Validation of a new high-throughput method to determine urinary S-phenylmercapturic acid using low-temperature partitioning extraction and ultra high performance liquid chromatography-mass spectrometry.

A new highly sensitive and environmentally friendly analytical method, using low-temperature partition extraction and ultra-high-performance liquid chromatography with tandem mass spectrometry, without the use of a labeled analyte, was developed and validated to determine and quantify urinary S-phenylmercapturic acid in urine samples. The World Health Organization, in its guidelines for air quality in Europe, recognizes that benzene is carcinogenic to humans and there is no safe level of exposure. Urinary S-phenylmercapturic acid is a sensitive and specific biological marker of exposure to benzene. The new analytical method, extraction, and analysis, were linear in the working range between 0.1 and 200.0 µg/L, precise (relative standard deviation lower than 6.0%), accurate (97.0-105.0%), and sensitive. The method's limits of detection and quantification were 0.02 and 0.084 µg/L, respectively. The recovery with the low-temperature partition extraction was 96.1%, with relative standard deviation less than 3.8%. The method is simple, accurate, and reproducible, and has been successfully applied in the evaluation of nonoccupational exposure to benzene, by urinary S-phenylmercapturic acid in urine samples.

Biotechnological potential of yeast isolates from cachaça: the Brazilian spirit.

This study identified phenotypic traits appropriate for biotechnological applications of 118 yeasts isolated from cachaça distilleries. Different properties were verified: capacity to use alternative carbon sources; ability to tolerate high concentrations of sucrose, ethanol, methanol, aluminum and zinc as well as different pH values and foam production. Pichia guilliermondii and Pichia anomala strains were identified as the most promising ones for application in the second-generation biofuel industry, showing ability to grow on high glycerol concentrations. Other isolates, identified as Saccharomyces cerevisiae, produced bioethanol comparable to the industrial strains, and were therefore ideal for use in the first-generation ethanol industry. Some of these strains also showed high resistance to aluminum, as observed in sugarcane juice, and to inter-cycle washings with diluted sulphuric acid, as performed in the industrial bioethanol production process. In summary, yeast isolates from cachaça distilleries displayed robustness and phenotypic plasticity, which makes them interesting for biotechnological applications.

Strategies to select yeast starters cultures for production of flavor compounds in cachaça fermentations.

In this work, we have used classical genetics techniques to find improved starter strains to produce cachaça with superior sensorial quality. Our strategy included the selection of yeast strains resistant to 5,5',5″-trifluor-D: ,L: -leucine (TLF) and cerulenin, since these strains produce higher levels of higher alcohols and esters than parental strains. However, no clear relationship was observed when levels of flavoring compounds were compared with the levels expression of the genes (BAT1, BAT2, ATF2, EEB1 genes) involved with the biosynthesis of flavoring compounds. Furthermore, we determined the stability of phenotypes considered as the best indicators of the quality of the cachaça for a parental strain and its segregants. By applying the principal component analysis, a cluster of segregants, showing a high number of characteristics similar to the parental strain, was recognized. One segregant, that was resistant to TLF and cerulenin, also showed growth stability after six consecutive replications on plates containing high concentrations of sugar and ethanol. "Cachaça" produced at laboratory scale using a parental strain and this segregant showed a higher level of flavoring compounds. Both strains predominated in an open fermentative process through seven cycles, as was shown by mitochondrial restriction fragment length polymorphisms analysis. Based on the physical chemical composition of the obtained products, the results demonstrate the usefulness of the developed strategies for the selection of yeast strains to be used as starters in "cachaça" production.

Biochemical and molecular characterization of Saccharomyces cerevisiae strains obtained from sugar-cane juice fermentations and their impact in cachaça production.

Saccharomyces cerevisiae strains from different regions of Minas Gerais, Brazil, were isolated and characterized aiming at the selection of starter yeasts to be used in the production of cachaça, the Brazilian sugar cane spirit. The methodology established took into account the screening for biochemical traits desirable in a yeast cachaça producer, such as no H2S production, high tolerance to ethanol and high temperatures, high fermentative capacity, and the abilities to flocculate and to produce mycocins. Furthermore, the yeasts were exposed to drugs such as 5,5',5"-trifluor-D,L-leucine and cerulenin to isolate those that potentially overproduce higher alcohols and esters. The utilization of a random amplified polymorphic DNA-PCR method with primers based on intron splicing sites flanking regions of the COX1 gene, as well as microsatellite analysis, was not sufficient to achieve good differentiation among selected strains. In contrast, karyotype analysis allowed a clear distinction among all strains. Two selected strains were experimentally evaluated as cachaça producers. The results suggest that the selection of strains as fermentation starters requires the combined use of biochemical and molecular criteria to ensure the isolation and identification of strains with potential characteristics to produce cachaça with a higher quality standard.

Isolation of Saccharomyces cerevisiae strains producing higher levels of flavoring compounds for production of "cachaça" the Brazilian sugarcane spirit.

In Brazil, spontaneous fermentation and open vessels are still used to produce cachaça (the Brazilian sugarcane spirit) and this fermentation is characterized by mixed cultures with continuous succession of yeast species. This work shows the development of a methodology for isolation of yeasts, particularly Saccharomyces cerevisiae, used in the production of cachaça. According to the proposed strategy, the strains were selected for their ability to adapt to stress conditions encountered during fermentation of the sugarcane juice such as high sucrose concentration; high temperatures and high alcohol concentration; for their capacity to flocculate; and for their higher fermentative ability. For strains with such characteristics, specific procedures were employed to select for 5,5,5-trifluoro-DL-leucine (TFL) and cerulenin-resistant strains, since these characteristics are related to a higher capacity of production of the flavoring compounds isoamyl alcohol and caproic acid, respectively. The effectiveness of such a selection strategy was documented. Taken together, the results obtained present the development of a new strategy to isolate yeast strains with appropriated characteristics to be used in the cachaça industry. Moreover, the results obtained offer an explanation for the great variability in terms of chemical composition found in products obtained even in a single distillery.

Desenvolvimento de metodologia analítica para a determinação de indicador biológico de exposição ao benzeno / Development of analytic methodology for the determination of biological indicator of exhibition to the benzene

Os limites de exposição ocupacional ao benzeno, um agente carcinogênico, vêm diminuindo drasticamente nos útimos anos. Por outro lado, a concentração de benzeno em ambientes não ocupacionais tem aumentado devido à emissão biogênica e antropogênica, como exaustão de motores e gasolina e fumaça de cigarro. Indicadores Biológicos de Exposição (IBE) são utilizados como ferramentas importantes na avaliação da exposição humana ao benzeno. Com a diminuição dos limites de exposição, se faz necessário o desenvolvimento de metodologias analíticas com sensibilidade adequada para a determinação do IBE em fluidos biológicos que se correlacionem com baixas concentrações de benzeno absorvido pelo organismo. A utilização do fenol urinário como IBE ao benzeno, embora reconhecida mundialmente, tem a desvantagem de não apresentar boa correlação com a concentração de benzeno ambiental quando esta é menor do que 10 ppm (32 mg/mÝ). Os ácidos trans, trans-mucômico e S-fenilmercaptúrico, metabólitos do benzeno encontrados na urina, estão entre os compostos mais estudados como IBE ao benzeno. O ácido trans, trans-mucônico foi determinado na urina de indivíduos expostos ao benzeno utilizando as técnicas de Eletroforese Capilar (CE) e HPLC, ambas com detecção no UV. Na determinação por HPLC foi adaptada uma metodologia da literatura utilizando coluna analítica com fase reversa. Na determinação por CE foi proposta uma metodologia empregando duas condições analíticas alternativas: uma que utiliza um capilar especial com cela ótica de alta sensibilidade e a outra que utiliza um capilar comum, mas com adição de um modificador orgânico ao eletrólito. As duas condições apresentaram grandes vantagens, como análise rápida (15 minutos) e baixo limite de detecção (25µg/L). Foram analisadas amostras de urina de indivíduos fumantes e não fumantes onde a sensibilidade da metodologia proposta foi suficiente para diferenciar estatisticamente os dois grupos avaliados.