Orientador: Rosiane Lopes da Cunha / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-23T01:16:34Z (GMT). No. of bitstreams: 1 Kuhn_KatiaRegina_D.pdf: 3588047 bytes, checksum: 56035122a0c4826c829bf99675b3f00c (MD5) Previous issue date: 2013 / Resumo: A encapsulação de compostos bioativos vem sendo utilizada como uma alternativa para minimizar a degradação destes ingredientes durante o processamento, armazenamento e/ou processo digestivo, permitindo o aumento da vida de prateleira dos alimentos e a liberação controlada destes compostos. Nesse sentido, o objetivo geral deste trabalho foi produzir microgéis a partir da extrusão de emulsões O/A contendo isolado protéico de soro de leite (IPS) e gelana em uma solução gelificante de cloreto de cálcio visando a encapsulação e liberação controlada de óleo e hidrolisado protéico da linhaça. Na primeira parte deste estudo, a influência das condições de homogeneização (pressão e número de passagens) no preparo de emulsões estabilizadas por IPS foi avaliada com o intuito de obter sistemas mais estáveis e com menor oxidação lipídica. Todos os sistemas foram estáveis à cremeação e um aumento da pressão de homogeneização para 800 bar e do número de passagens para até 3 vezes, diminuiu o tamanho médio de gota das emulsões. Condições extremas de homogeneização levaram à formação de agregados protéicos de alta massa molecular (>200 kDa), favorecendo o aumento na viscosidade das emulsões. Com o aumento da pressão, uma distribuição de tamanho de gotas bimodal, indicando que coalescência pode ter ocorrido, e um aumento na formação de produtos primários da oxidação foram observados. Na segunda etapa do trabalho, avaliou-se o potencial do isolado protéico da linhaça (IPL) como agente emulsificante em sistemas puros e mistos com proteínas do soro de leite preparados sob alta pressão de homogeneização. As emulsões estabilizadas por IPL ou IPSIPL mostraram-se cineticamente instáveis e o aumento da concentração de IPL e das condições de homogeneização melhoraram a estabilidade dos sistemas puros, o que foi atribuído à sua maior viscosidade. No entanto, a maior estabilidade foi obtida com a adição de IPS nas emulsões contendo menor concentração de IPL (0,14% m/v) e utilizando condições mais drásticas de homogeneização. Por último, microgéis de IPS e gelana foram produzidos a partir da gelificação iônica de emulsões visando a encapsulação de compostos bioativos da linhaça. Os microgéis foram avaliados quanto à estabilidade, resistência e liberação destes compostos bioativos através da simulação in vitro do processo digestivo. Os resultados mostraram que óleo e hidrolisado protéico da linhaça foram encapsulados e que os microgéis resistiram às condições gástricas, mas foram desintegrados no meio intestinal. Além disso, a adição de hidrolisado diminuiu o tamanho das partículas e parece ter auxiliado na encapsulação do óleo de linhaça. Sendo assim, os microgéis produzidos poderiam ser utilizados para a proteção e liberação controlada dos compostos bioativos encapsulados / Abstract: The encapsulation of bioactive compounds has been used as an alternative to minimize degradation of these ingredients during processing, storage and/or digestive process, allowing increased shelf life of foods and the controlled release of these compounds. In this way, the general purpose of this work was to produce microbeads from extrusion of the O/W emulsions containing whey protein isolate (WPI) and gellan into a gelling solution of calcium chloride aiming the encapsulation and controlled release of oil and protein hydrolysate from flaxseed. In the first part of this study, the influence of homogenization conditions (pressure and number of passes) in the preparation of emulsions stabilized by WPI was assessed in order to obtain more stable systems and decreased lipid oxidation. All the systems were stable to creaming and an increase of homogenization pressure to 80 MPa and the number of passes up to 3 times, decreased the mean droplet size of the emulsions. Extreme homogenization conditions led to the formation of high molecular weight protein aggregates (>200 kDa), favoring the increase in viscosity of the emulsions. Increasing pressure, a bimodal droplets size distribution, indicating droplets coalescence, and an increase in the formation of primary oxidation products were observed. In the second step of the work, the potential of flaxseed protein isolate (FPI) as an emulsifying agent was evaluated in pure systems and mixed with whey proteins prepared under high pressure homogenization. The emulsions stabilized by FPI or WPI-FPI were kinetically unstable and the increase of FPI concentration and homogenization conditions improved the stability of pure systems, which was attributed to its higher viscosity. However, the greatest stability was achieved with the WPI addition in the emulsions containing the lowest FPI concentration (0.14% w/v) and using more drastic homogenization conditions. Finally, WPI-gellan microgels were produced by ionic gelation of the emulsions aiming the encapsulation of bioactive compounds from flaxseed. Microgels were evaluated in relation to stability, resistance and release of these bioactive compounds by simulating in vitro digestion process. The results showed that oil and protein hydrolysate from flaxseed were encapsulated and that microbeads resisted to gastric conditions, but were disintegrated in intestinal medium. Furthermore, the hydrolysate addition decreased the particle size and seems to have contributed for the flaxseed oil encapsulation. Thus, microbeads produced could be used for protection and controlled release of the encapsulated bioactive compounds / Doutorado / Engenharia de Alimentos / Doutora em Engenharia de Alimentos

Orientadores: Aline Carvalho da Costa, Rubens Maciel Filho, Francisco Maugeri Filho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-19T22:59:39Z (GMT). No. of bitstreams: 1 Andrade_RafaelRamosde_D.pdf: 13239438 bytes, checksum: 3878b0150588cf72948be14b3f7dec47 (MD5) Previous issue date: 2012 / Resumo: O objetivo deste trabalho é modelar um processo de produção de etanol a partir da fermentação de hidrolisado enzimático de bagaço concentrado com melaço de cana-de-açúcar. Para este propósito, foram realizadas 23 fermentações em batelada com e sem reciclo de células em várias temperaturas: 30, 32, 34, 36 e 38 °C. O reciclo visa avaliar a influência da exposição da levedura S. cerevisiae ao hidrolisado contendo inibidores (especialmente ácido acético) por longos períodos de tempo, e com altas concentrações celulares. Para cada temperatura a primeira fermentação foi realizada com inóculo fresco e realizaram-se no mínimo 4 reciclos. Inicialmente avaliou-se um modelo desenvolvido em trabalho anterior (ANDRADE, 2007) para fermentação de melaço de cana. Este modelo não foi capaz de descrever novas fermentações na presença de alterações da matéria-prima, nem mesmo quando o substrato era melaço de uma safra diferente. Devido ao grande número de parâmetros (11) no modelo, desenvolveu-se uma metodologia de re-estimação, na qual os parâmetros mais sensíveis eram reajustados, e os menos sensíveis mantidos fixos, tornando a re-estimação mais simples. Para isso fez-se uma análise de sensibilidade paramétrica através de planejamentos de Plackett-Burman usando o software STATISTICA, variando os parâmetros cinéticos e calculando seus efeitos nos perfis de concentração de células, substrato e etanol. Os parâmetros mais relevantes nos perfis foram ?máx, Pmáx, Yx e Yp/x, os quais foram escolhidos para serem reajustados quando se alterava o substrato. Com o reajuste desses 4 parâmetros, foi possível descrever os dados experimentais da fermentação de melaço de nova safra com precisão. O mesmo algoritmo de re-estimação foi utilizado para ajuste dos parâmetros do modelo de fermentações de caldo hidrolisado concentrado com melaço. Além dos 4 parâmetros definidos como mais importantes (?máx, Pmáx, Yx ,Yp/x), re-estimou-se também o parâmetro Xmáx, já que no desenvolvimento do modelo original não foi considerado reciclo de células, havendo baixa concentração de biomassa. Acrescentou-se, ainda, ao modelo original, um termo para levar em conta a inibição por ácido acético, logo estimaram-se também os parâmetros relacionados. Após re-estimação dos parâmetros usando um algoritmo de otimização quasi-Newton desenvolvido em COMPAQ VISUAL FORTRAN, realizou-se nova análise de sensibilidade para verificar se os parâmetros mais sensíveis continuavam sendo os mesmos. Neste caso, o ?máx deixou de ser xviii relevante se a análise fosse feita no tempo final de fermentação, porém realizando análise de sensibilidade em diferentes tempos, mostra-se que ?máx tem efeito nos perfis do modelo no início da fermentação. Foi possível desenvolver um modelo preciso que descreveu as fermentações em batelada com e sem reciclo de células em função da temperatura mantendo 11 dos 13 parâmetros (ajustados a partir de dados de fermentação de hidrolisado concentrado com melaço) fixos. Apenas os parâmetros Yx e Yp/x tiveram valores diferentes para as fermentações com reciclo / Abstract: The objective of this paper is to model a process of ethanol production from sugarcane bagasse hydrolysate concentrated with molasses. For this purpose, experimental data were obtained from 23 batch fermentations with and without cells recycle in different temperatures: 30, 32, 34, 36 e 38 °C. The recycle aims to evaluate the influence of exposing the yeast S. cerevisiae to the hydrolysate inhibitors (specifically acetic acid) for long time periods, and under high cell concentrations. For each temperature the first fermentation was performed with fresh inoculum and at least 4 recycles were carried out. Initially a model developed in a previous work (ANDRADE, 2007) for sugarcane molasses fermentation was evaluated. This model was not able to describe new fermentations in the presence of changes of raw material, not even when the substrate was molasses from a different harvest. Due to the large number of parameters (11) in the model, a re-estimation methodology was proposed, in which the most sensitive parameters were adjusted and the less sensitive were kept fixed, making the re-estimation easier. A parametric sensitivity analysis through Plackett-Burman designs was performed, using the software STATISTICA, by varying the kinetic parameters and calculating their influence on the profiles of cell, substrate and ethanol concentrations. The most relevant parameters in the profiles were ?max, Pmax, Yx and Yp/x, which were chosen to be re-estimated when there was change in the substrate. With the re-estimation of these four parameters, it was possible to describe the experimental data from fermentation of molasses from a new harvest accurately. The same re-estimation algorithm was used to adjust the parameters of the fermentation of hydrolysate concentrated with sugarcane molasses. In addition to the 4 parameters defined as most important (?max, Pmax, Yx, Yp/x), Xmax was also re-estimated, since the development of the original model did not consider recycling of cells, which resulted in a low biomass concentration. A term to account for the inhibition by acetic acid was added to the original model, so the related parameters were also estimated. After the parameters re-estimation using a quasi-Newton optimization algorithm developed in COMPAQ VISUAL FORTRAN, a new sensitivity analysis was carried out to verify if the most sensitive parameters remained the same. In this case, ?max had no influence if xx the analysis was performed at the end of the fermentation, but by performing the sensitivity analysis at different times, ?max was shown to influence the model profiles at the beginning of fermentation. It was possible to develop an accurate model that described the batch fermentations with and without cells recycle as a function of temperature keeping 11 of the 13 parameters (adjusted from data of fermentation of hydrolyzate concentrated with molasses) fixed. Only the parameters Yx and Yp/x had different values for the fermentation with recycle / Doutorado / Desenvolvimento de Processos Químicos / Doutor em Engenharia Química

Biostimulants offer an innovative approach to potentially improve crop yield and quality under abiotic stresses. Particularly, plant-derived protein hydrolysates (PH), a mixture of amino acids and soluble peptides from enzymatic or chemical hydrolysis of agricultural waste, are gaining global interest due to their sustainability and positive effects on crops. However, a functional role of the PH in crop yield and quality remains uncertain and is proposed to be associated with its phytohormone-like activities or serve as an additional nitrogen (N) source. Besides, the effects of PH on crop yield and quality are limited in intensive production systems such as greenhouse facilities. The purposes of this research are to examine the effects and mechanisms of PH on crops and to assess the potential of PH application to reduce fertilizer use in crop production. The specific objectives were to; 1) elucidate the hormone-like activities of PH in the adventitious rooting formation of cuttings, 2) evaluate the effects of different PH application methods on greenhouse crop yield and quality under different N levels when plants are grown with a commercial growing medium, and 3) examine the effects of PH application methods on yield and quality of hydroponically grown lettuce under different N levels and forms. Three conclusions were that 1) <a>the hormonal effects of PH are attributed to brassinosteroid-mediated processes, and PH has overlapping functions with auxin during adventitious rooting of cuttings in a plant species-specific manner</a>, 2) root application of PH (PH-R) effectively improves nutrient uptake compared to foliar spray of PH (PH-F), subsequently, increases the lettuce and tomato yield and quality regardless of N levels while PH-R did not change the chemical properties of growing media, and 3) PH-R effectively increases root growth, and subsequently, improving shoot yield and quality with significant PH × N levels and PH × NO₃:NH₄ratios interactions. Also, PH-R counteracted the negative effects of low NO₃:NH₄ratios on lettuce yield. The outcomes provide the optimization of PH and N fertilization in modern sustainable greenhouse production and the development of a new strategy for producing high-quality greenhouse crops with improved nutrient use efficiency.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24678号 / 農博第2561号 / 新制||農||1100(附属図書館) / 学位論文||R5||N5459(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 菅原 達也, 教授 舟場 正幸 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

The heavy reliance on live feeds is currently restricting the growth and sustainability of the aquaculture industry, therefore, the overall goal of this research was to improve the utilization of formulated dry diets at first feeding of larval fish. This was done with a specific focus on the production and provision of the optimal dietary protein form and composition. Chapter 2 aimed to provide an efficient protein source for larval fish by using same-species muscle and endogenous enzymes to produce hydrolysates and by providing a series of diets with increasing molecular weight protein fragments through larval development. Largemouth Bass (Micropterus salmoides) (LMB) muscle was mixed with the digestive enzymes from adult LMB and hydrolyzed for 1.5, 3, and 6 h, respectively. Five diets were produced, an intact diet containing non-hydrolyzed muscle and four diets with 37% muscle hydrolysate inclusion. The molecular weight profile of those diets were formulated to vary based on the inclusion level of each hydrolysate. To account for gut development, one group of larval LMB was fed a weekly series of diets with an increasing molecular weight profile. The initial inclusion of the hydrolysates significantly improved the total length of the larval LMB; however, neither the hydrolysate inclusion nor the series of dietary molecular weight profiles improved the overall growth of larval LMB. The inclusion of hydrolysates significantly decreased the occurrence of skeletal deformities. The results from this study suggest that the inclusion of same-species hydrolysates can improve the initial growth of first-feeding LMB, but further research is necessary to determine the optimal molecular weight profile, hydrolysate inclusion level, and physical properties of feeds to improve the overall growth performance during the larval stage. Chapter 3 compared the effect of dietary inclusion of a fish muscle hydrolysate produced from species-specific muscle and enzymes to hydrolysates produced from those of a different species, in diets for larval Walleye (Sander vitreus). Four intact and hydrolyzed protein products were produced from each combination of Walleye muscle and endogenous enzymes, and muscle and endogenous enzymes from Nile Tilapia (Oreochromis niloticus). The hydrolyzed products were continuously mixed for 3 h during the hydrolysis, (at 22oC and 28oC for Walleye and Tilapia enzymes, respectively), and the pH was adjusted throughout the process to mimic gastric and intestinal digestion conditions. Four diets were produced with the dietary protein supplied as a 50/50 ratio of the intact and hydrolyzed muscle from the respective muscle/enzyme combination. There was a significant interaction effect between muscle and enzyme source on the growth of larval Walleye. At the conclusion of the study, the larval Walleye that received the diet with muscle hydrolysate produced with Walleye muscle and Walleye endogenous enzymes had a significantly higher average weight than all other groups, and significantly higher postprandial levels of total free amino acids and indispensable amino acids in the muscle. Each hydrolysate-based diet led to a significant reduction in skeletal deformities and survival, compared to a group fed with a commercial diet. The results from this study suggest that species-specific muscle and enzymes produce a more optimal dietary protein source for larval fish than non-species-specific products, but further research should focus on improving the physical properties of the formulated diets to improve survival of fish larvae. Chapter 4 proposed a practical controlled hydrolysis method to utilize the endogenous enzymes within the fish body for the breakdown of tissues proteins, and to produce a species-specific meal that is tailored to the nutritional requirements and absorptive capacity of fish larvae. Four Zebrafish (Danio rerio) meals were produced from whole-body adult Zebrafish, three hydrolysates that were hydrolyzed for 1, 2, and 3 h, respectively, and an unhydrolyzed meal. From these meals, three diets were produced, each defined by their supply of dietary protein. The Unhydro diet was solely based on the unhydrolyzed Zebrafish meal. The 50% Hydro diet was based on 50% Zebrafish hydrolysate mix and 50% unhydrolyzed Zebrafish meal. The 100% Hydro diet was 100% based on the Zebrafish meal hydrolysate. The hydrolysate mix contained equal parts of the 1, 2, and 3 h hydrolysates. Proteomic analysis showed that the proposed hydrolysis method was able to efficiently hydrolyze the protein within Zebrafish body. The feeding trial found no significant differences in the final weight, total length, or survival between the Unhydro, 50% Hydro, and 100% Hydro groups, but the 50% Hydro group did express a significant upregulation of PepT1 at 24 h after feeding, compared to the Unhydro group. The growth results paired with PepT1 gene expression potentially indicate Zebrafish larvae to be adapted to dry feeds at first feeding and able to utilize dietary protein in different molecular forms efficiently for growth. Overall, the proposed hydrolysis method provides a practical and cost-effective approach to producing species-specific fishmeal hydrolysates. Further research is necessary to determine whether the produced hydrolysates can improve the growth of larval fish in other fish models. Further insight into behavioral and physiological responses in fish to imbalanced dietary amino acid profiles was provided in Chapter 5. The objective of this study was to determine how stomachless fish respond to diets deficient in the main limiting IDAA (lysine, methionine, and threonine), using Zebrafish as a model species. Six semi-purified diets were formulated for this study. The CG diet contained casein and gelatin as its only protein sources, while FAA50 diet had 50% of is dietary protein supplied with crystalline amino acids. Both were formulated to contain identical, balanced amino acid profiles. The remaining diets were supplied with the same amino acid mix as the FAA50 diet, but with minor adjustments to create deficiencies of the selected IDAA. The (-) Lys, (-) Met, and (-) Thr diets had lysine, methionine, and threonine withheld from the free amino acid (FAA) mix, respectively, and the Def diet was deficient in all three. The fish were fed to apparent satiation three times a day, and each feeding was carefully observed to ensure all feed added to the tanks was consumed. The results showed that although the singular deficiency of the three main limiting amino acids did not induce significant changes in feed intake, the combined deficiency of the three IDAA significantly increased the feed intake of juvenile Zebrafish. This increased feed intake prevented the IDAA deficiencies from significantly reducing growth, however, the feeding efficiency was also reduced. There was also an observed upregulation of neuropeptide Y (NPY), an orexigenic hormone, in the Def group, compared to the FAA50 group. The outcomes of this study provide insight into the behavioral and physiological response to dietary amino acid imbalances of stomachless fish and suggests stomachless fish increase their feed intake when challenged with IDAA-deficient diets, and that the regulation of NPY might play a role in this response. Chapter 6 assessed the postprandial FAA dynamics in the plasma, liver, and muscle of three species; 1) Largemouth Bass – warm-water, stomach-possessing carnivorous species; 2) Walleye – cool-water, stomach-possessing carnivorous species; and 3) Zebrafish– tropical, stomachless omnivorous species. Two diets were formulated for this study, a diet based on intact casein and gelatin (CG), and a diet with 50% of its protein supplied in FAA form (FAA50). Forty-two fish from each species were utilized, with one group of 21 receiving the CG diet, and the other 21 receiving the FAA50 diet. All fish were starved for 24 hours prior to the final feeding before sampling. Three fish were sampled at each time point, with three samples (plasma, liver, and muscle) taken from each fish. Samples were taken prior to feeding (0 h) and then at 0.5, 1, 2, 3, 6, and 12 h after feeding, for all species. A significant three-way interaction was observed between the diet, species, and postprandial time on the total FAA, IDAA, and DAA levels in the plasma, liver, and muscle, indicating that the postprandial FAA patterns were significantly different between species and in response to the different diets. In stomach-possessing species, dietary amino acids from the FAA50 diet were absorbed more rapidly than those from the CG diet, resulting in fewer correlations with the dietary IDAA profiles. The absorption of FAA in cool-water Walleye was more gradual and prolonged than the warm-water LMB, leading to more significant correlations with the dietary IDAA and more sustained peaks. The postprandial peaks of FAA typically occurred at the same time in the stomachless Zebrafish fed with the CG or FAA50 diet. The levels of FAA were noticeably lower after feeding with the FAA50 diet in Zebrafish, compared to the CG diet. These results provide a reference for differences in the FAA dynamic patterns of three species with differing physiological characteristics, when fed diets with intact protein or supplemented with FAA. The findings presented in this dissertation provide support and novel methods for the production and inclusion of species-specific protein hydrolysates as an ideal protein source in formulated diets for first-feeding larval fish. This research contributes to the development of larval diets that can release the limitations of growth placed on the aquaculture industry by the reliance on live feeds, particularly within the hatchery sector. This research also provides further understanding of dietary protein utilization and delivers new fish nutrition knowledge that will benefit the aquaculture industry as a whole.

Les hydrolysats protéiques ont une haute valeur ajoutée pour des secteurs industriels variés, de par leurs propriétés nutritives, fonctionnelles et / ou nutraceutiques. Pour améliorer les propriétés des hydrolysats, l’ultrafiltration est utilisée. Cependant, le manque d’outils de modélisation lié à la complexité des mélanges est un verrou pour une mise en œuvre rationnelle du procédé. Ces travaux ont permis de valider une stratégie de prédiction basée sur des caractéristiques classiques des hydrolysats et un étalonnage expérimental de la membrane d’ultrafiltration. Cette méthode permet de prédire les rendements et enrichissements en fraction(s) ou peptide(s) cible(s), ainsi que la productivité du procédé. Le modèle global de prédiction de l’ultrafiltration obtenu est alors utilisé afin d’optimiser la mise en œuvre de ce procédé. La démarche d’optimisation consiste à maximiser l’enrichissement de fractions ou de peptides cibles en minimisant la consommation d’eau et la durée du procédé / Protein hydrolysates are high added value mixtures for various industrial areas, thanks to their nutritive, functional or nutraceutical properties. To enhance hydrolysates performances, fractionation processes such as ultrafiltration are used. However, the lack of tools to predict ultrafiltration performances is a major bottleneck for a rational implementation of the process. This research thesis work enables to validate a prediction strategy based on classical characteristics of hydrolysates and an experimental calibration of the membrane. Yields and enrichment factors in targeted peptides or fractions during ultrafiltration as well as the productivity of the process can be predicted. This global methodology of performances prediction is then used to optimize the implementation modes of ultrafiltration. The multiobjective optimization approach consists in maximizing the enrichment in targeted peptides or fractions while water consumption and / or process duration is minimized

A utilização de resíduos da agroindústria na produção de biomoléculas de interesse industrial agrega valor ao produto, por não utilizar meios sintéticos, reduzindo custos totais de produção e promovendo o desenvolvimento de tecnologias mais limpas e sustentáveis. Os exopolissacarídeos, ou simplesmente EPS são polímeros naturais, excretados por algumas espécies de bactérias e fungos. Possuem grande interesse no setor industrial, dado a versatilidade da biomolécula na utilização nas áreas médica, farmacêutica e química apresentando potencial de geração de diversos produtos. O presente trabalho teve como objetivo estudar a obtenção do exopolissacarídeo lasiodiplodana utilizando hidrolisados obtidos a partir de subprodutos agrícolas pelo fungo Lasiodiplodia theobromae. Foram realizados Delineamentos Compostos Centrais Rotacionais (DCCR) 22 com três repetições nos pontos centrais para avaliação de condições ótimas de produção de hidrolisados de farelos de arroz e milho com concentrações estimáveis de açúcares totais, açúcares redutores e proteínas totais. Os hidrolisados obtidos foram utilizados em ensaios fermentativos para obtenção da lasiodiplodana em meio de cultivo (LAS-M) e aderida à biomassa celular (LASC), bem como produção de células. Para elaboração dos ensaios foram utilizados DCCR 23 e 22 em hidrolisados de farelos de arroz e milho, respectivamente. As variáveis escolhidas para o DCCR 23 foram pH, volume de meio em frasco e adição de sais suplementares, enquanto o DCCR 22 apresentaram como variáveis o estudo da agitação e temperatura. Os delineamentos estudados geraram diversos modelos significativos para a produção de hidrolisados e obtenção de frações de lasiodiplodana. As frações de LAS-M e LAS-C obtidas foram caracterizadas parcialmente quanto a seus conteúdos de carboidratos e proteínas, sendo também analisadas por espectroscopia de infravermelho (FTIR) e análises cristalográficas de Raios-X. Os hidrolisados de farelo de arroz apresentaram concentrações aproximadas para açúcares totais (40 g/L), açúcares redutores (10 g/L) e proteínas (14 g/L) enquanto o hidrolisado de farelo de milho apresentou 170, 70 e 14 g/L dos respectivos componentes. Os hidrolisados foram tratados e utilizados em ensaios fermentativos apresentando concentrações de LAS-M de 7,8 e 5,73 g/L, LAS-C 4,93 e 3,0 g/L e biomassa celular de 10,68 e 4,47 g/L respectivamente para hidrolisados de farelos de arroz e milho, apresentando consumo dos açúcares totais de 94 e 84%. As frações de lasiodiplodana LAS-M apresentaram em sua composição concentrações de açúcares redutores correspondentes a 83,62 e 92,16%, enquanto as frações LAS-C apresentaram 82,19 e 88,42% para os respectivos hidrolisados. As análises de FTIR e Raios-X demonstraram que os biopolímeros obtidos neste trabalho apresentaram estruturas semelhantes aos citados na literatura. Evidenciou-se que os hidrolisados utilizados nas condições estudadas proporcionaram condições adequadas para o fungo L. theobromae sintetizar o exopolissacarídeo lasiodiplodana. / The utilization of agro-industrial wastes for industrial-interest biomolecules production generates add-value products, reducing total production costs and promoting the development of cleaner and more sustainable technologies. Exopolysaccharides, or simply EPS are natural polymers, excreted by some species of bacteria and fungi. They present a great interest from industrial sector, given the versatility of the biomolecule and its use in medical, pharmaceutical and chemical sectors, generating a diversity of products. The present work had as objective to study the exopolysaccharide lasiodiplodan by fermentation of hydrolysates obtained from agricultural by-products from filamentous fungi Lasiodiplodia theobromae. Rotational Central Compound Designs (DCCR) 22 were performed for the evaluation of optimum conditions for the production of hydrolysates from rice and corn brans with estimated concentrations of total sugars, reducing sugars and total proteins. The optimized hydrolysates were used in fermentative assays for obtaining lasiodiplodan from culture medium (LAS-M) and cell biomass (LAS-C), as well as cell production. For the elaboration of the tests, DCCR 23 and 22 were performed using rice bran and corn bran hydrolysates, respectively. The chosen variables for the DCCR 23 were pH, medium volume in flask and addition of supplemental salts, while the DCCR 22 presented agitation and temperature as variables. The studied designs presented several significant models for the hydrolysates production and its lasiodiplodan fractions. Obtained fractions of LAS-M and LAS-C were partially characterized due to carbohydrate and protein contents, as well as X-ray crystallographic analysis and Fourier-Transform Infrared Spectroscopy - FTIR. Rice bran hydrolysates presented estimated concentrations for total sugars (40 g / L), reducing sugars (10 g / L) and proteins (14 g / L) while corn bran hydrolysates showed 170, 70 and 14 g / L respectively. Treated rice and corn bran hydrolysates used for fermentative assays presented respective concentrations for LAS-M (7.8 and 5.73 g/L), LAS-C (4.93 and 3.0 g/L) and cellular biomass (10.68 and 4.47 g/L) and total sugar consumption of 94 and 84%, respectively. The lasiodiplodan fractions LAS-M presented reducing sugars compositions of 83.62 and 92.16%, while LAS-C fractions presented 82.19 and 88.42% for rice and corn bran hydrolysates. FTIR and X-ray anlysis showed that the present obtained biopolymers presented similar structures to those mentioned in the literature. It was evidenced that the hydrolysates used under the studied conditions provided adequate conditions for L. theobromae to synthesize the exopolysaccharide lasiodiplodan.

A utilização de resíduos da agroindústria na produção de biomoléculas de interesse industrial agrega valor ao produto, por não utilizar meios sintéticos, reduzindo custos totais de produção e promovendo o desenvolvimento de tecnologias mais limpas e sustentáveis. Os exopolissacarídeos, ou simplesmente EPS são polímeros naturais, excretados por algumas espécies de bactérias e fungos. Possuem grande interesse no setor industrial, dado a versatilidade da biomolécula na utilização nas áreas médica, farmacêutica e química apresentando potencial de geração de diversos produtos. O presente trabalho teve como objetivo estudar a obtenção do exopolissacarídeo lasiodiplodana utilizando hidrolisados obtidos a partir de subprodutos agrícolas pelo fungo Lasiodiplodia theobromae. Foram realizados Delineamentos Compostos Centrais Rotacionais (DCCR) 22 com três repetições nos pontos centrais para avaliação de condições ótimas de produção de hidrolisados de farelos de arroz e milho com concentrações estimáveis de açúcares totais, açúcares redutores e proteínas totais. Os hidrolisados obtidos foram utilizados em ensaios fermentativos para obtenção da lasiodiplodana em meio de cultivo (LAS-M) e aderida à biomassa celular (LASC), bem como produção de células. Para elaboração dos ensaios foram utilizados DCCR 23 e 22 em hidrolisados de farelos de arroz e milho, respectivamente. As variáveis escolhidas para o DCCR 23 foram pH, volume de meio em frasco e adição de sais suplementares, enquanto o DCCR 22 apresentaram como variáveis o estudo da agitação e temperatura. Os delineamentos estudados geraram diversos modelos significativos para a produção de hidrolisados e obtenção de frações de lasiodiplodana. As frações de LAS-M e LAS-C obtidas foram caracterizadas parcialmente quanto a seus conteúdos de carboidratos e proteínas, sendo também analisadas por espectroscopia de infravermelho (FTIR) e análises cristalográficas de Raios-X. Os hidrolisados de farelo de arroz apresentaram concentrações aproximadas para açúcares totais (40 g/L), açúcares redutores (10 g/L) e proteínas (14 g/L) enquanto o hidrolisado de farelo de milho apresentou 170, 70 e 14 g/L dos respectivos componentes. Os hidrolisados foram tratados e utilizados em ensaios fermentativos apresentando concentrações de LAS-M de 7,8 e 5,73 g/L, LAS-C 4,93 e 3,0 g/L e biomassa celular de 10,68 e 4,47 g/L respectivamente para hidrolisados de farelos de arroz e milho, apresentando consumo dos açúcares totais de 94 e 84%. As frações de lasiodiplodana LAS-M apresentaram em sua composição concentrações de açúcares redutores correspondentes a 83,62 e 92,16%, enquanto as frações LAS-C apresentaram 82,19 e 88,42% para os respectivos hidrolisados. As análises de FTIR e Raios-X demonstraram que os biopolímeros obtidos neste trabalho apresentaram estruturas semelhantes aos citados na literatura. Evidenciou-se que os hidrolisados utilizados nas condições estudadas proporcionaram condições adequadas para o fungo L. theobromae sintetizar o exopolissacarídeo lasiodiplodana. / The utilization of agro-industrial wastes for industrial-interest biomolecules production generates add-value products, reducing total production costs and promoting the development of cleaner and more sustainable technologies. Exopolysaccharides, or simply EPS are natural polymers, excreted by some species of bacteria and fungi. They present a great interest from industrial sector, given the versatility of the biomolecule and its use in medical, pharmaceutical and chemical sectors, generating a diversity of products. The present work had as objective to study the exopolysaccharide lasiodiplodan by fermentation of hydrolysates obtained from agricultural by-products from filamentous fungi Lasiodiplodia theobromae. Rotational Central Compound Designs (DCCR) 22 were performed for the evaluation of optimum conditions for the production of hydrolysates from rice and corn brans with estimated concentrations of total sugars, reducing sugars and total proteins. The optimized hydrolysates were used in fermentative assays for obtaining lasiodiplodan from culture medium (LAS-M) and cell biomass (LAS-C), as well as cell production. For the elaboration of the tests, DCCR 23 and 22 were performed using rice bran and corn bran hydrolysates, respectively. The chosen variables for the DCCR 23 were pH, medium volume in flask and addition of supplemental salts, while the DCCR 22 presented agitation and temperature as variables. The studied designs presented several significant models for the hydrolysates production and its lasiodiplodan fractions. Obtained fractions of LAS-M and LAS-C were partially characterized due to carbohydrate and protein contents, as well as X-ray crystallographic analysis and Fourier-Transform Infrared Spectroscopy - FTIR. Rice bran hydrolysates presented estimated concentrations for total sugars (40 g / L), reducing sugars (10 g / L) and proteins (14 g / L) while corn bran hydrolysates showed 170, 70 and 14 g / L respectively. Treated rice and corn bran hydrolysates used for fermentative assays presented respective concentrations for LAS-M (7.8 and 5.73 g/L), LAS-C (4.93 and 3.0 g/L) and cellular biomass (10.68 and 4.47 g/L) and total sugar consumption of 94 and 84%, respectively. The lasiodiplodan fractions LAS-M presented reducing sugars compositions of 83.62 and 92.16%, while LAS-C fractions presented 82.19 and 88.42% for rice and corn bran hydrolysates. FTIR and X-ray anlysis showed that the present obtained biopolymers presented similar structures to those mentioned in the literature. It was evidenced that the hydrolysates used under the studied conditions provided adequate conditions for L. theobromae to synthesize the exopolysaccharide lasiodiplodan.

Hidrolisados enzimáticos e aminoácidos livres têm sido reportados como fontes adequadas de proteína para o tratamento clínico de pacientes que por diversas razões, não conseguem digerir a proteína intacta. Os hidrolisados enzimáticos têm sido preferidos por fornecer peptídeos de composição bem definida, promovendo uma absorção mais efetiva pelo organismo. Isto se deve ao fato de que di e tripeptídeos podem ser diretamente absorvidos por sistemas independentes daqueles usados pelos aminoácidos, e a taxa máxima de captação na absorção de di e tripeptídeos é maior do que a dos aminoácidos livres. As proteínas do leite, soja e peixe são as mais utilizadas, devido ao alto valor nutricional, disponibilidade comercial e custo moderado. Em nosso trabalho, caracterizamos seis hidrolisados enzimáticos de \"minced\" sob o ponto de vista químico, físico-químico e nutricional, visando atender grupos populacionais que necessitem de uma alimentação especial. A composição química (proteína, lípides, umidade e o conteúdo de resíduo mineral) e o perfil de aminoácidos dos hidrolisados foram determinados. A caracterização do peso molecular destes hidrolisados protéicos foi realizada por ultrafiltração em membranas de exclusão molecular de 30, 10 e 3Kd. Os seis hidrolisados apresentaram um rendimento variando entre 63,4 e 94,2%, dependendo do sistema enzimático utilizado. Os hidrolisados liofilizados continham teores protéicos semelhantes entre si, em média 74,2% e um teor lipídico muito reduzido, da ordem de 1,2%. Todos os hidrolisados apresentaram uma composição em aminoácidos semelhantes entre si e que atende as recomendações da FAO 1991, para crianças entre 3 e 8 anos e para adultos. Os hidrolisados 1 (pepsina 1:10.000/bromelina) e 4 (pepsina 1:10.000/Aspergillus oryzae) são indicados para o tratamento de pacientes com encefalopatia hepática, pois apresentaram uma relação de Fischer elevada de 3: 1. O processo de ultrafiltração mostrou ser um método de baixo custo e eficiente para separar as frações peptídicas com diferentes pesos moleculares e pouco interferiu na distribuição dos aminoácidos. No hidrolisado 5 (pepsina 1:60.000/Streptomyces griseus), a fração com peso molecular menor que 3 KDa, apresentou uma porcentagem elevada (57%) de peptídeos com baixo peso molecular, podendo ser utilizada no tratamento de pacientes com alergias alimentares. Esta mesma fração apresentou uma elevada concentração de fenilalanina livre, sugerindo o emprego de um processo de remoção deste aminoácido, para posterior formulação de dietas para pacientes fenilcetonúricos. / Protein hydrolysates and free amino acids have been used for the nutritional management of individuais who are not able to digest intact protein. Studies within the past two decades have shown that dipeptides and tripeptides may serve efficiently and safely to improve protein nutrition and that in certain instances they may be superior to free amino acids. Owing to their superior nutritional quality, hydrolysates of cow\'s milk protein, soy protein and fish protein are the most widely usado In our work, a minced fish was hydrolyzed, employing six different systems of two sequential enzymes. lhe resultant fish protein hydrolysates (FPH) were characterized for protein, fat, dry matter and ash contents and their amino acid composition was analyzed either. lhe six FPH were submitted to an ultrafiltration through a series of membranes with molecular cut-offs (MWCO) of 30, 10 and 3 KDa. lhe results showed yields of protein hydrolysates using different proteolytic enzymes varying from 63,4 to 94,2%. lhe protein contents on a dry basis of the six hydrolysates were similar, on average, 74,2%. lhe lipid contents of the six hydrolysates were low (mean of 1,2%). Although decreases of some amino acids were observed after hydrolysis, adequate amounts of essential amino acids in relation to the reference pattern of FAO (1991) were found for children from 3-8 years and adults. lhe hydrolysates 1 . (pepsin 1:10.0001 bromelain) and 4 (pepsin 1:10.000/Aspergillus oryzae) present a high Fischer ratio (molar ratio of VaI + Leu + lIe to lyr + Phe) and seem to be interesting for clinicai treatment of patients with liver diseases. lhe separation of peptides with different molecular weights by ultrafiltration seems to be a low cost and efficient method and does not interfere substantially with the amino acids distribution. lhe hydrolysate 5 (pepsin 1:60.000/Streptomyces griseus) contained a high percentage of low molecular weight peptides (57%), lower than 3 KDa. lhe same fraction contains a high free phenylalanine content, suggesting the removal of this amino acid in order to formulate diets for PKU patients.

Hemp seed proteins (HSP) were investigated for physicochemical and functional properties in model food systems. In addition, the HSP were enzymatically digested and the released peptides investigated as potential therapeutic agents. Membrane isolated HSP (mHPC) were the most soluble with >60% solubility at pH 3-9 when compared to a maximum of 27% for isoelectric pH-precipitated proteins (iHPI). However, iHPI formed emulsions with smaller oil droplet sizes (<1 µm) while mHPI formed bigger oil droplets. The iHPI was subjected to enzymatic hydrolysis using different concentrations (1-4%) of six proteases (pepsin, pancreatin, flavourzyme, thermoase, papain and alcalase) to produce various HSP hydrolysates (HPHs). HPHs had strong in vitro inhibitions of angiotensin converting enzyme (ACE) and renin activities, the two main enzyme systems involved in hypertension. Oral administration of the HPHs to spontaneously hypertensive rats led to fast and persistent reductions in systolic blood pressure. The HPHs also inhibited in vitro activities of acetylcholinesterase (AChE), a serine hydrolase whose excessive activities lead to inadequate level of the cholinergic neurotransmitter, acetylcholine (ACh). Inadequate ACh level in the brain has been linked to neurodegenerative diseases such as dementia and Alzheimer’s disease (AD); therefore, AChE inhibition is a therapeutic target. The 1% pepsin HPH was the most active with up to 54% AChE inhibition at 10 µg/mL peptide concentration. The 1% pepsin HPH (dominated by <1 kDa) was subjected to reverse-phase HPLC peptide purification coupled with tandem mass spectrometry, which led to identification of several peptide sequences. Some of the peptides inhibited activities of both animal and human AChE forms with LYV being the most potent against human AChE (IC50 = 7 µg/ml). Thus the LYV peptide may serve as a useful template for the development of future potent AChE-inhibitory peptidomimetics. In conclusion, several novel AChE-inhibitory peptides were discovered and their amino acid sequences elucidated for the first time. Results from this work identified HSP products that could serve as functional ingredients in the food industry. The work also produced and confirmed the in vitro AChE-inhibitory activities of several new peptide sequences that may serve as therapeutic agents for AD management. / October 2015