Littérature scientifique sur le sujet « Polysaccharide oxidation »

This review covers methods for modifying the structures of polysaccharides. The introduction of hydrophobic, acidic, basic, or other functionality into polysaccharide structures can alter the properties of materials based on these substances. The development of chemical methods to achieve this aim is an ongoing area of research that is expected to become more important as the emphasis on using renewable starting materials and sustainable processes increases in the future. The methods covered in this review include ester and ether formation using saccharide oxygen nucleophiles, including enzymatic reactions and aspects of regioselectivity; the introduction of heteroatomic nucleophiles into polysaccharide chains; the oxidation of polysaccharides, including oxidative glycol cleavage, chemical oxidation of primary alcohols to carboxylic acids, and enzymatic oxidation of primary alcohols to aldehydes; reactions of uronic-acid-based polysaccharides; nucleophilic reactions of the amines of chitosan; and the formation of unsaturated polysaccharide derivatives.

Enzymatic conversion of polysaccharides into lower-molecular-weight, soluble oligosaccharides is dependent on the action of hydrolytic and oxidative enzymes. Polysaccharide monooxygenases (PMOs) use an oxidative mechanism to break the glycosidic bond of polymeric carbohydrates, thereby disrupting the crystalline packing and creating new chain ends for hydrolases to depolymerize and degrade recalcitrant polysaccharides. PMOs contain a mononuclear Cu(II) center that is directly involved in C–H bond hydroxylation. Molecular oxygen was the accepted cosubstrate utilized by this family of enzymes until a recent report indicated reactivity was dependent on H2O2. Reported here is a detailed analysis of PMO reactivity with H2O2 and O2, in conjunction with high-resolution MS measurements. The cosubstrate utilized by the enzyme is dependent on the assay conditions. PMOs will directly reduce O2 in the coupled hydroxylation of substrate (monooxygenase activity) and will also utilize H2O2 (peroxygenase activity) produced from the uncoupled reduction of O2. Both cosubstrates require Cu reduction to Cu(I), but the reaction with H2O2 leads to nonspecific oxidation of the polysaccharide that is consistent with the generation of a hydroxyl radical-based mechanism in Fenton-like chemistry, while the O2 reaction leads to regioselective substrate oxidation using an enzyme-bound Cu/O2 reactive intermediate. Moreover, H2O2 does not influence the ability of secretome from Neurospora crassa to degrade Avicel, providing evidence that molecular oxygen is a physiologically relevant cosubstrate for PMOs.

Safe coating formulated from biopolymer can be an alternative for better packaging for fruits. Among biopolymers used for safe coating, polysaccharides attracted more attention due to its biocompatibility and edibility. However, polysaccharide-based materials have weaknesses such as low water barrier and mechanical properties which result in lower capability on preserving the coated fruits. Hence, the incorporation of nanoparticles (NPs) such as zinc oxide (ZnO) is expected to increase the ability of polysaccharide-based coating for the enhancement of fruit shelf life. In this review paper, the basic information and the latest updates on the incorporation of ZnO NPs into the polysaccharide-based safe coating for fruit are presented. Various research has investigated polysaccharide-ZnO nanocomposite safe coating to prolong the shelf life of fruits. The polysaccharides used include chitosan, alginate, carrageenan, cellulose, and pectin. Overall, polysaccharide-ZnO nanocomposites can improve the shelf life of fruits by reducing weight loss, maintaining firmness, reducing the ripening process, reducing respiration, reducing the oxidation process, and inhibiting microbial growth. Finally, the challenges and potential of ZnO NPs as an active agent in the safe coating application are also discussed.

The specific polysaccharide of Streptococcus pneumoniae type 11F (American type 11) is composed of 2-acetamido-2-deoxy-D-glucose (one part), D-glucose (one part), D-galactose (two parts), ribitol (one part), phosphate (one part), and O-acetyl (two parts). Hydrolysis, dephosphorylation, periodate oxidation, methylation, optical rotation, and 1H and 13C nuclear magnetic resonance studies showed that the polysaccharide is an unbranched linear polymer of a ribitol-phosphate substituted repeating tetrasaccharide unit having the structure:[Formula: see text]The specific capsular polysaccharides of S. pneumoniae type 11B and 11C (American types 76 and 53) were found to have the same tetrasaccharide repeating unit as the 11F polysaccharide, but differed from it in their mode of O-acetylation and the replacement of the ribitol phosphate by glycerol phosphate in the 11C specific polysaccharide.

Zwitterionic capsular polysaccharide extracts, ∼8 kDa in mass, from Streptococcus pneumoniae type 1 (Spt1) have shown unique T-cell activating properties. Oligomers of the trisaccharide repeating unit of the Spt1 capsular polysaccharide [→3)-4-NH2-α-d-QuipNAc-(1→4)-α-d-GalpA-(1→3)-α-d-GalpA-(1-]n of defined length are needed to further investigate this response. An approach towards iteratively extendable trisaccharide building blocks of the zwitterionic capsular polysaccharides of Spt1 is described. Key elements include the comparison of pre-glycosylation oxidation and post-glycosylation oxidation approaches using thioglycoside donors to the target trisaccharide, the optimisation of the post-glycosylation oxidation approach, and the conversion of the trisaccharide to building blocks tailored for iterative glycosylation. The construction and evaluation of stereotunable 2-N-3-O-oxazolidinone donors for the common bacterial 2-acetamido-4-amino-2,4,6-trideoxy-α-d-galactopyranoside motif is also described, as is a key intermediate for their efficient synthesis.

Abstract The lignin oxidation in the oxygen delignification/bleaching process catalysed by Mn(II) substituted heptamolybdopentavanadophosphate polyanion (HPA-5-MnII) was studied using monomeric lignin model compounds and Eucalyptus globulus dioxane lignin adsorbed on the bleached sulphite pulp. The analysis of reaction products after lignin models oxidation in the presence HPA-5-MnII and the parent heptamolybdopentavanadophosphate polyanion (HPA-5) showed similar qualitative and quantitative patterns, indicating the identical lignin oxidative delignification mechanisms with both catalysts. The higher delignification selectivity of the pulp in the reaction system HPA-5-MnII/O2 than with HPA-5/O2 was explained by the protective effect of MnII in the heteropolyanion towards oxidative degradation of polysaccharides. The study of lignin adsorbed on bleached sulphite pulp, in the HPA-5-MnII/O2 delignification system, showed the cleavage of β-O-4 linkages as the major contribution to the lignin depolymerisation. The formation of Cα-O-polysaccharide linkages in β-O-4 structures of lignin during the delignification was observed.

Salmonella madelia reported to express the O-antigenic factors 1, 6, 14, and 25, defined in the Kauffmann–White classification system, was found to produce three different homogeneous lipopolysaccharides, which differed in having three structurally distinct O-polysaccharide components. The O-polysaccharide fraction obtained by mild acetic acid hydrolysis of the S. madelia lipopolysaccharide was analyzed by chemical composition, nitrous acid deamination, periodate oxidation, methylation, and 1H and 13C nuclear magnetic resonance methods and was demonstrated to be composed of three polysaccharides, PS(I), PS(II), and PS(III), which had the structures of repeating oligosaccharide units:[Formula: see text]Key words: Salmonella madelia, lipopolysaccharide, structure, analysis, nuclear magnetic resonance.

AbstractIdeally, a bioscaffold should mimic the characteristics of an extracellular matrix of a living organ of interest. The present study deals with the formation of composite scaffolds of collagen with gum arabic. Collagen was cross-linked with oxidized gum arabic having aldehyde groups to form a porous block. By changing the oxidation level of gum arabic, incorporation of the polysaccharides into the scaffold could be varied resulting in scaffolds with variable polysaccharide to protein content. A series of scaffolds were made by altering collagen concentration and oxidation level of gum arabic. The scaffolds were tested for their physical properties, stability, biocompatibility and ability to support the cell growth. Results implied that variable polysaccharide incorporation into the scaffolds was possible depending on the oxidation level of gum arabic which could influence the swelling behavior. The scaffolds showed non-toxic behavior towards the mesenchymal stem cells and nucleus pulposa cells using viability assay in culture conditions up to 30 days; the growth of cells was seen at all combinations of gels. Nucleus pulposa cells were able to maintain their phenotype in the GACO gels. The studies show that these scaffolds are potential candidates in applications, such as tissue engineering, and can be designed to match the requirement of different cell/tissues as per their ECM.

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.

Accumulation of non-biodegradable plastics is causing high levels of environmental pollution. Currently, edible and biodegradable films and coatings for food and nutraceutical applications are a fast emerging technology with increased attention among researchers and consumers, which acts as an alternative to these synthetic plastics. Edible coatings are most commonly developed from polysaccharides and proteins. Among polysaccharides, seaweed-based polysaccharides play a vital role. These seaweed-based polysaccharides, which are utilized in the development of edible coatings and films, include Agar, Alginate, and carrageenan. Alginates are extracted from brown algae, while agar and carrageenan are extracted from red algae varieties. These developed coatings and films are commonly applied to extend the shelf life and maintain the desired quality level in food. These films or coatings can be applied to foods such as fruits, vegetables, meat, poultry, seafood, and dairy products. They improve the quality of the product by retarding moisture loss, reducing lipid oxidation and discoloration, sealing in volatile flavors, and functioning as carriers of food additives such as antimicrobial and antioxidant agents. This book chapter discusses the application of seaweed-based biodegradable films and coatings for food and nutraceuticals.

Genomic DNA extraction is the starting point for various downstream molecular biology applications viz. PCR, restriction analysis, hybridisation etc. Numerous problems like DNA degradation, co-isolation of viscous polysaccharides, polyphenols and other secondary metabolites causing damage to DNA, inhibiting restriction enzymes, DNA polymerases etc, are routinely encountered during DNA isolation from plants. Quinone compounds resulting from oxidation of polyphenols lead brown the DNA preparations and can also damage proteins and DNA’s due to their oxidizing properties. This results in a poor yield of high molecular weight DNA. The protocol below explains the extraction of DNA via the CTAB method, involving three major steps viz lysis of cell wall and membranes, extraction of genomic DNA and precipitation of DNA.

Edible coatings are thin layers of edible materials formed directly on fruits, usually by immersing the fruits in a coating material solution, and they are one of the most intriguing food developments in recent years. Colorants, flavours, nutrients, and anti-browning and antimicrobial agents can all be carried by edible coatings, extending shelf life and reducing pathogen growth on food surfaces. To manage moisture transfer, gas exchange, or oxidative processes, edible coatings can be applied using various procedures such as dipping, spraying, or coating. Because these systems have a larger surface area, nanoparticles may help to improve the barrier characteristics and functionality of fruit preservation coatings. Antimicrobial nanoparticles (NPs) are employed as matrixes in edible coatings and films (ECF), which are then applied to fruits to extend shelf life and improve storage quality. Nano Chitosan is one of the most prevalent polysaccharides, protein, and lipid-based edible coatings. These are characterised by poor gas and water barrier qualities, and they are frequently used as moisture loss sacrifice agents. Therefore, the purpose of this book chapter is to study the effect of nano edible coatings such as chitosan/tripolyphosphate (TPP), chitosan-methyl cellulose/silica (SiO2), gelatin-fiber/titanium dioxide (TiO2), gelatin-chitosan/ (Ag/ZnO), Gelatin/kafirin to quality attributes and prolonging the shelf life of fruits.

Marine fish oil is the richest source of long-chain n-3 polyunsaturated fatty acids (LCn3PUFAs), particularly rich in EPA and DHA, with many health benefits. Lutein and curcumin are two bioactive compounds that prevent age-related macular degeneration and provide anti-cancer and anti-inflammatory functions. Directly adding fish oil (FO), lutein, and curcumin to meat during processing can produce products rich in DHA/ EPA, lutein, and curcumin and help improve human health without changing consumers' dietary habits. However, FO, lutein, and curcumin are unstable under processing and storage conditions and can cause undesirable quality issues such as lipid oxidation and fishy odor to the meat products. Encapsulation is a common strategy to overcome these challenges, and essential oils (EO) extracted from spices can mask fishy odor and inhibit lipid oxidation during encapsulating, processing, and storage. Thus, the FO and EOs were co-encapsulated first, and then lutein and curcumin were incorporated into the encapsulate to provide more functions to the final products. All the encapsulated FO-EO powders were stable at room temperature during the first ten days of storage, but GO produced the best protective effect among the EOs. Adding lutein or curcumin to the garlic EO-FO co-encapsulate significantly increased the MDA content in the encapsulation powders after ten days of storage. Similar results were also observed in the rosemary EO-FO group. The increased MDA content in the lutein- or curcumin-added FO-EO encapsulates might be due to the long dissolving time of the lutein or curcumin in the FO, which allowed prolonged air contacts to the fish oil. The encapsulation efficiency (EE%) of the final products increased significantly when rosemary and garlic EOs, lutein, and curcumin were used, but pepper black EO resulted in a decrease in the EE, probably due to the differences in the polarities of the essential oils.

Polysaccharides have the potential to be used as green packaging materials because of their biodegradability, biocompatibility and renewability. In this work, soluble soybean polysaccharide (SSPS) based films containing gelatin and curcumin were fabricated for oil packaging. These composite films were heat seable and can easily be made into pouches. These pouches were used as food packaging material in edible oil packaging. The structure, physical and mechanical properties of the SSPS-based composite films were investigated. When compared to neat SSPS and SSPS/gelatin films, the composite films containing curcumin showed excellent antioxidant activity and certain antibacterial performance. The high oxygen barrier properties of the composite films resulted in reduced oxidative rancidity of soybean oil packaged in the pouches made from the SSPS-based films. Our results indicated that blending of SSPS, gelatin and curcumin effectively improved heat sealability and activity for edible oils.

The results of experimental studies of the kinetics of oxidation of maltodextrin with sodium periodate and hydrogen peroxide are presented, and the effect of oxidation conditions on the properties of the polymer tanning agent is shown. The obtained dialdehyde derivatives of maltodextrin have been used for structuring gelatin and in the tanning process with positive results.

Iron oxide nanoparticles are of interest for drug delivery, since they can be targeted using a magnetic field. However, prior to using nanoparticles in vivo, they must be shown as relatively non-toxic to cells. We and others have shown that bare iron oxide nanoparticles are readily taken up by cells, where they catalyze production of highly toxic reactive oxygen species (ROS). This oxidative stress disrupts the cell cytoskeleton and alters cell mechanics. [1] Iron oxide nanoparticles under current development for in vivo biomedical applications are often coated with a polysaccharide (eg. dextran) or a polymer (eg. polyethylene glycol, PEG). Both the size and the surface coating of nanoparticle may play an important role in cell toxicity.

Emulsions including Pickering emulsions and nanoemulsions have enormous application potential for inhibiting lipid oxidation. Polysaccharides like κ-carrageenan can improve the gel-like behavior of protein-stabilized Pickering emulsions. Tea water-insoluble proteins and κ-carrageenan can be used for preparing fish oil-in-water Pickering emulsions and fish oil gels. The characteristics of Pickering emulsions and fish oil gels at different proportion of tea water-insoluble proteins and κ-carrageenan were analyzed by high-speed homogenization assisted with ultrasonic treatment. The physicochemical analysis and secondary structure of mixtures showed that the turbidity reduced, particle size unchanged except that of mixtures at 80:20 and zeta potential was unaltered with the addition of κ-carrageenan; meanwhile, the secondary structure of mixtures changed due to the addition of κ-carrageenan. The viscoelastic behavior enhanced compared with that stabilized by tea water-insoluble proteins only. Fish oil gels using mixture-stabilized PEs as a template had a solid-like behavior with a storage modulus of approximately 200 kPa. Besides, the effects of tocopherol nanoemulsions on lipid oxidation of fish sausages were assessed during 16-day storage at 4 °C. Nanoemulsions including 250 and 500 mg/kg tocopherol improved fish sausages’ quality during cold storage and effectively inhibited lipid oxidation due to low peroxide value and high polyunsaturated fatty acid content. The droplet diameter of nanoemulsions including tocopherol stored at 4 °C for 16 days was below 500 nm. The small droplets with even distribution, and high stability of tocopherol nanoemulsions might explain the potential mechanism of inhibiting lipid oxidation in fish sausages. Interestingly, nanoemulsions encapsulated with 250 mg/kg tocopherol were effective to delay the lipid oxidation and improve fish sausages quality during cold storage. These findings will be useful for developing new edible hydrocolloid systems such as Pickering emulsions and nanoemulsions to inhibit lipid oxidation in aquatic products and broaden the application of emulsions in food industry.

Innate immune responses in animals and plants involve receptors that recognize microbe-associated molecules. In plants, one set of this defense system is characterized by large families of TIR–nucleotide binding site–leucine-rich repeat (TIR-NBS-LRR) resistance genes. The direct interaction between plant proteins harboring the TIR domain with proteins that transmit and facilitate a signaling pathway has yet to be shown. The Arabidopsis genome encodes TIR-domain containing genes that lack NBS and LRR whose functions are unknown. Here we investigated the functional role of such protein, TLW1 (TIR LECTIN WOUNDRESPONSIVE1). The TLW1 gene encodes a protein with two domains: a TIR domain linked to a lectin-containing domain. Our specific aim in this proposal was to examine the ramifications of the TL1-glycan interaction by; A) The functional characterization of TL1 activity in the context of plant wound response and B) Examine the hypothesis that wounding induced specific polysaccharides and examine them as candidates for TL-1 interactive glycan compounds. The Weizmann group showed TLW1 transcripts are rapidly induced by wounding in a JA-independent pathway and T-DNA-tagged tlw1 mutants that lack TLW1 transcripts, fail to initiate the full systemic wound response. Transcriptome methodology analysis was set up and transcriptome analyses indicates a two-fold reduced level of JA-responsive but not JA-independent transcripts. The TIR domain of TLW1 was found to interact directly with the KAT2/PED1 gene product responsible for the final b-oxidation steps in peroxisomal-basedJA biosynthesis. To identify potential binding target(s) of TL1 in plant wound response, the CCRC group first expressed recombinant TL1 in bacterial cells and optimized conditions for the protein expression. TL1 was most highly expressed in ArcticExpress cell line. Different types of extraction buffers and extraction methods were used to prepare plant extracts for TL1 binding assay. Optimized condition for glycan labeling was determined, and 2-aminobenzamide was used to label plant extracts. Sensitivity of MALDI and LC-MS using standard glycans. THAP (2,4,6- Trihydroxyacetophenone) showed minimal background peaks at positive mode of MALDI, however, it was insensitive with a minimum detection level of 100 ng. Using LC-MS, sensitivity was highly increased enough to detect 30 pmol concentration. However, patterns of total glycans displayed no significant difference between different extraction conditions when samples were separated with Dionex ICS-2000 ion chromatography system. Transgenic plants over-expressing lectin domains were generated to obtain active lectin domain in plant cells. Insertion of the overexpression construct into the plant genome was confirmed by antibiotic selection and genomic DNA PCR. However, RT-PCR analysis was not able to detect increased level of the transcripts. Binding ability of azelaic acid to recombinant TL1. Azelaic acid was detected in GST-TL1 elution fraction, however, DHB matrix has the same mass in background signals, which needs to be further tested on other matrices. The major findings showed the importance of TLW1 in regulating wound response. The findings demonstrate completely novel and unexpected TIR domain interactions and reveal a control nexus and mechanism that contributes to the propagation of wound responses in Arabidopsis. The implications are to our understanding of the function of TIR domains and to the notion that early molecular events occur systemically within minutes of a plant sustaining a wound. A WEB site (http://genome.weizmann.ac.il/hormonometer/) was set up that enables scientists to interact with a collated plant hormone database.