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Miniaturizované techniky pro analýzu průmyslových kvasinek / Miniaturized techniques for analysis of industrial yeastsObalil, Jiří January 2008 (has links)
Carotenoids are natural pigments that have antioxidation and antimutagenic abilities. They are produced with the help of new technological methods. For example, carotenoid yeast Rhodotorula glutinis produces -carotene with the yield of up to 6 – 10 mg/g of the dry substance. The method of the mass spectrometry with the nanoelectrospray in the positive mode was optimized for the determination of -carotene, lycopene and astaxanthin in this project. Ionizing voltage of 4 kV and the sample flow rate of 15 – 80 nl/min through the spray silica fused capillary with the internal diameter of 25 µm were found to be the optimum parameters of the analysis. A mixture of chloroform with the addition of ammonia was used as a spray solvent for both standard and cellular samples. During the process of ionization by nanoelectrospray, -carotene and lycopene form cation radical [M] • + with the molecular mass to charge ratio (m/z) of 536, while asthaxanthin forms the protonated molecule [M + H]+ with the m/z of 597. The partial lysis of individual Rhodotorula glutinis cells was demonstrated under microscope in the organic solvents tetrahydrofuran and dimethylsulfoxide. Chloroform, acetone, acetonitrille, methanol and isopropanol did not affect the cells after a 15 min treatment.
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Síntese, caracterização e reatividade de antiradicais de dupla função / Synthesis , Characterization and Reactivity of Dual Function of AntioxidantsPapa, Thiago Bueno Ruiz 29 October 2015 (has links)
A deterioração oxidativa de alimentos é o fator determinante na qualidade sensorial e nutricional do produto, podendo ocorrer em alimentos complexos tanto na fase contínua como na fase dispersa. Em geral, a formação de radicais e espécies reativas de oxigênio e nitrogênio é o evento primário que ocorre priori ao progresso da oxidação de componentes e estruturas sensíveis e está comumente associado a eventos na fase aquosa. A oxidação de lipídeos é frequentemente investigada em alimentos e em meio biológico separadamente dos eventos no meio aquoso e a proteção antioxidante somente avaliada na fase dispersa. Entretanto, a oxidação de proteínas e eventos na fase contínua vêm despertando considerável interesse e aparentemente antioxidantes eficientes na fase dispersa não protegem de forma eficaz as proteínas e os componentes na fase contínua. Um futuro progresso na proteção antioxidante de alimentos e sistemas biológicos origina de uma abordagem holística dos processos redox envolvidos em ambas as fases dispersa e contínua, bem como o uso combinado de espécies antioxidantes e antiredutoras para um superior efeito antiradical global. Neste sentido, foram preparados três novos compostos antiradicais de dupla função (antioxidante e antiredutor) com adequado balanço hidrofílico-lipofílico e aprimorada propriedade antiradical global. Os antiradicais diferulato de astaxantina, retinoato de quercetina e retinoato de epicatequina se mostraram melhores antioxidantes e antiredutores do que seus precursores isoladamente ou em misturas, apresentando superior eficiência na desativação do oxigênio singlete excitado, captação do radical 1-hidroxietila, menor efeito pró-oxidante em sistemas de oxidação catalisados por íons de ferro, e eficiência na redução do reativo estado triplete da safranina (E1/2 = 1,48 V vs. NHE). / The oxidative deterioration of foods is the determining factor in the sensory and nutritional quality of the product, which may occur in complex food both at the continuous and the dispersed phases. In general, the formation of radicals and reactive oxygen and nitrogen species is the primary event that occurs prior to the progress of oxidation of components and sensitive structures and is commonly associated with events in the aqueous phase. The oxidation of lipids is often investigated in food and biological media separately from the events in the aqueous phase and the measurement of the antioxidant protection is only evaluated in the dispersed phase. However, the oxidation of proteins and the events in the continuous phase have attracted considerable interest and apparently, efficient antioxidants in dispersed phase do not effectively protect proteins and components in the continuous phase. A further progress in the antioxidant protection of food and biological systems arise from a holistic approach of the redox processes involved in both the dispersed and continuous phases as well as the combined use of antioxidant and antireductant species for a superior overall antiradical effect. In this sense three new antiradical compounds with dual function (antioxidant and antireductant) were prepared with proper hydrophilic-lipophilic balance and global antiradical properties. The antiradicals astaxanthin diferulate, epicatechin retinoate, and quercetin retinoate are shown to be better antioxidant and antireductant than their precursors, isolated or in mixture, exhibiting enhanced singlet excited oxygen deactivation, 1-hydroxyethyl radical scavenging, lower pro-oxidative effect in the oxidative system catalysed by iron ions and an efficient reduction of the reactive triplet state of safranine (E1 / 2 = 1.48 V vs. NHE).
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Extraction biocompatible par les champs électriques pulsés des molécules d'intérêt de la microalgue verte Haematococcus pluvialis (Flotow 1844) / Biocompatible extraction by pulsed electric fields of molecules of interest from the green microalga Haematococcus pluvialis (Flotow 1844)Gateau, Hélène 21 December 2017 (has links)
Les champs électriques pulsés (CEP) offrent un réel intérêt dans le cadre de la traite des microalgues. En effet, ils permettent l'extraction sélective des composés hydrosolubles ou l'utilisation de solvants biocompatibles pour récolter les molécules hydrophobes. La viabilité des microalgues peut ainsi être conservée. L'objectif de ce travail de thèse est de définir des conditions de traitement permettant à la fois l'extraction des composés d'intérêt et le maintien de la viabilité des microalgues. Le modèle d'étude est la microalgue verte Haematococcus pluvialis. Au stade végétatif, celle-ci contient près d'un tiers de son poids de matière sèche en protéines et en conditions stressantes, elle accumule de l'astaxanthine, un caroténoïde à haute valeur ajoutée.L'application de CEP de 1 kV.cm-1 permet de collecter 50 % des protéines extractibles par broyage avec des microbilles. La mesure de cinq paramètres biologiques suite à ce traitement a mis en évidence que les cellules retrouvaient un état physiologique comparable à celui de microalgues non traitées au bout de 72 h. Cette condition de traitement constitue donc un bon compromis entre l'extraction des protéines et la survie des microalgues, ce qui renforce la faisabilité d'une traite de microalgues par CEP.Dans le cadre de l'extraction de l'astaxanthine, la paroi très résistante des kystes constitue le principal verrou à lever. Une optimisation des conditions de traitement (en particulier de la force des impulsions) et du stade cellulaire traité représentent les deux principales perspectives à étudier pour que l'utilisation des CEP dans le cadre de l'extraction de l'astaxanthine soit pertinente. / Pulsed electric fields (PEF) offer a real interest for microalgae milking. Indeed, they allow the selective extraction of water-soluble compounds or the use of biocompatible solvents to harvest the hydrophobic molecules. The viability of microalgae can thus be maintained. The aim of this PhD thesis work is to define the treatment conditions allowing both the extraction of compounds of interest and the maintenance of the microalgae viability. The biological model is the green microalga Haematococcus pluvialis. At the vegetative stage, it contains nearly one third of its dry matter weight in proteins and under stressful conditions, it accumulates astaxanthin, a high added value carotenoid.The application of PEF of 1 kV.cm-1 allows to collect 50% of the proteins extractable by bead milling. The measurement of five biological parameters highlights that treated cells recover a physiological state comparable to that of untreated microalgae after 72h. This treatment condition constitutes therefore a good compromise between the protein extraction and the survival of the microalgae, which reinforces the feasibility of microalgae milking by PEF.Within the context of astaxanthin extraction, the high resistance of the cell wall of the cysts constitutes the main limitation. Optimization of the treatment conditions (particularly pulse strength) and the cellular stage to treat represent the two main perspectives to study for the use of PEF for astaxanthin extraction to be relevant.
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Camar?o Litopenaeus vannamei: componentes de import?ncia nutricional na carne e nos res?duos do beneficiamentoSeabra, Larissa Mont Alverne Juca 23 November 2011 (has links)
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Previous issue date: 2011-11-23 / Universidade Federal do Rio Grande do Norte / Farming of marine shrimp is growing worldwide and the Litopenaeus vannamei (L. vannamei) shrimp is the species most widely cultivated. Shrimp is an attractive food for its nutritional value and sensory aspects, being essential the maintenance of this attributes throughout storage, which takes place largely under freezing. The aim of this research was to evaluate quality characteristics of Litopenaeus vannamei shrimp, during freezing storage and to verify the effect of rosemary (Rosmarinus officinalis) adding. Considering the reutilization of processing shrimp wastes, total carotenoids analysis were conducted in waste of Litopenaeus vannamei shrimp and in the flour obtained after dryer. Monthly physicochemical and sensorial analysis were carried out on shrimp stored at 28,3 ? 3,8?C for 180 days. Samples were placed in polyethylene bags and were categorized as whole shrimp (WS), peeled shrimp (PS), and PS with 0,5% dehydrated rosemary (RS). TBARS, pH, total carotenoid and sensorial Quantitative Descriptive Analysis (QDA) were carried out. Carotenoid total analysis was conducted in fresh wastes and processed flour (0 day) and after 60, 120 and 180 days of frozen storage. After 180 days, RS had lower pH (p = 0.001) and TBARS (p = 0.001) values and higher carotenoids (p = 0.003), while WS showed higher carotenoid losses. Sensory analysis showed that WS were firmer although rancid taste and smell were perceived with greater intensity (p = 0.001). Rancid taste was detected in RS only at 120 days at significantly lower intensity (p = 0.001) than WS and PS. Fresh wastes had 42.74μg/g of total carotenoids and processed flour 98.51μg/g. After 180 days of frozen storage, total carotenoids were significantly lower than 0 day (p<0,05). The addition of rosemary can improve sensory quality of frozen shrimp and reduce nutritional losses during storage. Shrimp wastes and flour of L. vannamei shrimp showed considerable astaxanthin content however, during storage it was observed losses in this pigment / O cultivo do camar?o marinho cresce mundialmente, sendo o Litopenaeus vannamei (L. vannamei), a esp?cie mais cultivada na atualidade. O camar?o ? um alimento atrativo por seu valor nutricional e aspectos sensoriais, sendo essencial, a manuten??o desses atributos ao longo do armazenamento. Os objetivos da presente pesquisa foram avaliar caracter?sticas de qualidade do camar?o L. vannamei adicionado de alecrim (Rosmarinus officinalis) e armazenado sob congelamento, assim como realizar a determina??o da concentra??o de caroten?ides totais dos res?duos do camar?o e da farinha obtida ap?s secagem. As amostras foram colocadas em embalagens de polietileno e estocadas como Camar?o Inteiro (CI), Camar?o Descascado (CD) e Camar?o descascado adicionado de 0,5% de alecrim desidratado (CA). An?lises de TBARS, pH e caroten?ides totais foram realizadas, assim como an?lise sensorial utilizando An?lise Descritiva Quantitativa (ADQ). As an?lises f?sico-qu?micas e sensoriais do camar?o L. vannamei armazenado sob congelamento (-28,3?3,8?C), foram realizadas mensalmente, por um per?odo de 180 dias. Com rela??o ?s an?lises dos res?duos, estas foram realizadas no material in natura e na farinha rec?m-processada (dia 0) e aos 60, 120 e 180 dias de armazenamento sob congelamento. No final do per?odo de armazenamento CA apresentou menor pH (p = 0,001) e valores de TBARS (p = 0,001) e concentra??o mais alta de caroten?ides totais (p = 0,003), enquanto CI demonstrou maior perda de caroten?ides. A an?lise sensorial mostrou maior firmeza para CI, por?m estas amostras apresentaram sabor e cheiro de ran?o em maior intensidade (p = 0,001). Os provadores perceberam sabor de ran?o nas amostras CA apenas aos 120 dias de armazenamento, e em menor intensidade do que nas amostras de CI e CD (p = 0,001). No que se refere ?s an?lises dos res?duos, observou-se concentra??o de 42,74μg/g de caroten?ides totais nos res?duos frescos e 98,51μg/g na farinha rec?m-processada. Ap?s 180 dias de armazenamento sob congelamento, os teores de caroten?ides totais diminu?ram significativamente quando comparados ao dia 0 (p<0,05). A adi??o de alecrim pode melhorar a qualidade sensorial do camar?o congelado e reduzir perdas de seu valor nutricional ao longo do armazenamento sob congelamento. Os res?duos e a farinha do camar?o L. vannamei, apresentaram concentra??o consider?vel de astaxantina, que foi reduzida ao longo do congelamento
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Análise por injeção em batelada com detecção amperométrica da astaxantina e da capacidade antioxidante em alimentosOliveira, Gracy Kelly Faria 17 March 2017 (has links)
CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nesta tese, apresenta-se o desenvolvimento de métodos eletroquímicos que
empregam a análise por injeção em batelada (BIA) com detecção amperométrica para
determinação da capacidade antioxidante de amostras de chá e extratos vegetais e
determinação de astaxantina em salmão. O primeiro método foi desenvolvido para
determinar a capacidade antioxidante de amostras de chá e extratos de plantas com
base na medida do consumo de radicais DPPH, uma vez que este radical é eletroativo.
Com este método, mediu-se a concentração eficaz ou o valor CE50 que corresponde
à concentração necessária de amostra ou padrão capaz de consumir 50% de radicais
DPPH. Para a determinação exata do CE50, as amostras foram incubadas com radical
DPPH durante 1 h, pois muitos compostos polifenólicos tipicamente encontrados nas
plantas e responsáveis pela atividade antioxidante exibem cinética lenta. O sistema
BIA com detecção amperométrica utilizando um eletrodo de carbono vítreo apresentou
alta precisão (DPR = 0,7%, n = 12), baixo limite de detecção (1 μmol L-1) e seletividade
do radical DPPH (livre de interferências de antioxidantes). Estes resultados
contribuíram para baixos limites de detecção para os antioxidantes ácido gálico e butilhidroxitolueno
(0,015 e 0,19 μmol L-1, respectivamente). O segundo método
desenvolvido baseou-se na oxidação eletroquímica do antioxidante astaxantina em
eletrodo de carbono vítreo para sua determinação amperométrica em amostras de
salmão. O método consistiu na injeção de 80 μL de extrato da amostra diretamente
no eletrodo de carbono vítreo imerso em solução composta por acetona,
diclorometano e água (80:10:10 v/v) contendo 0,1 mol L-1 HClO4. Este método
apresentou vantagens que incluem alta precisão (DPR de 2,4%), frequência analítica
de 240 h-1 e baixo limite de detecção (0,3 μmol L-1, que corresponde a 0,1 μg g-1) para
a análise de amostras de salmão extraídas com acetona. Valores de recuperação
entre 83 e 97% foram obtidos. Além disso, ambos métodos propostos são promissores
pois trazem a possibilidade do uso de sistemas portáteis uma vez que a
instrumentação usada (micropipeta eletrônica e potenciostato) está disponível
comercialmente. / In this thesis, we present the development of electrochemical methods using batch
injection analysis (BIA) with amperometric detection to determine the antioxidant
capacity of tea samples and plant extracts and determination of astaxanthin in salmon.
The first method was developed to determine the antioxidant capacity of tea samples
and plant extracts based on the extent of the consumption of DPPH radicals, since this
radical is electroactive. In this method the effective concentration or EC50 value
corresponding to the required concentration of sample or standard capable of
consuming 50% of DPPH radicals was measured. For the exact determination of the
EC50, the samples were incubated with DPPH radical for 1 h, since many polyphenolic
compounds typically found in plants and responsible for the antioxidant activity exhibit
slow kinetics. The BIA system with amperometric detection using a vitreous carbon
electrode showed high precision (RSD = 0.7%, n = 12), low detection limit (1 μmol L-1)
and DPPH radical selectivity (free of antioxidant interferences). These results
contributed to low detection limits for the antioxidants gallic acid and butylhydroxytoluene
(0.015 and 0.19 μmol L-1, respectively). The second method developed
was based on the electrochemical oxidation of the antioxidant astaxanthin on a glassy
carbon electrode for its amperometric determination in salmon samples. The method
consisted of the injection of 80 μL of sample extract directly into the vitreous carbon
electrode immersed in a solution composed of acetone, dichloromethane and water
(80:10:10 v/v) containing 0.1 mol L-1 HClO4. This method presented advantages that
include high accuracy (RSD of 2.4%), analytical frequency of 240 h-1 and low detection
limit (0.3 μmol L-1, corresponding to 0.1 μg g-1) for The analysis of salmon samples
extracted with acetone. Recovery values between 83 and 97% were obtained. In
addition, both proposed methods are promising because they bring the possibility of
using portable systems since the instrumentation used (electronic micropipette and
potentiostat) is commercially available. / Tese (Doutorado)
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Síntese, caracterização e reatividade de antiradicais de dupla função / Synthesis , Characterization and Reactivity of Dual Function of AntioxidantsThiago Bueno Ruiz Papa 29 October 2015 (has links)
A deterioração oxidativa de alimentos é o fator determinante na qualidade sensorial e nutricional do produto, podendo ocorrer em alimentos complexos tanto na fase contínua como na fase dispersa. Em geral, a formação de radicais e espécies reativas de oxigênio e nitrogênio é o evento primário que ocorre priori ao progresso da oxidação de componentes e estruturas sensíveis e está comumente associado a eventos na fase aquosa. A oxidação de lipídeos é frequentemente investigada em alimentos e em meio biológico separadamente dos eventos no meio aquoso e a proteção antioxidante somente avaliada na fase dispersa. Entretanto, a oxidação de proteínas e eventos na fase contínua vêm despertando considerável interesse e aparentemente antioxidantes eficientes na fase dispersa não protegem de forma eficaz as proteínas e os componentes na fase contínua. Um futuro progresso na proteção antioxidante de alimentos e sistemas biológicos origina de uma abordagem holística dos processos redox envolvidos em ambas as fases dispersa e contínua, bem como o uso combinado de espécies antioxidantes e antiredutoras para um superior efeito antiradical global. Neste sentido, foram preparados três novos compostos antiradicais de dupla função (antioxidante e antiredutor) com adequado balanço hidrofílico-lipofílico e aprimorada propriedade antiradical global. Os antiradicais diferulato de astaxantina, retinoato de quercetina e retinoato de epicatequina se mostraram melhores antioxidantes e antiredutores do que seus precursores isoladamente ou em misturas, apresentando superior eficiência na desativação do oxigênio singlete excitado, captação do radical 1-hidroxietila, menor efeito pró-oxidante em sistemas de oxidação catalisados por íons de ferro, e eficiência na redução do reativo estado triplete da safranina (E1/2 = 1,48 V vs. NHE). / The oxidative deterioration of foods is the determining factor in the sensory and nutritional quality of the product, which may occur in complex food both at the continuous and the dispersed phases. In general, the formation of radicals and reactive oxygen and nitrogen species is the primary event that occurs prior to the progress of oxidation of components and sensitive structures and is commonly associated with events in the aqueous phase. The oxidation of lipids is often investigated in food and biological media separately from the events in the aqueous phase and the measurement of the antioxidant protection is only evaluated in the dispersed phase. However, the oxidation of proteins and the events in the continuous phase have attracted considerable interest and apparently, efficient antioxidants in dispersed phase do not effectively protect proteins and components in the continuous phase. A further progress in the antioxidant protection of food and biological systems arise from a holistic approach of the redox processes involved in both the dispersed and continuous phases as well as the combined use of antioxidant and antireductant species for a superior overall antiradical effect. In this sense three new antiradical compounds with dual function (antioxidant and antireductant) were prepared with proper hydrophilic-lipophilic balance and global antiradical properties. The antiradicals astaxanthin diferulate, epicatechin retinoate, and quercetin retinoate are shown to be better antioxidant and antireductant than their precursors, isolated or in mixture, exhibiting enhanced singlet excited oxygen deactivation, 1-hydroxyethyl radical scavenging, lower pro-oxidative effect in the oxidative system catalysed by iron ions and an efficient reduction of the reactive triplet state of safranine (E1 / 2 = 1.48 V vs. NHE).
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Produção de astaxantina por Mucor javanicus (UCP 69), a partir de meio definido e utilizando resíduo industrial (milhocina e quirera de milho)Silveira, Aline Alves Barbosa da 08 August 2007 (has links)
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Previous issue date: 2007-08-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES# / #2075167498588264571# / #600 / With the recent substitution of synthetic pigments for the natural carotenoids, research has been effected in the direction to make possible the production of these substances through alternative considered biological sources. In this work was studied the production of astaxanthin for the sample of Mucor javanicus (CPU - 69), using the media definite Hesseltine and Anderson (1957) modified by Andrade (2000) and using different corn waste media (corn steep liquor .CSL and .quirera.) in three different concentrations (4, 7 and 10%), pH 6,5, 120 rpm, 25ºC. It was analyzed, also, the influence of the time of culture of the sample (48h, 72h and 96 h) and the presence and absence of blue Light. To the ending of the fermentative process, the astaxanthin, was extracted in solution of Hexano/methanol (1: 1, v/v), centrifuged in 2000 rpm/10 minutes and analyzed by UV-visible spectrophotometry (470 nm). All the parameters studied in the experiments had been combined through a factorial design 33 and analyzed in Software Statistica 5,0. With Hesseltine and Anderson the condition that more favored the increase of the income of the astaxanthin was with in the time of 96h (26,7 g/g) in the absence of light and with the presence of light (37,7 g/g) increase almost 41%. With CSL the best condition for the production of the astaxanthin was with in the time of 96h (55,8 g/g) in the absence of light and with the presence of light (28,0 g/g) increase almost 100%. The .quirera. in the concentration of 7%, presented astaxanthin income better, in the time of 96h, with light (18,4 g/g) increase 37%, when compared with the cultures growth in the absence of light (13,4 g/g). The best income of astaxanthin with CLS and .quirera. was in the concentration of 7%, 96h, with light (33,8 g/g), increase almost 47% in when compared with the cultures growth in the absence of light (22,9 g/g). The concentrations that more favored the income of astaxanthin was: CSL 4%, .quirera. 7% and CSL with .quirera. 7%, both in the presence of blue light, demonstrate the blue light intervenes directly with the astaxanthin synthesis. Also, the wastes utilizes have been potential for the astaxanthin production. The analyses in Statistica 5,0, demonstrate that other studies are necessary for attainment of the maximum productivity of astaxanthin for M. javanicus, in alternative means. / Com a recente substituição dos pigmentos sintéticos pelos carotenóides naturais, pesquisas têm sido desenvolvidas para viabilizar uma maior produção destas substâncias, através de fontes biológicas alternativas. Neste trabalho foi estudada a produção de astaxantina por uma amostra de Mucor javanicus utilizando o meio definido Hesseltine e Anderson (1957) modificado por Andrade (2000) e meios utilizando resíduos de milho (milhocina e quirera), em concentrações distintas (4%, 7% e 10%), pH 6,5, 120 rpm, 25ºC. Foram analisada também a influência do tempo de cultivo da amostra durante 48h, 72h e 96h, na presença e ausência de luz azul. Ao término do processo fermentativo, a astaxantina foi extraída em solução de Hexano/metanol e analisada por espectroscopia UV- visível (470 nm). Todos os parâmetros estudados nos experimentos foram combinados através de um planejamento fatorial 33 e analisados no Software Statistica 5.0. No meio Hesseltine e Anderson o melhor rendimento de astaxantina foi verificado no tempo de 96h (26,7 g/g), na ausência de luz e quando se utilizou luz azul (37,7 g/g), obteve-se um aumento de 41%. A melhor condição para a produção da astaxantina com a milhocina deu-se na concentração de 4%, 96h, com luz (55,8 g/g), aumentando em quase 100%, quando comparada com as culturas crescidas na ausência de luz (28,0 g/g). A quirera na concentração de 7% apresentou melhor rendimento de astaxantina, no tempo de 96h, com luz (18,4 g/g), aumentando 37%, quando comparada com as culturas crescidas na ausência de luz (13,4 g/g). O melhor rendimento de astaxantina com milhocina e quirera deu-se na concentração de 7%, 96h, com luz (33,8 g/g), aumentando em quase 47%, quando comparada com as culturas crescidas na ausência de luz (22,9 g/g). As concentrações que mais favoreceram ao aumento do rendimento da astaxantina foram: milhocina 4%, quirera 4% e milhocina com quirera 7%, todos na presença de luz azul, demonstrando que a luz azul interfere diretamente na síntese de astaxantina. Bem como que os resíduos utilizados possuem potencial para a produção de astaxantina. As análises no Statistica 5.0, demonstram a necessidade da realização de outros estudos para obtenção da produtividade máxima de astaxantina por M. javanicus, nos meios alternativos.
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Proteção antioxidante promovida por astaxantina sobre citocromo c, incorporado em vesículas e desafiado com SIN-1 / Antioxidant Protection Promoted by Astaxanthin over Cytochrome c Incorporated in Vesicles and Challenged with SIN-1Mano, Camila Marinho 16 September 2008 (has links)
A astaxantina (AST) é um carotenóide derivado do β-caroteno produzido por algas e cianobactérias, mas que também pode ser encontrada em animais marinhos. Em animais, é reportada como interceptadora de radicais de oxigênio mais eficiente que o β-caroteno. O objetivo central dessa dissertação foi avaliar a capacidade antioxidante da AST em lipossomos enriquecidos com citocromo c (cit c) desafiados com 3-morfolinosidnonimina (SIN-1), um doador de óxido nítrico, em diferentes microambientes (pH e composição das vesículas). Diferenças na interação destas vesículas com o cit c periférico, com reflexos na atividade antioxidante da AST também foram avaliadas. O SIN-1 gera, por termólise, quantidades equimolares de radical superóxido e óxido nítrico, quando há oxigênio no meio. Vesículas unilamelares de fosfatidilcolina (PC), PC contendo 5% ou 10% de fosfatidilglicerol (PG), com ou sem AST, foram incubadas com SIN-1 e/ou cit c. Medidas do índice de lipoperoxidação pelo teste das substâncias reativas ao ácido tiobarbitúrico (TBARS) revelaram que SIN-1 não causa aumento de TBARS, enquanto o cit c foi capaz de aumentar significativamente este índice. Este fato pode ser explicado pela atividade peroxidásica do cit c. Apenas em vesículas de PCPG10%, ao realizar a incubação do cit c concomitantemente com SIN-1, o índice de TBARS foi maior ao observado em vesículas incubadas apenas com cit c. É conhecido que a interação entre cit c e membranas aniônicas pode alterar a conformação da proteína, aumentando sua atividade peroxidásica. A presença da AST fez com que os índices de lipoperoxidação chegassem a valores próximos aos do controle. A alteração no pH do meio revelou que a AST possui ação antioxidante mais pronunciada em pHs 7,4 e 8,0, em comparação com pHs levemente ácidos. A presença de PG evidenciou ainda mais esta tendência e em pH 6,2, a AST apresentou inclusive pequena atividade próoxidante. Estes resultados podem ser discutidos à luz de alterações da permeabilidade da membrana e da reatividade de espécies reativas induzidas por mudanças da fluidez e de pH. O efeito dos produtos gerados por SIN-1 sobre o cit c foi estudado em condições de normóxia e hipóxia. Resultados de EPR e de fluorescência demonstram que a presença do radical superóxido previne lesões oxidativas causada por peróxido orgânico (t-butOOH) tanto no cit c quanto nas membranas, pois é capaz de reduzir o ferro hemínico do cit c. Através de CD e espectrofotometria UV-Vis e EPR, foi observado que a incubação com SIN-1 promove alterações estruturais no cit c causando ruptura na sexta coordenação do ferro hemínico, levando à geração de uma espécie de cit c com rombicidade menor em comparação ao cit c nativo e que apresenta maior atividade peroxidásica. Este trabalho contribui com informações para entendimento do mecanismo antioxidante da AST em diferentes microambientes, além de demonstrar o efeito paradoxal do superóxido que é capaz de proteger o cit c, através da redução do ferro hemínico, mas também pode expor a proteína à oxidação promovida por peroxinitrito. / Astaxanthin (AST) is a β-carotene derived carotenoid, produced by algae and cyanobacteria, but can also be found in marine animals. In phytoplankton it has the function to absorb light radiation for photosinthesys occurence. In animals AST acts as a scavenger of oxygen free radicals, even more efficiently than β-carotene itself. The main objective of this work is to evaluate the antioxidant capacity of AST over cytochrome c (cyt c) incorporated in liposomes and challenged with 3-morpholinosidnonimine (SIN-1), a nitric oxide donor, under different experimental conditions, namely vesicles composition and pH. Distinct interactions between cyt c and vesicles affecting the AST antioxidant activity were also evaluated. SIN-1 spontaneously generates equal amount of nitric oxide and superoxide anion when oxygen is present. Unilamellar vesicles made from phosphatidylcholine (PC) or PC with 5% or 10% of phosphatidylglycerol (PG), with or without AST, were incubated with SIN-1 and/or cyt c. The extent of lipid peroxidation was evaluated by the classical method of thiobarbituric reactive substances (TBARS). Control experiments with SIN-1 alone showed no increase in TBARS content, whereas cyt c significantly increased TBARS. Concomitant addition of cyt c, SIN-1 to PCPG10% vesicles led to lipid peroxidation indices even higher than those found when cyt c was incubated with PCPG10% vesicles. A peroxidase activity of cyt c resulting from the interaction between this protein and anionic membranes can explain this result. In this system, the presence of AST inhibited formation of TBARS, whose levels were near the control values. Astaxanthin was found to exhibit a more effective antioxidant capacity under basic pH (7.4 and 8.0), in comparison with pH 6.2 and 6.8. In the presence of PG, this trend became more evident. Interestingly, at pH 6.2, AST showed a slight pro-oxidant activity. These results can be explained by differences in membrane permeability and reactivity of reactive species, caused by pH and membrane fluidity alterations. The effects of products of SIN-1 decomposition on cyt c structure and its peroxidase activity were investigated under hypoxia and normoxia. EPR and fluorescence experiments revealed that superoxide anion radical, due to its ability to reduce heme iron, prevents oxidative damage of cyt c and membrane lipids by peroxide-derived free radicals. By means of CD and UV-Visible spectroscopy, we have found that concomitant incubation of SIN-1 and cyt c promoted structural alterations in the protein which changes the irons sixth axial coordination, leading to generation of a less rhombic cyt c, which is reportedly a better peroxidase than native cyt c. This work contributes with information aiming to better understand the antioxidant mechanism of AST under different membrane microenvironments and unveil a paradoxal effect of superoxide ion, which can protect cyt c from oxidative lesions by transferring electron to ferricyt c, but can also expose cyt c to oxidation by peroxynitrite.
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Experimental Studies on the Regulation of Pigment Dynamics in Phytoplankton and Copepods by Dissolved Inorganic NutrientsVan Nieuwerburgh, Lies January 2004 (has links)
<p>This thesis examines the role of dissolved inorganic nutrients in generating changes in phytoplankton community and pigment composition and if such changes can affect the production of the antioxidant astaxanthin in the ecosystem via pelagic copepods. The background of my studies is the possible relationship between eutrophication and a reproductive disturbance in Baltic populations of Atlantic salmon (M74), which is associated with astaxanthin and thiamine deficiencies and oxidative stress. In the southern Baltic Sea, changes in nutrient loads correlate with observed trends of flagellates replacing diatoms in the phytoplankton. Copepods are the main producers of astaxanthin and a major link between phytoplankton and higher trophic levels. In laboratory and field experiments in the Baltic Sea proper and the Norwegian Sea, I show that astaxanthin synthesis in copepods is fast and depends on pigment composition of the phytoplankton diet. Among single-species diets, a diatom and a green algal cyst yielded the highest astaxanthin levels in copepods, and another diatom species, a green alga and a cyanobacterium the lowest. In nutrient-generated phytoplankton blooms in mesocosms, copepods grazing on diverse communities dominated by weakly silicified diatoms produced more astaxanthin compared with copepods grazing on communities dominated by strongly silicified diatoms. This suggests that diatoms invested in defence mechanisms and escaped grazing at surplus Si. A nutrient-starved diatom culture subjected to intraspecific competition exhibited decreased pigment levels, increased thiamine levels and increased oxidative stress. </p><p>My results suggest that diatoms are beneficial for astaxanthin and thiamine production compared to other phytoplankton groups, but not under all circumstances. Copepod growth and development also responded to inorganic nutrient availability and affected total astaxanthin production per volume seawater, with highest production when the copepods grazed on diatoms. From an ecosystem perspective, increased N and P loads seem to promote high astaxanthin production, but not when diatoms disappear completely.</p>
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Pigment and Thiamine Dynamics in Marine Phytoplankton and CopepodsWänstrand, Ingrid January 2004 (has links)
Based on a field study and several mesocosm experiments, I evaluated the use of pigments as chemotaxonomical biomarkers for phytoplankton community composition in the Baltic Sea and I examined effects of inorganic nutrients on the dynamics of carotenoids and thiamine (vitamin B1) at the phytoplankton–copepod level in marine pelagic food webs. My results show that HPLC pigment analysis combined with CHEMTAX data processing was an accurate alternative to microscopic analysis of Baltic Sea phytoplankton. Experimental supply of N, P and Si affected copepod growth and biochemical status via changes in biomass and composition of their phytoplankton diet. Net population growth rates were generally higher when phytoflagellates dominated (low Si:N ratio) and lower when diatoms dominated (high Si:N ratio). Copepod body concentrations of astaxanthin decreased with fertilization. Correlations with reduced under-water irradiance were consistent with the photo-protective function of this antioxidant. Thiamine concentrations in phytoplankton also decreased with fertilization. In copepods, low Si:N ratios resulted in higher thiamine concentrations than high Si:N ratios. Thiamine concentration and degree of phosphorylation were useful as indicators of thiamine shortage both in phytoplankton and copepods. The concentrations of thiamine and astaxanthin in the copepod communities were positively correlated. As copepods constitute a major link between pelagic primary producers and higher trophic levels, fertilization effects may be responsible for astaxanthin and thiamine deficiencies in salmon suffering from the M74 syndrome, which appeared concurrently with large-scale eutrophication in the Baltic Sea. As both thiamine and astaxanthin are deficient in M74-affected salmon, there is a need for physiological and molecular investigations of possible interactions between the two compounds in living cells.
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