Ces travaux de thèse CIFRE, réalisés au sein du Laboratoire de Biotechnologie et Chimie Marines de l’Université de Bretagne-Sud et du Groupe Olmix, s’inscrivent dans le cadre d’un projet national de valorisation de biomasses d’algues vertes, le projet ULVANS. Ce projet est le fruit d’une collaboration entre cinq industriels bretons et deux laboratoires universitaires. Les objectifs de ces travaux de thèse concernent i) la caractérisation approfondie de la matière première, en particulier des polysaccharides matriciels solubles (ulvanes) et des protéines, ii) la mise au point d’un procédé d’extraction assistée par enzymes des métabolites algaux, iii) la caractérisation biochimique et moléculaire des hydrolysats enzymatiques, avec pour objectif la compréhension de l’effet des enzymes commerciales sur les algues, iv) l’évaluation des activités antioxydantes et antivirales des hydrolysats, v) l’étude de l’influence des paramètres d’hydrolyse en vue de déterminer les conditions optimales pour l’extraction des métabolites d’intérêt et enfin vi) de conclure, à partir des éléments fournis par l’étude, sur la viabilité d’un tel procédé à l’échelle industrielle. En conclusion de ces travaux, l’hydrolyse enzymatique apparait comme un procédé intéressant pour le bioraffinage des algues vertes. Bien que les préparations enzymatiques commerciales utilisées ne soient pas spécifiques des composés algaux, les protéases ont conduit à une augmentation significative des rendements d’extraction, alors que l’effet des carbohydrases reste modéré. L’étude de l’influence des paramètres d’hydrolyses a confirmé les résultats préliminaires et montré que la température, la concentration d’enzyme et la méthode de broyage présentaient peu d’effets sur les activités des protéases testées. Un résultat majeur de cette étude aura été la mise en évidence d’activités anti-herpétiques dans les hydrolysats. La caractérisation des fractions actives a montré que les activités étaient liées à la présence de protéines ou glycoprotéines dans les extraits. Ce résultat présente un intérêt majeur car à l’heure actuelle, a priori, aucune activité de ce type n’a été identifiée chez Ulva sp. / This PhD Thesis works, conducted in the Laboratoire de Biotechnologie et Chimie Marines of the Université de Bretagne-Sud and in the Olmix Group, was included in a national project for the upgrading of green seaweed biomasses, the Ulvans project. This project results in the collaboration of five industrial companies and two academic research laboratories. The objectives of this thesis works had been i) the characterization of the raw material, particularly the soluble matrix polysaccharides (ulvans) and proteins, ii) the development of an enzyme-assisted extraction process of algal metabolites, iii) the biochemical and molecular characterization of the enzymatic hydrolyzates, with the aim of understanding the effect of enzymes on algae thallus, iv) the screening of antioxidant and antiviral activities of hydrolyzates, v) the study of the influence of hydrolysis parameters to determine the optimum conditions for the extraction of metabolites of interest and finally vi) to conclude, according to the results provided by this study, on the viability of the industrial upscaling of the process. In conclusion of this work, enzymatic hydrolysis appeared to be an effective process for biorefinery of green seaweeds. Although commercial enzymatic preparations were not specific of algal compounds, protéases led to a significant increase in the extraction yields, whereas the effect of carbohydrases were moderate. The study of hydrolysis parameters confirmed the preliminary results and showed that the temperature, the concentration of enzyme and the grinding method had no effect on the protease used. A major result of this study has been the highlighting of anti-oxidant and anti-herpetic compounds in hydrolyzates. The antiviral activity of ulvans had several times been demonstrated but the biochemical characterization of actives fractions showed that the activity could be associated to proteins or glycoproteins. This result is very interesting because, a priori, any antiviral activity has also been related to this type of compounds in Ulva sp.

Orientadores: Maria Aparecida Silva, Adriano Viana Ensinas / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-22T07:39:18Z (GMT). No. of bitstreams: 1 Albarelli_JulianaQueiroz_D.pdf: 4345487 bytes, checksum: 3b35163c3127eaa2e83d7a92e58466c2 (MD5) Previous issue date: 2013 / Resumo: Nesta tese, utilizou-se o simulador comercial Aspen Plus® para simular o processo de produção conjunta de açúcar, etanol e eletricidade em uma usina convencional e a produção de etanol e eletricidade em uma destilaria autônoma. A integração dos processos e diferentes usos para o bagaço excedente foram estudados. Foram avaliadas modificações nos processos convencionais para aumentar a produção de eletricidade e a produção de etanol de segunda geração, utilizando como matéria prima o bagaço ou fração fina do bagaço composta por células de parênquima (fração-P). Os casos estudados também foram avaliados economicamente e quanto à captação de água, propondo alternativas para minimização desta. Os resultados indicaram que a integração energética dos processos convencionais minimizou a quantidade de vapor necessária, disponibilizando uma maior quantidade de bagaço para outros usos. O uso do bagaço excedente, em conjunto com a palha, como combustível em um sistema de cogeração com a instalação de uma turbina de condensação, permitiu o aumento da eletricidade disponível em 244%, quando utilizada a maior quantidade de palha admitida para o caso estudado para a destilaria autônoma. Os casos que estudaram a fração-P apresentaram maior produção de energia elétrica e produção de etanol/t material lignocelulósico do que os casos de segunda geração a partir do bagaço. Todos os casos apresentaram captação de água superior à permitida no Estado de São Paulo, 1 m3/t de cana de açúcar, sendo desta forma considerada a concentração da vinhaça e o aproveitamento, após tratamento, da água de lavagem do material pré-tratado, para os casos de segunda geração. A análise econômica mostrou que é possível aumentar a lucratividade dos processos convencionais, a longo prazo, com modificações no sistema de cogeração resultando em pouca alteração no tempo de retorno do investimento. O uso da fração-P para produção de etanol de segunda geração, tendo como pré-tratamento LHW e utilizando custo baixo para as enzimas, se apresentou igualmente atrativo, do ponto de vista econômico, ao melhor caso estudado de modificações no sistema de cogeração. Menores tempo de retorno, rentabilidade do investimento e lucratividade, a longo prazo, foram encontrados para os casos que consideraram a produção conjunta de açúcar e etanol. A viabilidade econômica dos processos de segunda geração apresentou-se altamente dependente do custo das enzimas / Abstract: In this thesis, the commercial simulator Aspen Plus® was used to simulate the conventional mill producing sugar, ethanol and electricity and the autonomous distillery producing ethanol and electricity. The integration of processes and different uses for the surplus bagasse were studied. It was evaluated changes in conventional processes to increase electricity production and the production of second generation ethanol using bagasse or bagasse fine fraction composed by parenchyma cells (fraction-P). The cases were economically evaluated and water uptake was analyzed, alternatives were proposed to its minimization. The results indicated that the energy integration of the conventional processes minimized the amount of steam, it provided a larger amount of bagasse for other uses. The use of bagasse, together with sugarcanetrash, as a fuel to the cogeneration system considering the installation of a condensation turbine led to an increase of 244% in the available electricity when using the higher amount of sugarcane-trash studied for the autonomous distillery. The cases that studied the P-fraction showed higher production of electricity and ethanol/t lignocellulosic material than the cases of second generation that used bagasse. All cases presented water uptake higher than allowed in the state of São Paulo, 1 m3/t de sugarcane, therefore, it was considered the concentration of vinasse and recovery of wash water from the pre-treated material, for the second generation cases. The economic analysis showed that it is possible to increase the profitability of the conventional processes, in the long term, with changes in the cogeneration system resulting in little change in the payback time of the investment. The use of P-fraction for production of second generation ethanol, with the LHW pretreatment and considered low cost of the enzymes, was equally attractive, from an economic point of view, to the best studied case of changes in the cogeneration system. Lower payback time, return on the investment and long-term profitability were found for cases that considered the joint production of sugar and ethanol. The economic viability of the second generation process presented itself highly dependent to the enzyme cost / Doutorado / Engenharia Química / Doutor em Engenharia Química

Orientadores: Gustavo Henrique Goldman, Juliana Velasco de Castro Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T05:49:45Z (GMT). No. of bitstreams: 1 Borin_GustavoPagotto_M.pdf: 4025117 bytes, checksum: 5d652481786ac0f6cf4259069d181514 (MD5) Previous issue date: 2015 / Resumo: A parede celular vegetal é uma estrutura recalcitrante, composta por polissacarídeos complexos que podem ser quebrados em açúcares fermentáveis. A desconstrução desse material complexo pode ser feita por diversos tipos de enzimas hidrolíticas, que são produzidas naturalmente por uma variedade de microrganismos. Entre eles, o fungo Trichoderma reesei se destaca pela capacidade de produzir e secretar estas enzimas em grandes quantidades. Embora alguns trabalhos utilizando abordagens de proteômica e transcriptômica já tenham sido realizados com esse fungo, ainda não são conhecidos em detalhes os mecanismos moleculares responsáveis pela degradação da parede e a regulação gênica envolvida nesse sistema lignocelulolítico. O presente trabalho tem como objetivo principal a análise da expressão gênica global de T. reesei, crescido por 6, 12 e 24 horas em bagaço e colmo de cana-de-açúcar como fontes únicas de carbono, pela técnica de sequenciamento high-throughput de RNA (RNA-Seq). No transcriptoma de T. reesei foram identificadas sendo hiper-expressas as principais celulases, hemicelulases e proteínas acessórias relacionadas direta ou indiretamente com a desconstrução da parede vegetal. De modo geral, as celulases e hemicelulases apresentaram uma expressão maior do que outras enzimas, e o nível dos seus transcritos foi crescente ao longo do tempo tanto em colmo quanto no bagaço. A grande maioria dos genes de CAZymes e proteínas acessórias hiper-expressos foram compartilhados pelos dois substratos, o que demonstra que a estratégia usada por T. reesei para degradar a parede celular do colmo e do bagaço é similar. Adicionalmente, vários fatores de transcrição, proteínas de função desconhecida e transportadores supostamente envolvidos na assimilação dos açúcares liberados também foram hiper-expressos nas condições amostradas. Para validação do RNA-Seq, foi realizado PCR em tempo real de diversos genes hiper-expressos que codificam para enzimas hidrolíticas, reguladores transcricionais, proteínas acessórias e genes ainda não caracterizados. Para isso, a análise temporal foi ampliada para 30 minutos, 2, 4, 6, 12 e 24 horas de crescimento após o inóculo, o que permitiu uma análise mais detalhada da expressão desses genes. Como objetivo secundário, foi analisado o secretoma deste fungo e os açúcares concomitantemente liberados no sobrenadante. Estas análises indicaram que a desconstrução da parede celular já se inicia dentro de 6 horas pós inoculo, com a liberação de monômeros (principalmente xilose e glicose) dos polissacarídeos e secreção de diversas CAZymes. Ensaios enzimáticos também foram realizados, mostrando atividades celulo e hemicelulolíticas. Assim, descrevemos pela primeira vez o arsenal de enzimas transcritas e secretadas por T. reesei RUT C30, desde pontos inicias de crescimento, em bagaço explodido e colmo de cana-de-açúcar. Por fim, este trabalho também permitiu a identificação de vários genes, com função predita ou não, que podem abrir caminho para a descoberta de novos atuantes na resposta do fungo ao substrato lignocelulósico / Abstract: Plant cell wall is a recalcitrant structure composed of complex polysaccharides which can be broken down into fermentable sugars. The deconstruction of this complex material can be made by a variety of hydrolytic enzymes which are naturally produced by a variety of microorganisms. Among them, stands out the fungus Trichoderma reesei, able to produce and secrete those enzymes in large quantities. Although some studies using transcriptomics and proteomics approaches have been performed with this fungus, the molecular mechanisms responsible for the degradation of the cell wall and gene regulation involved in this lignocellulosic system are not well known. This work has as main objective the analysis of global gene expression of T. reesei grown at 6, 12 and 24 hours in sugarcane bagasse and culm as sole carbon sources by high-throughput RNA sequencing technology (RNA-Seq). In the T. reesei transcriptome, it was identified the major cellulases, hemicellulases and accessory proteins directly or indirectly related to the deconstruction of plant cell wall. In general, cellulases and hemicellulases exhibited higher expression than other enzymes, and the level of their transcripts was increased over the time in both culm and bagasse cultures. Most of up-regulated CAZymes and accessory proteins were shared between the two substrates, which demonstrates the strategy used by T. reesei to degrade the bagasse and culm cell wall is similar. Furthermore, several transcription factors, proteins of unknown function and transporters supposedly involved in the assimilation of sugars were also up-regulated in the sampled conditions. To validate the RNA-Seq data, real-time PCR of several up-regulated genes encoding hydrolytic enzymes, transcriptional regulators, accessory proteins and proteins not yet characterized was carried out. The time points was extended to 30 min, 2, 4, 6, 12 and 24 hours of growth after inoculation, allowing a more detailed analysis of the expression of these genes. As a secondary objective, T. reesei secretome and the sugars released in the supernatant were analyzed. It was shown that the sugarcane cell wall deconstruction begins within the first 6 hours post inoculation, releasing sugar monomers (mainly xylose and glucose) from polysaccharides due to the secretion of several hydrolytic enzymes. Enzymatic assays were also performed, showing cellulosic and hemicellulosic activities. Finally, this is the first study showing the arsenal of enzymes transcribed and secreted by T. reesei grown on steam exploded sugarcane bagasse and culm, at early time points. It was possible identify several genes, with predicted function or not, that can open new paths to discover novel players on the fungus response to lignocellulosic substrate / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

In diploma thesis the process of enzymatic hydrolysis of waste paper as a source for the production of liquid biofuels is discused. It follows directly the homonymous diploma thesis from Ing. Brummer, and it is based on the findings, which were solved and decided in previous work. In the theoretical part there is a summarization of basic information on the enzymatic hydrolysis of waste paper and the associated influences of various factors of the rate and degree of hydrolysis. Higher attention is paid to a waste cardboard and its pretreatment methods due to the maximalization of the yield of hydrolysis. The next part summarizes options of the fermentative production of biofuels, focusing on the method of simultaneous saccharification and fermentation, where the further appropriate organism for ethanol fermentation is discussed. The last part is about the technological process from the raw material input to the separation of ethanol. In the experimental section the pre-treatment of waste paper in order to maximize the efficiency of hydrolysis was examined. The best results were achieved using a vibratory mill. In addition, various parameters for simultaneous saccharification and fermentation were optimized using enzymes from Novozymes® company and the yeast Saccharomyces cerevisiae. The conversion rate of waste paper cellulose to reducing sugars was observed by spectrophotometric method by Somogyi - Nelson and the amount of produced ethanol was quantified using HPLC / RI. As a part of this thesis some conditions (amount of enzyme, substrate, nutrients, yeasts, temperature, pH, type of buffer) were optimized to maximize the effectiveness of the overall process. All experiments were carried out on corrugated cardboard, which was chosen as the most promising material for hydrolysis that was among the waste paper pulp in diploma thesis by Ing. Brummer.

In the diploma thesis are discussed the process of enzymatic hydrolysis of waste paper as a source for the production of bioethanol by bacteria Zymomonas mobilis. In the theoretical part summarize basic information about particular methods of hydrolysis, about paper used as a raw material for enzymatic hydrolysis, about possibilities of the fermentative production of bioethanol focusing on the method of simultaneous saccharification and fermentation comparison with enzymatic hydrolysis and fermentation. Suitable microorganisms for ethanolic fermentation and simultaneous saccharification and fermentation and their advantages and disadvantages, are further discussed in this part as well. The theoretical part ends with the suggestion of the technological process for production of bioetanol. It covers all necessary steps from the input of raw material to the separation of produced ethanol. In the experimental part various parameters of hydrolysis, fermentation and simultaneous saccharification and fermentation were optimized using enzymes from Novozymes® company and the Zymomonas mobilis CCM2770 and Zymomonas mobilis LMG457 bacterium. The conversion rate of paper cellulose to gluckose and production of ethanol were observed by HPLC/RI method. Type of buffer, quantity of cells, enzyme and substrate were optimized in order to maximize the efficiency of the process. All experiments were performed on paper containing high amount of cellulose and for comparison on standard medium which contains gluckose. The highest yields was achieved with the use of Novozymes® Cellulosic ethanol enzyme Kit. The strain Zymomonas mobilis LMG457 has demonstrated as a better producer.

The potential to use acidic hydrogen peroxide in the presence of ferrous ions (Fenton’s reagent) as a pre-treatment when producing microfibrillar cellulose (MFC) from a fully bleached birch (Betula verucosa) kraft pulp was investigated and the properties of the produced MFC was compared to the properties of a MFC produced with enzymatic pre-treatment with a monocomponent endoglucanase (FiberCare® R). The mechanical treatment to MFC was performed in a laboratory colloid mill or in a pilot high-pressure homogeniser and the pre-treated pulps as well as the produced MFCs were chemically and morphologically characterised. Additionally, the MFCs produced in the colloid mill were evaluated as strength enhancers in test sheets representing the middle ply of paperboard. From the chemical characterisation, it was concluded that the Fenton pre-treatment caused a decrease in the degree of polymerisation (DP) and an increase in both carboxyl- and carbonyl groups. The increase in carbonyl groups could not be explained by the formation of new reducing end groups due to depolymerisation which indicates that carbonyl groups are introduced along the cellulose chain. The enzymatic pre-treatment as performed in this study caused less impact on the cellulosic material, i.e. resulted in a pulp with a higher DP and a much lower amount of carbonyl- and carboxylic groups compared with the Fenton pre-treated pulps. In the subsequent mechanical treatment in a colloid mill, the Fenton pre-treated pulps were easier to process mechanically i.e. reached a higher specific surface area and a higher surface charge at a given mechanical treatment time compared to enzymatic pre-treated pulps and pulps not subjected to any pre-treatment. These findings were confirmed when MFCs were produced by homogenisation at high pressure in multiple passes; the birch kraft pulp was either pre-treated with Fenton’s reagent or the combined mechanic and enzymatic pre-treatment methodology used at the Centre Technique du Papier (CTP, France). By size fractionation, rheological measurements and scanning electron microscopy, it was revealed that Fenton pre-treatment resulted in MFC suspension containing a significantly higher proportion of small sized material (< 0.2 mm). When the MFCs were evaluated as strength enhancers in test sheets produced from a furnish consisting of a spruce (Picea abies) chemithermomechanical pulp, MFC and a retention system containing cationic starch and an anionic silica sol, Fenton pre-treated MFCs increased the strength properties more than the enzymatic pre-treated MFCs. Addition of 5 wt% Fenton pre-treated MFC resulted in an increase in z-directional strength of about 50%, an increase in tensile stiffness index of about 25% and an increase in tensile index of 35% compared to test sheets prepared without MFC addition. / The potential to use acidic hydrogen peroxide in the presence of ferrous ions (Fenton’s reagent) as a pre-treatment when producing microfibrillar cellulose (MFC) from a bleached birch kraft pulp was investigated and the properties of the produced MFC was compared to the properties of a MFC produced with enzymatic pre-treatment. Additionally, the MFCs evaluated as strength enhancers in test sheets representing the middle ply of paperboard. From the chemical characterisation, it was concluded that the Fenton pre-treatment caused a decrease in the degree of polymerisation (DP) and an increase in both carboxyl- and carbonyl groups. In the subsequent mechanical treatment in a colloid mill, the Fenton pre-treated pulps were easier to process mechanically indicating a potential to lower the energy consumption. When the MFCs were evaluated as strength enhancers in test sheets, Fenton pre-treated MFCs increased the strength properties more than the enzymatic pre-treated MFCs at a given mechanical treatment time. Addition of 5 wt% Fenton pre-treated MFC resulted in an increase in z-directional strength of about 50%, an increase in tensile stiffness index of about 25% and an increase in tensile index of 35% compared to test sheets prepared without MFC addition.

This thesis project is a contribution to addressing the pressing need for sustainable alternatives to fossil fuels and traditional plastics. The work focuses on enzymatic saccharification of microalgae biomass to generate a sugar-rich stream as raw material for biofuels and bioplastics production. Microalgae and cyanobacteria are highlighted for their ability to convert CO2 into valuable biomass components in the presence of light.  To enhance biomass conversion efficiency, the enzymatic hydrolysis of microalgae and cyanobacteria is explored by utilizing various enzymes to break down complex polymers into valuable sugars. Additionally, the cultivation of cyanobacteria is studied to optimize the overall process. Results indicate challenges, such as measurement uncertainty and the need for biomass pretreatment, suggesting areas for further research.  The primary objective of this thesis work is to optimize enzymatic hydrolysis processes by determining the optimal combinations of enzymes, to enhance biomass conversion efficiency. It also underscores the importance of microorganisms in transitioning to a more environmentally friendly future by offering sustainable alternatives to conventional products.

<p>Valorizacija permeata kao sporednog proizvoda industrije mleka je od izuzetnog ekolo&scaron;kog, ekonomskog i tehnolo&scaron;kog značaja.<br />Cilj doktorske disertacije je razvoj tehnolo&scaron;kog procesa prerade permeata, kao sporednog proizvoda dobijenog nakon ultrafiltracije mleka tokom proizvodnje feta sira i svežeg (&bdquo;mladog&ldquo;) sira. Ispitana je mogućnost enzimske hidrolize laktoze u permeatu kori&scaron;ćenjem enzima &beta;-galaktozidaze izolovanog iz Kluyveromyces lactis u koncentraciji 0,1, 0,3 i 0,5 g/100g na temperaturama 20&ordm;, 30&ordm; i 40 &ordm;C. Praćene su promene sadržaja laktoze, D&ndash;galaktoze i D&ndash;glukoze u vremenskim intervalima tokom 60 minuta. Posebna faza istraživanja obuhvatila je matematičko modelovanje i kinetiku procesa hidrolize laktoze u permeatu pod dejstvom &beta; &ndash;galaktozidaze i primenu hidrolizovanog permeata u proizvodnji mlečnih napitaka po odabranoj formulaciji. Predložen je tehnolo&scaron;ki proces proizvodnje napitka na bazi hidrolizovanog permeata sa dodatkom voćnih baza. Utvrđeni su parametri kvaliteta i trajnosti napitaka tokom 60 dana skladi&scaron;tenja.<br />Na temperaturi 40&deg;C dodatkom enzima &beta; -galaktozidaze u koncentraciji 0,1g/100g za 60 minuta postiže se 100% stepen hidrolize prisutne laktoze u permeatu. Sa većom koncentracijom enzima, 0,3 g/100g odnosno 0,5g/100g, na istoj temperaturi, isti efekat se postiže za 20 minuta.<br />Ispitivanjem kinetike hidrolize laktoze potvrđena je kinetika prvog reda. Generalno posmatrano visoki koeficijenti determinacije pokazuju dobro poklapanje eksperimentalnih rezultata i matematičkog modela reakcije prvog reda. Vrednosti se kreću od 0,974 (temperatura 20&deg;C) do preko 0,990 (na temperaturama 30&deg;C i 40&deg;C) pri koncentraciji enzima 0,1g/100g.<br />Proizvedeni napici od hidrolizovanog permeata su delaktozirani i ne sadrže mlečnu mast. Od ukupnih &scaron;ećera u svim napicima vi&scaron;e od 50% čini glukoza: 50,16% - napitak &scaron;umsko voće, 50,42% - napitak pomorandža/&scaron;argarepa, 54,65% - napitak multivitamin, odnosno 55,13% - napitak crveno voće.<br />Najveći sadržaj vitamina C nakon proizvodnje imao je napitak sa dodatkom voćne baze multivitamin 0,3972 mg/100g, zatim &scaron;umsko voće 0,2887 mg/100g i pomo-randža/&scaron;argarepa 0,1999 mg/100g.<br />Najveću vrednost antioksidativne aktivnosti nakon proizvodnje pokazali su uzorci napitka sa multivitaminom i &scaron;umskim voćem. Tokom perioda skladi&scaron;tenja dolazi do smanjenja DPPH vrednosti. Najmanji pad je u napitku sa pomorandžom / &scaron;argarepom (smanjenje za 17%), a najveći u napitku sa &scaron;umskim voćem (za 39%). Analizirani uzorci sadrže ukupnih polifenola u intervalu od 47,84 do 120,38 mg GAE/l u zavisnosti od vrste napitka, odnosno dodatih voćnih baza.<br />Generalno može se zaključiti da se prime-njenim tehnolo&scaron;kim procesom dobijaju napici stabilnog fizičko-hemijskog sastava tokom 60 dana skladi&scaron;tenja, visoke nutritivne i niske energetske vrednosti.</p> / <p>Valuation of the permeate as a by-product of the dairy industry is of great ecological, economic and technological importance.<br />The aim of the PhD thesis is the development of the technological process of refining permeate, as a by-product obtained after ultrafiltration of milk during the production of feta cheese and fresh cheese. The possibility of enzymatic hydrolysis of the lactose in the permeate using the enzyme &beta;-galactosidase isolated from Kluyveromyces lactis in a concentration of 0.1, 0.3 and 0.5 g / 100 g at a temperature of 20&deg;, 30&deg; and 40&deg; C was examined. Changes in the content of lactose, D-galactose and D-glucose at intervals of 60 minutes were monitored. A special stage of the research included mathematical modeling and kinetics of lactose hydrolysis in the permeate under the influence of &beta;-galactosidase and application of hydrolyzed permeate in the production of dairy products under the selected formulation. A technological process of producing a beverage on the basis of hydrolyzed permeate with the addition of fruit bases was suggested. Quality and durability parameters were determined for drinks during the 60 days of storage.<br />Addition of the enzyme &beta;-galactosidase at a concentration of 0.1 g / 100 g for 60 minutes at a temperature of 40 &deg; C a 100% degree of hydrolysis of lactose is achieved, present in the permeate. With a higher concentration of enzyme, 0.3 g / 100 g or 0.5 g / 100g, at the same temperature, the same effect can be achieved in 20 minutes.<br />By examining the kinetics of lactose hydrolysis the first order kinetics was confirmed. Generally high coefficients of determination show good correspondence between the experimental results and the mathematical model of the first order reaction. Values range from 0.974 (at a temperature of 20&deg; C) up to over 0.990 (at temperatures 30&deg; C and 40&deg; C) at a an enzyme concentration of 0.1g / 100g.<br />Beverages produced from hydrolyzed permeate are lactose-free and fat-free products. More than half of the total sugar content in all beverages consists of glucose: 50.16%-forest fruit beverage, 50.42%-beverage orange/carrot, 54.65% beverage multivitamin and 55.13% - beverage red fruit.<br />The highest vitamin C content after production was in a beverage with the addition of fruit base multivitamin (0.3972 mg/100g), followed by forest fruit (0.2887 mg/100g) and orange/carrot (0.1999 mg/100g).<br />Beverage samples with multivitamin and forest fruits showed the highest value of antioxidant activity after production. During the storage period there is a reduction of DPPH values. The smallest decrease was in the beverage with orange/carrot (decreased 17%), and the biggest in the beverage with forest fruit (39%). The content of polyphenols in analyzed samples ranges from 47.84 to 120.38 mg GAE/L depending on the type of beverage and added fruit base.<br />Overall it can be concluded that the applied technological process gives beverages of stable physical and chemical content during the 60 days of storage, of high nutritional value and low energy.</p>

Resumo: --- / Doutor

A possível escassez dos recursos fósseis, juntamente com o aumento imprevisível dos respectivos preços, levou, nas últimas décadas, a um aumento considerável de iniciativas dedicadas não só à procura de fontes alternativas de produtos químicos e polímeros a partir de fontes renováveis, mas também de fontes alternativas de energia - em particular a biomassa vegetal. O estudo desenvolvido no presente trabalho está inserido neste contexto. A despolimerização de celulose de sisal pode ocorrer via hidrólise, ácida ou enzimática, podendo resultar nos açúcares fermentescíveis necessários para a produção do chamado etanol celulósico e, em etapas intermediárias do processo, em micro e nanopartículas, que podem atuar como reforço de matriz polimérica baseada, por exemplo, em quitosana. O estudo aqui relatado está relacionado à análise do material celulósico não reagido durante a hidrólise, e do licor que contém principalmente glicose. As reações de hidrólise ácida e enzimática de polpa de sisal (constituída de celulose e hemicelulose) foram exploradas. Uma importante característica que envolve a hidrólise ácida de biomassa é a possibilidade de utilização de diversos ácidos, pois a princípio, necessita-se apenas de uma fonte de prótons no meio aquoso para que a reação ocorra. Neste contexto, em uma primeira etapa, uma série de reações de hidrólise ácida de polpa de sisal, previamente tratada com solução alcalina (mercerizada) ou não, foi feita com ácido sulfúrico (0,9 - 4,6 molL-1, 100&deg;C, 6h de reação). Em uma segunda etapa, o ácido sulfúrico foi substituído por ácido oxálico, e os tempos de reação foram maiores (18h) que aqueles considerados para o ácido sulfúrico, tendo em vista o menor valor do pKa do ácido oxálico. Reações de hidrólise enzimática foram realizadas com o uso de um complexo enzimático comercial (Accellerase 1500 - Genencor), e dois diferentes pré-tratamentos, ambos visando à eliminação de hemiceluloses, foram avaliados, sendo: mercerização e tratamento com solução de ácido oxálico 0,9 molL-1. Para acompanhar os processos, em determinados intervalos de tempo, foram retiradas alíquotas do meio reacional, sendo que os licores foram analisados por cromatografia líquida de alta eficiência (CLAE), a fim de avaliar a natureza e o teor dos produtos da hidrólise. As polpas residuais (não hidrolisadas), suspensas no licor, foram avaliadas por microscopia eletrônica de varredura, massa molar média por viscosimetria capilar, índice de cristalinidade por difração de raios X e tamanhos médios das fibras a partir de um analisador de fibras (MorFi - analisador de tamanho médio de fibras por imagem), e espalhamento de luz (FOQELS). Para todas as reações de hidrólise ácida estudadas, as massas molares médias das polpas residuais diminuíram até dez vezes logo nos primeiros minutos de reação e os valores de índice de cristalinidade mostraram que as regiões não cristalinas da celulose são primeiramente hidrolisadas, sendo as regiões cristalinas uma grande barreira frente à hidrólise. Os resultados mostraram que o aumento da concentração do catalisador ácido elevou consideravelmente a porcentagem de hidrólise, principalmente no caso do ácido oxálico que, quando usado na concentração de 0,9 molL-1, não foi capaz de hidrolisar com eficiência as cadeias de celulose, mas apenas eliminou as hemiceluloses presentes na polpa, motivo que levou à sua aplicação como agente de pré-tratamento para a polpa frente à hidrólise enzimática. Os rendimentos das reações mostraram que o ácido sulfúrico chega a ser aproximadamente 25% mais eficiente que o ácido oxálico em termos de produção de glicose. Entretanto, o ácido oxálico possui a grande vantagem de ser proveniente de fontes renováveis e, se usado nas concentrações adequadas, pode ser uma excelente opção como pré-tratamento da polpa de celulose para as reações de hidrólise. Os resultados de hidrólise enzimática mostraram que a polpa que passou pelo pré-tratamento da mercerização foi mais eficiente como material de partida do que aquela tratada com ácido oxálico, já que a primeira levou a concentrações de glicose até 2,5 vezes maiores, nas mesmas condições de concentração de enzima, temperatura e tempo de reação. As reações de hidrólise ácida e enzimática de material lignocelulósico são de grande importância no que diz respeito à produção de etanol de segunda geração e micro/nanofibras que podem ser incorporadas em materiais. Filmes de matriz de quitosana foram produzidos com a inserção de fibras de celulose sem tratamento, mercerizada, e residuais das reações de hidrólise ácida e enzimática, em diferentes concentrações (2,5, 7,5 e 15% em massa). Os filmes foram submetidos à solicitação de tração, e a morfologia foi acessada por microscopia eletrônica de varredura de emissão de campo (FEG-MEV). Os resultados mostraram que, no geral, o filme de quitosana (69 MPa), assim como os baseados em quitosana/celulose (75 MPa), apresentam resistência à tração superior ou no mesmo patamar de filmes similares descritos na literatura. Este trabalho forneceu resultados promissores e está largamente inserido no interesse atual de utilização de materiais provenientes de fontes renováveis preferencialmente àqueles de fontes fósseis. / The possible shortage of crude oil and the unpredictable increase in its prices have led to an impressive expansion of initiatives in the last decades dedicated not only to the search of alternative sources of chemicals and polymers, but also to suppliers of energy, both from vegetal biomass. The depolymerization of sisal cellulose may occur via acid or enzymatic hydrolysis, resulting in the fermentable sugars used in the production of the so-called cellulosic ethanol and also at the intermediate steps of the process, in micro and nanoparticles that may act as reinforcement in polymeric matrices, including those derived from cellulose. The study here reported is related to the analysis of the unreacted cellulosic material and to the liquor containing mainly glucose, from acid and enzymatic hydrolysis of sisal pulp formed by cellulose and hemicellulose. An important characteristic that involves the acid hydrolysis of biomass is the possibility of utilization of different acids, since only a source of protons in the media is required for the reaction to occur, in principle. In this context, a series of reactions of acid hydrolysis of sisal pulp was carried out under varying concentrations of sulfuric acid, from 0,9 to 4,6 molL-1, at 100&deg;C as a first step. In a second step, the acid catalyst was replaced by oxalic acid, and the reaction lengths were bigger than those considered for sulfuric acid due to the lower value of pKa of oxalic acid. The reactions of enzymatic hydrolysis were carried out with a commercial enzymatic complex (Accellerase 1500 - Genencor), and two different pretreatments, both aiming at the elimination of hemicelluloses, were essayed as follows: mercerization and treatment with oxalic acid 0,9 molL-1. To follow the processes of acid and enzymatic hydrolysis in determined time intervals, aliquots were withdrawn from the reaction media so as to be analyzed by High Performance Liquid Chromatography (HPLC) aiming at the evaluation of the nature and content of the hydrolysis products. The unreacted cellulose suspended in the liquor was characterized by Scanning Electron Microscopy, capillary viscometry, X ray diffraction, and average size of fibers by using a fiber analyzer and light scattering. For all acid hydrolysis reactions studied, the average molar mass of the unreacted cellulose decreased up to ten times in the first minutes of reaction, and the values of crystallinity index showed that the non-crystalline regions of cellulose are firstly hydrolyzed, and the crystalline regions act as barriers to the hydrolysis. The results of HPLC showed that an increase in concentration considerably increases the yield of hydrolysis, mainly in the case of oxalic acid as a catalyst, which was not able to hydrolyze the chains of cellulose when in low concentrations (0,9 molL-1). It only eliminated the hemicellulose present in the pulp, reason why this acid was used as a pretreatment agent in enzymatic hydrolysis at this concentration. The reaction yields showed that the sulfuric acid can be up to 25% more efficient than the oxalic acid in terms of glucose production. However, the oxalic acid has the great advantage of possibly being produced from natural resources as well as being an excellent choice as a pretreatment agent for the lignocellulosic biomass to be used in hydrolysis reactions if used in the adequate concentrations. The results of enzymatic hydrolysis showed that the mercerized pulp was more efficient as raw material than the one treated with oxalic acid, as the first led to higher glucose content at the same conditions of concentration, temperature and time of reaction. The reactions of acid and enzymatic hydrolysis of lignocellulosic materials are of great importance to the production of second generation ethanol and micro and nanofibers, which may be incorporated into biocomposites. Films of chitosan matrix were prepared with the addition of cellulose fibers (untreated, mercerized and residual from the acid and enzymatic hydrolysis reactions) under various concentrations (2,5, 7,5 e 15% wt%). The films were subjected to traction analysis and its morphology was accessed by field emission scanning electron microscopy (SEM-FEG). The results showed that, in general, chitosan films (69 MPa), just like films based on chitosan-cellulose (75 MPa) presented tensile strength values that are superior or the same as similar films described in literature. Therefore, the study here reported produced promising results and is widely inserted in the current interest of utilization of materials from renewable resources instead of those from fossil resources.