presentes no material insolúvel em água, podendo corresponder até 40% dos compostos fenólicos possíveis de serem obtidos a partir do bagaço de maçã. A extração em fase sólida do material solúvel em água quente, usando cartuchos C18, mostrou que, juntamente com os polifenóis, foram extraídos polissacarídeos, alguns dos quais apresentando um comportamento hidrofóbico a pH 7 e/ou a pH 3. O fracionamento por diálise e precipitação em etanol, e a análise das ligações glicosídicas por cromatografia em fase gasosa, ressonância magnética nuclear, cromatografia de exclusão molecular e fusão alcalina, permitiu inferir que os polifenóis ligados covalentemente aos polissacarídeos são responsáveis pela sua hidrofobicidade, constituindo complexos de xiloglucanas-polifenóis-polissacarídeos pécticos e complexos de arabinanas-polifenóis. Para compreender a formação dos complexos polissacarídeos-polifenóis, estabelecidos por ligações covalentes, foram estudadas as interações de arabinanas com o ácido 5-O-cafeoilquinico e floridzina. Através da sua difusão ao longo de membranas de diálise na presença ou ausência de polissacarídeo, verificou-se que as arabinanas lineares apresentaram 10 a 2 vezes maior retenção do ácido 5-O-cafeoilquinico e da floridzina do que as arabinanas ramificadas sugerindo que um maior grau de ramificação dos polissacarídeos limita a sua interação com os compostos fenólicos. A mesma tendência foi observada para a interação de arabinanas com procianidinas por calorimetria de titulação isotérmica, onde arabinanas lineares apresentaram maiores constantes de afinidade (Ka 540 M-1) do que arabinanas ramificadas (Ka 391 M-1). Esta abordagem demonstrou também que a ocorrência de polifenóis ligados covalentemente a arabinanas restringe possíveis interações adicionais com os polifenóis (Ka 85 M-1). A análise do material que precipitou após interação entre as arabinanas e as procianidinas mostrou que as procianidinas de maior grau de polimerização e os polissacarídeos menos ramificados são as estruturas que tendem a formar os agregados insolúveis. Com o objetivo de garantir a estabilidade do bagaço de maçã para posterior extração de compostos, este subproduto foi secado via tecnologia de micro-ondas por hidrodifusão e gravidade (MHG), utilizando potências de 300-900 W. A secagem por MHG mostrou-se um processo composto por três fases: aquecimento, secagem e ponto de queima. Esta metodologia apresentou uma estimativa de 80% de eficiência energética, bem como tempos de secagem (1-2.6 h) e fluxos de água (5,1-13,9 mL/min) até 4 vezes superiores às verificadas por secagem com ar quente (3,6-9,9 h e 1-3,5 mL/min). Pela técnica MHG foi possível obter em simultâneo com a secagem, extratos constituídos por polifenóis e polissacarídeos. Através da adição de etanol durante o processo de secagem, a massa de extrato foi incrementada. O bagaço secado mostrou-se estável por pelo menos 2 anos, após os quais foi possível obter extratos ricos em compostos fenólicos e polissacarídeos. Em conclusão, a adoção de diferentes mecanismos de extração e de fracionamento permitiu uma maior compreensão das estruturas e propriedades químicas dos compostos presentes no bagaço de maçã, especialmente aquelas derivadas de reações de oxidação dos compostos fenólicos. Isto, em conjunto com a eficiente e rápida secagem verificada pela tecnologia de MHG abre novas perspetivas de investigação e aplicações que podem culminar na possível mitigação do bagaço de maçã como um resíduo agroindustrial.
Programa Doutoral em Ciência e Tecnologia Alimentar e Nutrição