Xylooligosaccharides (XOS) are considered to be prebiotics. Prebiotics are defined as non-digestible food ingredients that benefit the host by stimulating the growth and activity of a limited number of bacteria, such as the Bifidobacterium genus, in the colon. Corn fiber separated from distillers dried grains with solubles (DDGS) could be a valuable feedstock for XOS production. The objective of the first chapter was to determine the efficacy for autohydrolysis to produce XOS using fiber separated from DDGS. Fiber was treated with deionized water in a Parr-reactor, at temperatures ranging from 140 to 220 °C to produce XOS. The maximum total yield of XOS in the solution was 18.6 wt% of the corn fiber at 180 °C. The objective of the second chapter was to evaluate and compare the prebiotic effect of XOS produced by autohydrolysis of DDGS fiber (XOS-D) with other substrates (FOS, commercial XOS (XOS-C), xylose, glucose and inulin) on intestinal bacteria, B. adolescentis, B. breve and Lactobacillus brevis. Bacterial growth on XOS-C was comparable with growth on FOS and inulin. XOS-D promoted bacterial growth more than that of control. Prebiotic potential of XOS produced from corn fiber was confirmed. The objective of third chapter is to determine the yield of XOS from corn fiber separated from ground corn flour (FC) and DDGS (FD) at different autohydrolysis temperatures and hold-times. The conditions for maximum XOS production for FD and FC were 180 °C with 20 min hold-time and 190 °C with 10 min hold-time, respectively. The fourth chapter focuses on production of XOS by enzymatic hydrolysis method for XOS production. Endo-1-4-xylanase enzyme was ineffective for corn fiber as well as corn fiber gum (CFG), despite evaluating a multitude of pretreatment methods and processing conditions. We have proposed use of Multifect Pectinase PE and Multifect Xylanase enzymes, based on work from other researchers. For commercial applications such as food industries, XOS would need to be isolated from liquor. The fifth chapter of this study focuses on literature review of purification methods used in XOS purification.

Submitted by FRANCIANE CRISTINA DE FIGUEIREDO null (francf_4@hotmail.com) on 2016-05-09T18:39:20Z No. of bitstreams: 1 Efeito prebiótico de xilo-oligossacarídeos produzidos a partir da hemicelulose do bagaço de cana-de-açúcar utilizando extração alcalina e hidrólise de xilanases fúngicas.pdf: 1560444 bytes, checksum: 8ceefe052a8a84e57864dcbc36d1e6d0 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-05-12T19:20:29Z (GMT) No. of bitstreams: 1 figueiredo_fc_me_rcla.pdf: 1560444 bytes, checksum: 8ceefe052a8a84e57864dcbc36d1e6d0 (MD5) / Made available in DSpace on 2016-05-12T19:20:29Z (GMT). No. of bitstreams: 1 figueiredo_fc_me_rcla.pdf: 1560444 bytes, checksum: 8ceefe052a8a84e57864dcbc36d1e6d0 (MD5) Previous issue date: 2016-04-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os xilo-oligossacarídeos (XOs) e fruto-oligossacarídeos (FOs) apresentam atividades prebióticas, favorecendo a melhora nas funções intestinais, ação imunológica, antimicrobiana e outros benefícios à saúde. A produção dos XOs pode ser realizada pela utilização de materiais lignocelulósicos (MLCs). O objetivo deste trabalho foi aperfeiçoar a extração de hemicelulose a partir do bagaço de cana-de-açúcar, além de comparar a produção de XOs com xilanases de Trichoderma reesei QM 9414 e Aspergilus fumigatus M51. Foram feitos testes de digestibilidade “in vitro” e também de fermentação “in vitro” com bactérias probióticas e patogênicas para avaliar o potencial prebiótico de XOs produzidos por tratamento alcalino e compará-los com XOs e FOs comerciais. Os pré-tratamentos alcalinos desenvolvidos com 10% de KOH a 70ºC (tratamento 1) e com 5% de KOH a 121ºC (tratamento 3) se mostraram similares ao método que utiliza 24% de KOH a 35ºC (método ZD) para extração de hemicelulose, porém são mais simplificados e econômicos. O tratamento 1 apresentou redução de 54,1% de KOH e 1,98% de etanol e o tratamento 3 reduziu em 76,2% o KOH e 40,9% o etanol. Estes três experimentos de extração de hemicelulose foram selecionados para os testes de hidrólise enzimática com xilanases dos fungos T. reesei e A. fumigatus para produção de XOs. A hidrólise com enzimas de T. reesei se mostrou mais vantajosa apenas para produção de xilose (54,9%) em 24 horas, utilizando-se hemicelulose produzida pelo tratamento número 3. Testes com enzimas de A. fumigatus apresentaram melhor rendimento de XOs (27,1%) em 24 horas e menor rendimento de xilose (19,6%), com hemicelulose do tratamento 1 (XOs LABI). Experimentos de fermentação “in vitro” demonstraram a capacidade de Lactobacillus e Bifidobacterium fermentarem XOs e FOs comerciais, destacando-se a preferência de B. breve e L. brevis por XOs comercial, enquanto B. lactis e L. acidophilus demonstraram preferência por FOs comercial. B. longum cresceu tanto nos XOs e FOs comerciais e B. animalis não cresceu em nenhum oligossacarídeo comercial. Salmonella enterica em 4 horas não cresceu quando os oligossacarídeos estavam presentes, diferentes dos meios com outras fontes de carboidratos, comprovando-se a seletividade destes prebióticos por linhagens probióticas. Os XOs LABI não foram capazes de estimular o crescimento da maioria das bactérias probióticas, embora tenham inibido o crescimento de S. enterica e resistido à ação de enzimas digestivas. Estes dados indicam a provável ação prebiótica dos XOs LABI e a necessidade de purificação do produto para eliminar possíveis resíduos da extração, os quais inibem fortemente o crescimento dos micro-organismos estudados. / The xylo-oligosaccharides (XOs) and fructo-oligosaccharides (FOs) have prebiotic activities, improving bowel function, immunological and antimicrobial activity and other health benefits. XOs production can be accomplished by the use of lignocellulosic materials (LCMs). The goal of this work was to improve the hemicellulose extraction from sugarcane bagasse and to compare the XOs production using xylanases from Trichoderma reesei QM 9414 and Aspergillus fumigatus M51. We performed "in vitro" digestibility tests and also "in vitro" fermentation with probiotic and pathogenic bacteria to evaluate the prebiotic potential of XOs produced by alkali treatment and compared it with commercial XOs and FOs. Alkaline pretreatments developed with 10% KOH at 70° C (treatment 1) and 5% KOH at 121° C (treatment 3) were similar to the method using 24% KOH at 35° C (ZD method) for hemicellulose extraction, but they were more simplified and economical. Treatment 1 presented a reduction of 54.1% KOH and 1.98% ethanol and treatment 3 reduced KOH in 76.2% and ethanol in 40.9%. These three hemicellulose extraction experiments were selected for XOs production in the enzymatic hydrolysis tests with fungal xylanases of T. reesei and A. fumigatus. Hydrolysis with T. reesei enzymes proved advantageous only for xylose production (54.9%) at 24 hours, using hemicellulose produced by treatment number 3. Testing with A. fumigatus enzymes showed better XOs production (27.1%) at 24 hours and lower release of xylose (19.6%), using hemicellulose obtained by treatment 1 (XOs LABI). Fermentation experiments "in vitro" showed the ability of Lactobacillus and Bifidobacterium to ferment commercial XOs and FOs, emphasizing the preference of L. brevis and B. breve for commercial XOs while B. lactis and L. acidophilus demonstrated preference for commercial FOs. B. longum grew in both XOs and commercial FOs and B. animalis did not grow in any commercial oligosaccharide. Regarding the cultivation of Salmonella enterica, in four hours there was no growth where oligosaccharides were present, opposed to medium with other sources of carbohydrate, confirming the selectivity of this prebiotic by probiotic bacteria. XOs LABI were not able to stimulate the growth of most probiotic bacteria, although they inhibited the growth of S. enterica and resisted the digestives enzymes. These results indicate the probable prebiotic action of XOs LABI and the need of purification to eliminate possible residues from the extraction, which strongly inhibit the growth of the microorganisms studied.

Cancer is a global risk for human wellness and health. Dietary habits could profoundly affect the risk of certain cancer, such as colorectal cancer (CRC) (Platz, 2000). CRC has listed the third leading cancer among male and female in the United States (Stewart, 2014). Surprisingly, the consumption of dietary fiber has an inverse correlation with the mortality of CRC (Song, 2018). However, most Americans do not consume enough dietary fiber to meet the recommended level of dietary fiber intake (Clemens, 2012; Lee-Kwan, 2017). Hence, it is reasonable to increase the nutrient density, i.e., dietary fiber, of current food model. Xylo-oligosaccharides (XOS), an emerging prebiotics, showed multiple advantages over fructo-oligosaccharides (FOS) and inulin. For example, Hsu et al. (2004) reported XOS are more effective than FOS on increasing Bifidobacterium level in rat cecum (Hsu, 2004). It is also suggested that dietary fiber and phenolic compounds have synergistic effect on promoting gut health (Uehara, 2001; Matsukawa, 2009). Therefore, the application of XOS into a polyphenol-dense food vehicle (strawberry puree) could be a viable way to promote gastrointestinal health and help reduce CRC risk. First, the effect of xylo-oligosaccharides (XOS) incorporation on the quality attributes of strawberry puree was investigated. Rheological properties, color and physicochemical characteristics and composition such as water activity, pH, and total soluble solids, were investigated to determine the maximum XOS content that can be incorporated into the strawberry pure without significantly altering the original properties of samples. Based on the collected data, adding xylo-oligosaccharides greater than 7.5% w/w will significantly change the quality attributes of the strawberry puree including its rheological characteristics, color profile, water activity, and total soluble solids. The addition of XOS at 2.5% and 5% w/w did not significantly alter overall quality attributes of strawberry puree. Second, the effects of heat treatment (HTST, 75℃, 15s and UHT, 121℃, 2s) and storage condition (storage time: 1, 15, and 36 days; storage temperature: 4℃ and 55℃) on the quality attributes of xylo-oligosaccharides enhanced strawberry puree was studied (texture, color water activity, total soluble solids, and pH). In addition, the physicochemical (total phenolic, tannin, flavonoids contents, and antioxidant activity) evolution was studied. A 9-point Hedonic test was conducted to evaluate the sensory properties (overall, texture, color, appearance, sweetness, aroma, flavor) of purees (with and without XOS, 5%w/w) under different thermal treatments. Briefly, all nutritional attributes were retained better under cold storage (4 ℃) as compared to high-temperature storage (55 ℃). The result revealed that thermal treatment and high storage temperature showed an inverse correlation with puree consistency. As for chemical analysis, intense thermal treatment (UHT) caused the most degradation in TPC, TFC and tannin level. However, such treatments (UHT) helped significantly increase the measurable antioxidant level. For other physicochemical properties, processing methods (HTST / UHT) and high storage temperature exhibited more significance in changing the color profiles of the specimen than XOS incorporation. Overall, the addition of XOS up to 5% w/w could increase nutritional value of strawberry puree as well as consumer preference without significantly compromising quality attributes. Based on instrumental and sensory analysis, HTST treated strawberry puree with XOS incorporation (5% w/w) meet the standard of sensorial attributes of fresh puree with improved shelf-life stability and fiber concentration.

Resumo: --- / Doutor

The engineering of bacterial surfaces has in recent yearsattracted a lot of attention with applications in manydifferent areas of bioscience. Here we describe the use of twodifferent surface display systems for the gram-positivebacteria Staphylococcus carnosus and Staphylococcus xylosus invarious biotechnological applications. Environmental microbiology currently attracts a lot ofattention since genetically engineered plants and bacteriamight be used as bioadsorbents for sequestration of toxicmetals. Bacterial surface display of metal-binding peptidesmight enable recycling of the biomass by desorption ofaccumulated heavymetals. In an attempt to recruitstaphylococcal display systems for bioremediation purposes,polyhistidyl peptides were successfullly displayed on thesurface of recombinant S. carnosus and S. xylosus cells.Whole-cell Ni2+-binding assays demonstrated that therecombinant cells had gained metal-binding capacity compared towild-type cells. Tailor-made, metal-binding staphylococci was created using apreviously constructed phage-display combinatorial proteinlibrary based on a fungal cellulose-binding domain (CBD)derived from the cellobiohydrolase Cel7A of Trichoderma reseii.Novel metal-binding CBDs were generated through a phagemediated selection procedure. Selected CBD variants, now devoidof cellulose binding, were randomly selected and sequenceanalysis of selected variants revealed a marked preference forhistidine residues at the randomized positions. Surface displayof these novel CBD variants resulted in recombinantstaphylococci with increased metal-binding capacity compared tocontrol strains, indicating that this could become a generalstrategy to engineer bacteria for improved binding to specificmetal ions. Directed immobilization of cells with surface displayedheterologous proteins have widespread use in modernbiotechnology. Among other things they could provide aconvenient way of generating biofilters, biocatalysts orwhole-cell diagnostic devices. It was therefore investigatedwhether directed immobilization of recombinant staphylococci oncotton fibers could be achieved by functional display of afungal cellulose-binding domain (CBD). Recombinant S. carnosuscells with surface anchored CBDs from Trichoderma reseii Cel6Awere found to efficiently bind to cotton fibers creating almosta monolayer on the fibrous support. The co-expression of thisCBD together with previously described metal-binding proteinson the surface of our staphylococci would create means fordeveloping effective bioadsorbents for remediationpurposes. The original plasmid vector, designed for heterologoussurface display on recombinant S. carnosus cells has exhibitedproblems related to structural instability, possibly due to thepresence of a phage f1 origin of replication in the vectorsequence. This would be a problem if using the vector systemfor library display applications. Therefore, novel surfacedisplay vectors, lacking the phage ori were constructed andevaluated by enzymatic and flow cytometric whole-cell assays.One such novel vector, pSCXm, exhibited dramatically increasedplasmid stability with the retained high surface density ofexpressed heterologous proteins characteristic for the originalS. carnosus display vector, thus making it potentially moresuitable for library display applications. The successful engineering of our staphylococcal displaysystem encouraged us to further evaluate the potential to usethe staphylococcal system for display of combinatorial proteinlibraries and subsequent affinity based selections using flowcytometric cell sorting. A model system of recombinant S.carnosus cells with surface displayed engineered protein Adomains was constructed. It was demonstrated that target cellscould be sorted essentially quantitatively from a moderateexcess of background cells in a single sorting-step.Furthermore, the possibility of using staphylococcal surfacedisplay and flow cytometric cell sorting also for specificenrichment of very rare target cells by multiple rounds ofcell-sorting and in between amplification was demonstrated. <b>Key words:</b>affibody, albumin binding protein, bacterialsurface display, cell immobilization, bioremediation,combinatorial protein engineering, flow cytometry,Gram-positive, metal binding, staphylococcal protein A,Staphylococcus carnosus, Staphylococcus xylosus, whole-celldevices

<p>The engineering of bacterial surfaces has in recent yearsattracted a lot of attention with applications in manydifferent areas of bioscience. Here we describe the use of twodifferent surface display systems for the gram-positivebacteria Staphylococcus carnosus and Staphylococcus xylosus invarious biotechnological applications.</p><p>Environmental microbiology currently attracts a lot ofattention since genetically engineered plants and bacteriamight be used as bioadsorbents for sequestration of toxicmetals. Bacterial surface display of metal-binding peptidesmight enable recycling of the biomass by desorption ofaccumulated heavymetals. In an attempt to recruitstaphylococcal display systems for bioremediation purposes,polyhistidyl peptides were successfullly displayed on thesurface of recombinant S. carnosus and S. xylosus cells.Whole-cell Ni2+-binding assays demonstrated that therecombinant cells had gained metal-binding capacity compared towild-type cells.</p><p>Tailor-made, metal-binding staphylococci was created using apreviously constructed phage-display combinatorial proteinlibrary based on a fungal cellulose-binding domain (CBD)derived from the cellobiohydrolase Cel7A of Trichoderma reseii.Novel metal-binding CBDs were generated through a phagemediated selection procedure. Selected CBD variants, now devoidof cellulose binding, were randomly selected and sequenceanalysis of selected variants revealed a marked preference forhistidine residues at the randomized positions. Surface displayof these novel CBD variants resulted in recombinantstaphylococci with increased metal-binding capacity compared tocontrol strains, indicating that this could become a generalstrategy to engineer bacteria for improved binding to specificmetal ions.</p><p>Directed immobilization of cells with surface displayedheterologous proteins have widespread use in modernbiotechnology. Among other things they could provide aconvenient way of generating biofilters, biocatalysts orwhole-cell diagnostic devices. It was therefore investigatedwhether directed immobilization of recombinant staphylococci oncotton fibers could be achieved by functional display of afungal cellulose-binding domain (CBD). Recombinant S. carnosuscells with surface anchored CBDs from Trichoderma reseii Cel6Awere found to efficiently bind to cotton fibers creating almosta monolayer on the fibrous support. The co-expression of thisCBD together with previously described metal-binding proteinson the surface of our staphylococci would create means fordeveloping effective bioadsorbents for remediationpurposes.</p><p>The original plasmid vector, designed for heterologoussurface display on recombinant S. carnosus cells has exhibitedproblems related to structural instability, possibly due to thepresence of a phage f1 origin of replication in the vectorsequence. This would be a problem if using the vector systemfor library display applications. Therefore, novel surfacedisplay vectors, lacking the phage ori were constructed andevaluated by enzymatic and flow cytometric whole-cell assays.One such novel vector, pSCXm, exhibited dramatically increasedplasmid stability with the retained high surface density ofexpressed heterologous proteins characteristic for the originalS. carnosus display vector, thus making it potentially moresuitable for library display applications.</p><p>The successful engineering of our staphylococcal displaysystem encouraged us to further evaluate the potential to usethe staphylococcal system for display of combinatorial proteinlibraries and subsequent affinity based selections using flowcytometric cell sorting. A model system of recombinant S.carnosus cells with surface displayed engineered protein Adomains was constructed. It was demonstrated that target cellscould be sorted essentially quantitatively from a moderateexcess of background cells in a single sorting-step.Furthermore, the possibility of using staphylococcal surfacedisplay and flow cytometric cell sorting also for specificenrichment of very rare target cells by multiple rounds ofcell-sorting and in between amplification was demonstrated.</p><p><b>Key words:</b>affibody, albumin binding protein, bacterialsurface display, cell immobilization, bioremediation,combinatorial protein engineering, flow cytometry,Gram-positive, metal binding, staphylococcal protein A,Staphylococcus carnosus, Staphylococcus xylosus, whole-celldevices</p>

Submitted by Bruna Rodrigues (bruna92rodrigues@yahoo.com.br) on 2016-09-26T14:51:02Z No. of bitstreams: 1 DissMAF.pdf: 3997675 bytes, checksum: 3238bd54da33e9d0c201deb533005fa2 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-26T18:48:09Z (GMT) No. of bitstreams: 1 DissMAF.pdf: 3997675 bytes, checksum: 3238bd54da33e9d0c201deb533005fa2 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-26T18:48:18Z (GMT) No. of bitstreams: 1 DissMAF.pdf: 3997675 bytes, checksum: 3238bd54da33e9d0c201deb533005fa2 (MD5) / Made available in DSpace on 2016-09-26T18:48:26Z (GMT). No. of bitstreams: 1 DissMAF.pdf: 3997675 bytes, checksum: 3238bd54da33e9d0c201deb533005fa2 (MD5) Previous issue date: 2015-03-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Xylooligosaccharides (XOS) are short-chain polymers of xylose (2 to 7 units) which can be produced by enzymatic hydrolysis of the xylan from the lignocellulosic feedstocks. XOS have a great potential as probiotic ingredients, and when they are incorporated in diets, they can provide many health benefits. The worldwide interest in the use of lignocellulosic residues is constantly growing, and in this scenario the soybean hull arises as a potential residue of the Brazilian agroindustry. The bioconversion of these residues to value-added products requires suitable pretreatments to deconstruct/disorganize the recalcitrant lignocellulosic complex, separating its main fractions: cellulose, hemicellulose, and lignin. In this context, this work did evaluate different biomass pretreatments aiming to produce XOS by the action of a Bacillus subtilis endoxylanase. Initially, the conditions for maximum catalytic activity of this enzyme were evaluated changing pH, buffer, and temperature. Among these parameters, 50 mM citrate buffer, pH 5.5, and 45 oC were the one that gave highest activity. The in nature soybean hull (previously chemically characterized) was hydrolyzed with soluble endoxylanase with different enzyme loads (40, 80, and 100 U/g biomass) under preestablished pH and temperature, producing around 55 mg RS/g dry biomass. This result, though little expressive, showed the viability of XOS production from soybean hull. However, this approach requires a suitable pretreatment of the lignocellulosic biomass to improve the endoxylanase accessibility to the C-5 fraction. Several pretreatments were performed in the soybean hulls, such as, enzymatic deproteinization, hydrogen peroxide/acetic acid pretreatment, and organosolv-ethanol pretreatment. For some pretreatments, reagent concentration and reaction time were evaluated, as well as, sequential pretreatment. Besides, enzymatic hydrolysis of the in nature soybean hull under microwave irradiation was also evaluated. The deproteinization of the soybean hull was not very efficient to the enzymatic hydrolysis of the remnant solid (production of 30 mg RS/dry biomass). However, this pretreatment allows the protein recovery as a high nutritional value hydrolysate. The pretreatment of the deproteinized soybean hulls with hydrogen peroxide solution (5 M, 1 h) removed 56% lignin without cellulose losses. However, this pretreatment did not contribute to an efficient action of the endoxylanase to the hemicellulose fraction (production of around 30 mg RS/g dry biomass). The organosolv-(50% v/v)ethanol pretreatment of the deproteinized soybean hulls promoted the removal of around 50% lignin, with low solubilization of hemicellulose (<17%), producing a poor substrate for the endoxylanase. The organosolv pretreatments with 50 and 70% (v/v) ethanol of the in nature soybean hull were able to solubilize around 30% hemicellulose, allowing the production of around 76 and 49 mg RS/g dry biomass, respectively, after hydrolysis with endoxylanase. Finally, the microwave action on the lignocellulosic biomass probably decreased the biomass recalcitrance, because the hydrolysis of the in nature soybean hulls catalyzed by the endoxylanase (100 IU / g of biomass) yielded approximately 100 mg of RS/g dry biomass. On the other hand, the hydrolysis performed in a reactor under conventional heating produced only 52 mg RS/g dry biomass. The results of this work did show that the combination of microwave irradiation and enzymatic hydrolysis might be a promising alternative to produce XOS. Keywords: soybean hulls; xylo-oligosaccharides; pretreatments / Xilo-oligossacarídeos (XOS) são polímeros de xilose de cadeia curta (2 a 7 unidades) que podem ser obtidos por hidrólise enzimática da xilana presente na fração de hemicelulose dos materiais lignocelulósicos. XOS possuem um grande potencial como ingredientes prebióticos, e quando incorporados na dieta, podem fornecer muitos benefícios à saúde. O interesse mundial no aproveitamento de resíduos lignocelulósicos é cada vez maior, e no cenário nacional a casca de soja se destaca como um potencial resíduo da agroindústria brasileira. Para viabilizar a bioconversão desses resíduos em produtos de interesse comercial (etanol 2G e XOS, por exemplo) são necessários pré-tratamentos, que atuam desconstituindo/desorganizando a estrutura altamente recalcitrante do complexo lignocelulósico e separando as frações principais da biomassa: celulose, hemicelulose e lignina. Neste contexto, este trabalho teve por objetivo avaliar diferentes pré-tratamentos da biomassa para produzir sequencialmente XOS por ação de uma endoxilanase de Bacillus subtilis. Inicialmente as condições de máxima atividade catalítica dessa enzima foram avaliadas variando pH, tampão e temperatura. Dentre as variáveis estudadas, as que contribuíram para uma melhor atividade da endoxilanase foram tampão citrato de sódio (50mM) pH 5,5 e 45 °C. A casca de soja in natura (previamente caracterizada quimicamente) foi hidrolisada com endoxilanase solúvel com diferentes cargas enzimáticas (40, 80 e 100 U/g casca) nas condições de pH e temperatura pré-estabelecidas, produzindo em média 55 mg de AR/g biomassa seca. Esse resultado, embora pouco expressivo, demonstrou a viabilidade da produção de XOS a partir de casca de soja, requerendo, entretanto, um pré-tratamento adequado para melhorar a acessibilidade da endoxilanase à fração C-5 da biomassa. Os pré-tratamentos avaliados foram a desproteinização enzimática da casca, prétratamento com peróxido de hidrogênio e ácido acético e pré-tratamento organossolve-etanol, variando nestes, as concentrações de solventes, tempo de reação e pré-tratamentos sequenciais. Adicionalmente, realizou-se a hidrólise enzimática da casca de soja in natura em reator micro-ondas. A desproteinização da casca de soja mostrou-se ineficiente para a hidrólise da fração sólida remanescente com endoxilanase (produção de 30 mg de AR/g biomassa seca), embora esse pré-tratamento permita a recuperação de proteínas como um hidrolisado de alto valor nutricional. O pré-tratamento com peróxido de hidrogênio (5 M, 1 h) para casca de soja desproteinizada removeu 56% de lignina sem perdas de celulose, entretanto, este pré-tratamento não contribuiu para uma eficiente atuação da endoxilanase sobre a fração hemicelulósica (produção de aproximadamente 30 mg de AR/g biomassa seca). O pré-tratamento organossolve-etanol 50% (v/v) da casca de soja desproteinizada removeu em torno de 50% de lignina com baixa solubilização de hemicelulose (< 17%), gerando, portanto, um líquido com baixa concentração de substrato para a ação da endoxilanase. Os pré-tratamentos organossolve-etanol 50 e 70% (v/v) da casca de soja in natura foram capazes de solubilizar em torno de 30% da hemicelulose, sendo possível a produção de 76 e 49 mg de AR/g de biomassa seca, respectivamente, após hidrólise com endoxilanase. Por fim, a ação das microondas sobre a biomassa lignocelulósica provavelmente reduziu a recalcitrância da biomassa, pois a hidrólise da casca in natura com endoxilanase (100 U/g de casca) produziu aproximadamente 100 mg de AR/g de biomassa seca, ao contrário da hidrólise conduzida em reator com aquecimento convencional que produziu em torno de 52 mg de AR/g de biomassa seca. Os resultados deste trabalho indicam que a combinação de irradiação micro-ondas e hidrólise enzimática pode ser uma alternativa promissora para a produção de XOS.