Incorporação de ferro pela levedura Saccharomyces Cerevisiae e sua influência na formação de biomassa e na capacidade fermentativa

Orientadora : Profa. Dra. Tania M. B. BonfimCo-orientadora : Drª Debora BrandDissertação(mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmacêuticas. Defesa: Curitiba, 26/03/2010Bibliografia: fls. 73-77Área de concentração: Insumos, medicamentos e correlatosResumo: O ferro consiste em um micronutriente essencial para o metabolismo dos organismos, entretanto, sua deficiência é observada principalmente em mulheres com idade fértil e em crianças. Sais de ferro suplementados à dieta possuem baixa biodisponibilidade e podem causar desconfortos gastrointestinais. Leveduras enriquecidas com ferro podem ser um veículo para a suplementação deste mineral à dieta, pois a ligação do ferro com macromoléculas presentes nas células da levedura torna este mineral biodisponível. O presente trabalho avaliou o emprego de sulfato ferroso, sulfato férrico, cloreto ferroso, cloreto férrico, nitrato férrico, citrato férrico e EDTA férrico para suplementar o cultivo em frascos da levedura de panificação Saccharomyces cerevisiae. A quantificação do ferro absorvido pela levedura foi avaliada por colorimetria. A concentração de biomassa obtida nos cultivos suplementados com 15 mg Fe l-1 e 25 mg Fe l-1 empregando sulfato ferroso, cloreto ferroso, citrato férrico, EDTA férrico e sem suplementação de ferro foram semelhantes. O meio com sulfato ferroso 100 mg Fe l-1 permitiu maior acúmulo de ferro intracelular, em torno de 5,43 mg Fe g-1 célula seca, após 20 horas de cultivo. Avaliando-se as características morfológicas microscópicas das células, foi observada a presença moderada ou intensa de pseudo-hifas nos cultivos suplementados com os sais cloreto férrico e cloreto ferroso nas concentrações de 15 mg Fe l-1 e 25 mg Fe l-1 e em todos os cultivos com concentrações de ferro igual ou acima de 50 mg Fe l-1. Os valores de produtividade em biomassa e em produto mostraram que o melhor desempenho da levedura de panificação Saccharomyces cerevisiae tanto no que se refere ao crescimento celular quanto na incorporação de ferro ocorreu quando se empregou o meio YM 1% suplementado com 100 mg Fe l-1 utilizando o sulfato ferroso. O cultivo da levedura de panificação Saccharomyces cerevisiae em reator empregando meio YM 1% suplementado com 100 mg Fe l-1 utilizando o sulfato ferroso obteve biomassa com cerca de 8 mg Fe g-1 de célula seca, que manteve sua capacidade fermentativa no que se refere ao deslocamento de água pela produção de gás carbônico ou pelo seu emprego em panificação. O pão produzido com a levedura obtida por cultivo em meio YM 1% suplementado com 100 mg Fe l-1 obteve 6 vezes mais ferro que o pão produzido com a levedura cultivada em meio YM 1% sem suplementação de ferro.Abstract: Iron is an essential micronutrient to organism metabolism, however, its deficiency is well documented especially in pregnant women and in children. Iron salts supplemented to the diet have low ioavailability and can cause gastrointestinal discomforts. Iron enriched yeasts can provide a supplementation of this micronutrient to the diet because this mineral has a better bioavailability when bounded to yeast cells macromolecules. This work studied the use of ferrous sulfate, ferric sulfate, ferrous chloride, ferric chloride, ferric nitrate, ferric citrate and ferric EDTA to supplement flasks cultures of baker’s yeast accharomyces cerevisiae. In order to quantify the absorbed iron by the yeast, colorimetric evaluation was applied. Biomass formation in cultivation supplemented with 15 mg Fe l-1 or 25 mg Fe l-1 employing ferrous sulfate, ferrous chloride, ferric citrate, ferric EDTA and without supplementation was similar. The media with ferrous sulfate 100 mg Fe l-1 allowed the better intracellular iron accumulation, around 5,43 mg Fe g-1 dry matter, after 20 hours of cultivation. When the microscopic morphological characteristics of the cells were evaluated, it was observed a moderate or intense pseudohyphae presence in cultivations supplemented with ferric chloride or ferrous chloride 15 mg Fe l-1 or 25 mg Fe l-1 and in all cultivations which iron concentration applied was equal or superior to 50 mg Fe l-1. Biomass productivity and product productivity values showed that the best performance of baker’s yeast Saccharomyces cerevisiae for cellular growth as well as for iron uptake took place when Yeast Media (YM) 1% supplemented with 100 mg Fe l-1 of ferrous sulfate was used. Baker’s yeast Saccharomyces cerevisiae was cultivated in reactor employing YM 1% supplemented with 100 mg Fe l-1 using ferrous sulfate and the resultant biomass had around 8 mg Fe g-1 dry matter. This biomass kept its fermentative power regarding bothwater displace measurement thought carbonic dioxide production as well as its bakery characteristics. The bread produced using the yeast obtained by cultivation in YM 1% supplemented with 100 mg Fe l-1 presented 6 times more iron than the bread produced using the yeast obtained by cultivation in YM 1% without iron supplementation

Seleção de leveduras vínicas autóctones produtoras da enzima b-glicosidade em mosto de uvas Vitis labrusca visando o aumento do teor de resveratrol livre

Orientadora : Profª. Tania Maria Bordin BonfimCoorientador : Gildo Almeida da SilvaTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmacêuticas. Defesa: Curitiba, 22/06/2016Inclui referências : f. 120-131Área de concentração: Insumos, medicamentos e correlatosResumo: O vinho tinto é uma das principais fonte de resveratrol na dieta. Diferentesestratégias têm sido adotadas em vinificação para obter vinhos enriquecidos comresveratrol pois consumidores pagariam mais em um produto funcional, visto queeste estilbeno possui múltiplas atividades biológicas benéficas. Leveduras com açãoda enzima ?-glicosidase, isoladas de uvas, podem aumentar a concentração deresveratrol livre em vinhos hidrolisando seu glicosídio, piceid, sem modificar acomposição ou características organolépticas finais do produto. O objetivo destetrabalho foi isolar, selecionar e identificar leveduras de uvas do estado do Paraná,com ação da enzima ?-glicosidase sobre o substrato piceid, visando o resveratrollivre, adequadas para a elaboração de vinhos. Um total de 308 leveduras obtidas dediferentes tempos de fermentação espontânea de mosto de uvas foram isoladas eavaliadas primeiramente utilizando os substratos celobiose, arbutin e esculin paraverificar a reação de hidrólise catalisada pela enzima ?-glicosidase. As 58 levedurascom capacidade hidrolítica foram identificadas por testes fenotípicos, espectrometriade massa MALDI-TOF/MS, reação em cadeia da polimerase (PCR) e polimorfismono comprimento de fragmentos de restrição (PCR-RFLP) da região ITS do DNAribossomal. A ação da enzima ?-glicosidase no substrato piceid foi verificada nasleveduras identificadas. Uma levedura Saccharomyces e 32 leveduras não-Saccharomyces hidrolisaram o substrato piceid em resveratrol e glucose. Destas,durante a fermentação, quatorze leveduras hidrolisaram o substrato piceid presenteno mosto de uva Vitis labrusca em resveratrol livre e glucose. Houve um aumento naconcentração inicial de resveratrol livre de 102% sem exibir diferença significativaentre quatorze diferentes leveduras. Quatro leveduras Hanseniaspora uvarumapresentaram características enológicas desejadas e foram utilizadas emfermentações com mosto de uva Vitis labrusca, tanto em monocultura quanto eminóculo misto. Os resultados sugerem que a ação da enzima ?-glicosidase deleveduras não depende somente da espécie avaliada, mas também dascaracterísticas individuais de cada levedura. Uma levedura Hanseniaspora uvarumisolada, quando utilizada em monocultura, inóculo misto ou inóculo misto sequencial,interferiu na elaboração de vinhos, aumentando a concentração de resveratrol livre.Palavras-chave: Levedura vínica. ?-glicosidase. Piceid. Seleção de levedura.Hanseniaspora uvarum.Abstract: The red wine is one of the main resveratrol sources in the diet. Differentstrategies have been adopted in winery in order to obtain resveratrol-enriched wines,since consumers would pay more for a functional wine, as this stilbene possesesvarious beneficial biological activities. Wine yeasts endowed with ?-glucosidaseactivity can increase the free resveratrol content in wines by hydrolyzing its glycoside,piceid, without modifying the product's final composition or organolepticcharacteristics. The objective of this work was to isolate, select and identify yeastsfrom native grapes grown in Parana state vineyards endowed with ?-glucosidaseactivity over the piceid substrate, aiming at free resveratrol, and adequate to be usedin winery. A total of 308 yeasts, from different steps of the spontaneous fermentationfrom grape must, were isolated and tested, firstly over the substrates cellobiose,arbutin, and esculin to verify the reaction catalyzed by the ?-glucosidase enzyme.The 58 yeasts with hydrolytic capability were identified by phenotypic analysis,matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) massspectrometry, polymerase chain reaction (PCR) and restriction fragment lengthpolymorphism (PCR-RFLP) from the ITS region of ribosomal DNA. The activity of the?-glucosidase over the substrate piceid was evaluated employing the identifiedyeasts. One Saccharomyces and 32 non-Saccharomyces yeasts hydrolyzed piceid toresveratrol and glucose. During the fermentation, fourteen out of the 33 yeastshydrolyzed the substrate piceid existing in Vitis labrusca grape must to freeresveratrol. A 102% increment from the initial free resveratrol was observed withoutsignificant difference between the fourteen different yeasts. Four Hanseniasporauvarum also presented desired oenological characteristics and were later employedin grape must fermentation, in both single and mixed cultures. The results suggestthat the ?-glucosidase activity is more dependent on the yeasts' individualcharacteristics than on their species. One isolated Hanseniaspora uvarum, whenemployed in single, mixed or sequential mixed fermentation, can yield the productionof resveratrol-enriched wines.Keywords: Wine yeast. ?-glucosidase. Piceid. Yeast selection. Hanseniasporauvarum

Iron enriched Saccharomyces cerevisiae maintains its fermenting power and bakery properties

Iron is an essential micronutrient in the metabolism of almost all living organisms; however, its deficiency is well documented especially in pregnant women and in children. Iron salts as a dietary supplement have low bioavailability and can cause gastrointestinal discomforts. Iron enriched yeasts can provide a supplementation of this micronutrient to the diet because this mineral has a better bioavailability when bonded to yeast cell macromolecules. These yeasts can be used as feed supplement for human and animals and also as baker's yeast. Baker's yeast Saccharomyces cerevisiae was cultivated in a reactor employing yeast media supplemented with 497 mg ferrous sulfate.L-1, and the resultant biomass incorporated 8 mg Fe.g-1 dry matter. This biomass maintained its fermenting power regarding both water displace measurement through carbonic dioxide production and bakery characteristics. The bread produced using the yeast obtained by cultivation in yeast media supplemented with iron presented six times more iron than the bread produced using the yeast obtained by cultivation without iron supplementation

Autochthonous yeasts with β-glucosidase activity increase resveratrol concentration during the alcoholic fermentation of Vitis labrusca grape must

AbstractSince red wine is the main dietary source of resveratrol, a well-known polyphenol that reduces coronary events in humans, different strategies have been employed in winery to achieve resveratrol-enriched wines. Yeasts-endowed β-glucosidase activity enhances free-resveratrol concentration in wine without modifying its composition or sensorial properties. Current assay screened 308 autochthonous yeast strains for β-glucosidase activity employing arbutin, esculin, cellobiose and piceid as substrates. The β-glucosidase-producer yeasts were evaluated in the must of Vitis labrusca Bordô grape to quantify resveratrol concentration before and after alcoholic fermentation. Fourteen yeasts increased the resveratrol concentration up to 102% without any significant difference and nine of these yeast strains also produced high ethanol contents. Four autochthonous Hanseniaspora uvarum β-glucosidase-producer strains showed adequate oenological characteristics and hydrolysed resveratrol-glucosides during the alcoholic fermentation of V. labrusca grape must

Characterization of autochthonous wine yeasts isolated in vineyards of the State of Paraná, Brazil

The transformation of grape must into wine is a complex microbiological process and is the product of the combined action of several genera and species of yeasts, dominated in the intermediate and final stages of fermentation by an alcohol-tolerant Saccharomyces sp. Current assay characterizes 42 autochthonous yeasts, isolated from the state of Paraná, southern Brazil, according to the following oenological properties: H2S production, fermentation rate, flocculation capacity, and killer phenotype (killer, sensitive and neutral characteristics). Current analysis is the first to evaluate killer phenotype in yeasts isolated from the State of Paraná, Brazil. With regard to their oenological traits, the yeasts evaluated were not suitable for winemaking and suggested that, depending on the harvest, the winemakers may face problems during the spontaneous wine production process.

Evaluation of phenolic compounds content and in vitro antioxidant activity of red wines produced from Vitis labrusca grapes

Wine production in the northern Curitiba, Paraná, Brazil, specifically the communes of Colombo and Almirante Tamandaré, is based mainly on the utilization of Vitis labrusca grapes var. Bordô (Ives). Total sugar content, pH, and total acidity were analyzed in red wine samples from 2007 and 2008 vintages following official methods of analysis. Moreover, total phenolic, flavonoid, and tannin contents were analyzed by colorimetric methodologies and the antioxidant activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical methodology. Phenolic compounds were identified by high performance liquid chromatography. The total phenolic content of wine samples presented concentrations varying between 1582.35 and 2896.08 mg gallic acid.L-1 since the major part corresponds to flavonoid content. In these compounds' concentration range, a direct relationship between phenolic compounds content and levels of antioxidant activity was not observed. Among the identified phenolic compounds, chlorogenic, caffeic, and syringic acids were found to be the major components. Using three principal components, it was possible to explain 81.36% of total variance of the studied samples. Principal Components Analysis does not differentiate between vintages