The interference of sulfide with the colorimetric determination of nitrite, by the method of Bendschneider and Robinson (1952), can be eliminated by passing the samples through a cationic exchange resin, Dowex 50W-X8, in the zinc form. This resin retains sulfide without affecting nitrite and the technique can be applied to automated methods. / Graduation date: 1980

Graduation date: 1997

Sulfite mutants representing five complementation groups, previously derived from an ethyl methanesulfonate-treated haploid strain of Saccharomyces cerevisiae were studied. Although the wildtype S. cerevisiae strain used (isogenic to X2180-1 A) had a basal tolerance for sulfite (7 μM free H₂SO₃), the sensitive and resistant mutants were found to tolerate less than 3 to 5.5, or greater than 19 μM free H₂SO₃, respectively. No apparent correlation was found between the response to sulfite and generation time in rich (YEPD) or minimal media. Resistant mutant 11-1 had an extended lag phase relative to wildtype. Mutant and wildtype proteins were labeled with ³⁵S-methionine to determine differences in banding patterns due to sulfite-specific induction or disappearance of polypeptides. No obvious differences following SDS-PAGE and autoradiography were observed upon induction with 0.213 μM free H₂SO₃. No consistent correlations were found between the sulfite phenotypes and responses to other reducing agents. Sensitive mutant 35-2 appeared to be three to ten times more sensitive to dithiothreitol than wildtype and sensitive mutant 47-9 was three to four times more sensitive to sodium nitrite and three to seven times more sensitive to sodium thiosulfate than wildtype. Log phase cells of sensitive mutant 33-2 were found to have significantly less glutathione than wildtype. Wildtype contained 62.6 nmol min⁻¹ mg protein⁻¹ (62.6 mU mg protein⁻¹) glutathione reductase (GR) and 2.78 nmol min⁻¹ mg protein⁻¹ (2.78 mU mg protein⁻¹) glutathione S-transferase (GST). Log phase cells of one resistant mutant showed a significantly higher level of GR than wildtype, 135%. The resistant mutants as well as some of the sensitive mutants had reduced GST levels. Survival rates of the mutants in buffer in the presence of sulfite did not correlate with their sensitive or resistant phenotypes, suggesting that survival and growth in the presence of sulfite are not necessarily related functions. Relative to wildtype, survival upon prolonged storage at 4°C was markedly reduced for two of the four sensitive mutants, one of which was 33-2, and was enhanced for one resistant and another sensitive mutant. / Graduation date: 1992

In an effort to understand the basis for sulfite detoxification in S. cerevisiae, the functions of two genes were analyzed. SSU1, which encodes a plasma membrane protein, was found to be required for efficient sulfite efflux. FZFl-4, a dominant allele of a transcriptional activator of SSUl, was also found to be involved in efficient sulfite efflux. Analysis of an SSUl promoter-lacZ fusion showed that FZFl-4 conferred sulfite resistance through hyperactivation of SSUl. Efflux assays in cells expressing multicopy SSUl or FZFl-4 suggested that Ssulp specifically mediates efflux of the free form of sulfite. Sulfite resistance, mediated by either FZFl-4 or multicopy SSUl, was found to be a useful marker for selecting transformants of industrial and laboratory strains of S. cerevisiae. FZFl-4 was found to be more efficient than multicopy SSUl, and in the case of the laboratory strains, was found to be about half as efficient a selectable marker as URA3. Sulfite transport was studied to clarify the mechanism of sulfite uptake in S. cerevisiae. The kinetics of uptake were saturable, indicating a carrier-mediated process. Uptake was significantly reduced in cells pretreated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) or 2,4-dinitrophenol (DNP), both of which dissipate proton gradients. Extracellular alkalization was observed during sulfite uptake. These findings suggest that an anionic form of sulfite, HSO₃, is taken up by carrier-mediated proton symport. As an alternative to costly disposal of spent cherry brine, a sulfite-containing waste stream generated during maraschino cherry processing, brine was tested as a substrate for ethanol production by S. cerevisiae. Initially, the toxic level of sulfite in brine was reduced by raising brine pH to 8.5 with Ca(OH)₂ to precipitate calcium sulfite. Because the alkalization was found to result in a 10-fold reduction of phosphorus, brine was subsequently titrated with phosphoric acid to pH 6.0 prior to inoculation with S. cerevisiae. All strains of S. cerevisiae tested were able to efficiently ferment all lots of Ca(OH)₂-treated and phosphorus-enriched brine. / Graduation date: 2000

Os sulfitos, representados pelo SO2, fazem parte de um importante grupo de aditivos utilizados, há séculos, como conservantes em frutas secas, sucos de frutas, vinhos. No Brasil não existem dados sobre sua utilização pelas indústrias de alimentos e pesquisas sobre o seu consumo pela população. Assim, o objetivo desta pesquisa foi estimar a ingestão de sulfitos em dois grupos de alunos do ensino médio, respectivamente de escola particular e pública. Os dados foram coletados por meio de R24, que permitiu relacionar as fontes de sulfitos presentes na dieta. Para o cálculo do consumo deste aditivo foi utilizado o MPL, definido pela legislação brasileira para cada uma das fontes. Nenhum dos escolares ultrapassou a IDA de 0,70 mg SO2/kg pc/dia, com média de consumo de 0,07 mg SO2/kg pc/dia (p<0,001), sem diferença estatisticamente significante (p = 0,643) entre as escolas particular e pública. Os grandes consumidores (consumo de mais de 50% da IDA, ou seja, 0,35 mg SO2/kg pc/dia, até o limite 0,52 mg SO2/kg pc/dia) representaram 4,5% da amostra pesquisada e alcançaram estes níveis de ingestão devido ao consumo acima de 500 mL/dia de sucos industrializados e similares, e, na escola particular, por associar ao seu consumo bebidas alcoólicas, como cerveja e vinho. / The sulphites, represented for the SO2, are part of an important group of additives, have used for centuries as preservatives in dry fruits, fruit juices, wines. In Brazil there are no data on use of sulphites by the food industry and research on their consumption by population. Thus, the objective of this research was to estimate the ingestion of sulphites in two groups of high school students, one of privative school and another of public school. The data were collected through 24-hour dietary recall, that allowed to relate the sources of sulphites in the diet. For calculation of the consumption of this additive the MPL, stabilished by the Brazilian legislation was used for each sources. None of students exceeded the ADI of 0.70 mg SO2/kg bw/day, with average of consumption of 0.07 mg SO2/kg bw/day (p < 0.001), without statistical difference (p = 0.643) between privative and public school. Heavy consumers (consumption of more than 50% of the ADI, or either, 0.35 mg SO2/kg bw/day, until 0.52 mg SO2/kg bw/dia) represented 4.5% of the searched sample and reached these levels of ingestion due to the consumption above 500 mL/day of fruit industrialized juices, and, in privative school, for associating with its consumption alcoholic beverages, as beer and wine.

Os sulfitos, representados pelo SO2, fazem parte de um importante grupo de aditivos utilizados, há séculos, como conservantes em frutas secas, sucos de frutas, vinhos. No Brasil não existem dados sobre sua utilização pelas indústrias de alimentos e pesquisas sobre o seu consumo pela população. Assim, o objetivo desta pesquisa foi estimar a ingestão de sulfitos em dois grupos de alunos do ensino médio, respectivamente de escola particular e pública. Os dados foram coletados por meio de R24, que permitiu relacionar as fontes de sulfitos presentes na dieta. Para o cálculo do consumo deste aditivo foi utilizado o MPL, definido pela legislação brasileira para cada uma das fontes. Nenhum dos escolares ultrapassou a IDA de 0,70 mg SO2/kg pc/dia, com média de consumo de 0,07 mg SO2/kg pc/dia (p<0,001), sem diferença estatisticamente significante (p = 0,643) entre as escolas particular e pública. Os grandes consumidores (consumo de mais de 50% da IDA, ou seja, 0,35 mg SO2/kg pc/dia, até o limite 0,52 mg SO2/kg pc/dia) representaram 4,5% da amostra pesquisada e alcançaram estes níveis de ingestão devido ao consumo acima de 500 mL/dia de sucos industrializados e similares, e, na escola particular, por associar ao seu consumo bebidas alcoólicas, como cerveja e vinho. / The sulphites, represented for the SO2, are part of an important group of additives, have used for centuries as preservatives in dry fruits, fruit juices, wines. In Brazil there are no data on use of sulphites by the food industry and research on their consumption by population. Thus, the objective of this research was to estimate the ingestion of sulphites in two groups of high school students, one of privative school and another of public school. The data were collected through 24-hour dietary recall, that allowed to relate the sources of sulphites in the diet. For calculation of the consumption of this additive the MPL, stabilished by the Brazilian legislation was used for each sources. None of students exceeded the ADI of 0.70 mg SO2/kg bw/day, with average of consumption of 0.07 mg SO2/kg bw/day (p < 0.001), without statistical difference (p = 0.643) between privative and public school. Heavy consumers (consumption of more than 50% of the ADI, or either, 0.35 mg SO2/kg bw/day, until 0.52 mg SO2/kg bw/dia) represented 4.5% of the searched sample and reached these levels of ingestion due to the consumption above 500 mL/day of fruit industrialized juices, and, in privative school, for associating with its consumption alcoholic beverages, as beer and wine.

Brettanomyces bruxellensis est un microorganisme qui est considéré comme la cause majeure des défauts microbiologiques du vin. L’importance de cette levure à l’échelle industrielle est liée au fait qu’elle est isolée à partir de substrats différents tels que la bière, le kombucha, les molasses utilisées pour la production de bioéthanol et autres. Ce projet a pour objectif d’étudier la diversité génétique de l’espèce en se basant sur une large population d’isolats provenant de niches écologiques et géographiques variées. Pour ce faire, une méthode de génotypage robuste (analyse microsatellite) a été optimisée et appliquée sur la population, mettant en évidence la coexistence de populations diploïdes et triploïdes à l’échelle globale. Puis, la relation entre regroupement génétique et traits physiologiques a été explorée. Notamment, l'étude de la tolérance aux sulfites a été effectuée sur un sous-ensemble de souches représentatif de la population. Les résultats obtenus mettent en évidence un lien entre groupes génétiques et comportement vis-à-vis des sulfites. Des expériences de compétition en présence de dioxyde de soufre montrent un avantage sélectif des souches tolérantes aux sulfites par rapport aux souches sensibles, suggérant ainsi une adaptation spécifique au principal antiseptique utilisé en œnologie. Ce travail contribue à une meilleure connaissance de cette levure d’altération du vin en termes de diversité génétique et phénotypique et permet d’émettre des hypothèses sur les stratégies évolutives d'adaptation au milieu anthropique de cette espèce modèle non conventionnelle. / Brettanomyces bruxellensis is a microorganism described as the first cause of microbial spoilage of wine. Its industrial relevance is highlighted by the fact that this yeast is isolated from different substrates such as beer, kombucha, bioethanol fermentation molasses and others. This project aims to explore the genetic diversity of the species by studying a large population of isolates from various geographical and ecological niches. For this purpose, a robust genotyping method (microsatellite analysis) was optimized and applied on the population, thus highlighting the coexistence of diploid and triploid populations worldwide. Further, the relation between genotypic clustering and physiological traits was studied. Namely, sulphite tolerance assay was performed on a subset of strains representative of the total population. The results reveal a link between genetic group and growth profile in the presence of sulphur dioxide. Competition experiments in presence of sulphites highlight a selective advantage of sulphite tolerant strains compared to sulphite sensitive ones, thus suggesting a specific adaptation to the main antimicrobial used in winemaking. This work contributes to a deeper understanding of this wine spoilage microorganism in means of genetic and phenotypic diversity and sheds light on putative evolutionary strategies for adaptation to human related environment of this non-conventional model yeast species.

No Brasil os dados sobre a utilização dos sulfitos pela indústria de alimentos e pesquisas sobre o seu consumo são escassos. Sendo assim, o objetivo desta pesquisa foi avaliar o nível de sulfitos em sucos de frutas e estimar seu consumo por escolares de 5ª. a 8ª. séries de escolas públicas, do ensino fundamental, do município de São Caetano do Sul-SP, Brasil. Foi aplicado questionário de freqüência de consumo de alimentos (QFCA) em 313 escolares, sendo 56,9 % do sexo feminino e 43,1 % do sexo masculino, com média de idade de 14,7 anos. Os alimentos sulfitados mais consumidos foram os néctares ou sucos de frutas, o açúcar refinado, as batatas fritas congeladas, o coco ralado, o suco de caju e as frutas secas e cristalizadas. Considerando os limites máximos permitidos (LMP) pela legislação brasileira, o consumo de SO2, em mg SO2/kg pc/dia, foi de 0,12 mg SO2/kg pc/dia, sendo que este valor médio corresponde a 17% da IDA (0,7 mg SO2/kg pc/dia). Já os resultados das análises dos sucos de frutas demonstram que a análise por injeção em fluxo (Flow injection analysis - FIA) modificada obteve resultados de sulfitos nas amostras analisadas com menor variação do que aqueles obtidos pelo método Monier-Williams (M-W) otimizado, e que nenhuma amostra ultrapassou os LMP. / In Brazil there are no data on use of sulphites by the food industry and research on their consumption by population. Thus, the objective of this research was to evaluate sulphites in fruits juices and estimate intake sulphites in a group of 5th 8th schoolchildren of public schools in São Caetano do Sul-SP, Brazil. It was applied a food frequency questionnaire (FFQ) at 313 schoolchildrens (56.9% female, 43.1% male and average age 14.7 years). Sulphited products identified were fruits juices, sugar, frozen chips, grated coconut, cashew juice and dried fruits. Considering maximum permitted levels (MPL) stabilished by Brazilian legislation, none of the students exceeded the ADI of 0.70 mg SO2/kg bw/day, with average of consumption of 0.12 mg SO2/kg bw/day (17 % of the ADI). The results of the fruits juices analysis demonstrated flow injection analysis (FIA) modified obtained better results than optimized Monier-Williams method (M-W) and no sample exceeded the MPL.

No Brasil os dados sobre a utilização dos sulfitos pela indústria de alimentos e pesquisas sobre o seu consumo são escassos. Sendo assim, o objetivo desta pesquisa foi avaliar o nível de sulfitos em sucos de frutas e estimar seu consumo por escolares de 5ª. a 8ª. séries de escolas públicas, do ensino fundamental, do município de São Caetano do Sul-SP, Brasil. Foi aplicado questionário de freqüência de consumo de alimentos (QFCA) em 313 escolares, sendo 56,9 % do sexo feminino e 43,1 % do sexo masculino, com média de idade de 14,7 anos. Os alimentos sulfitados mais consumidos foram os néctares ou sucos de frutas, o açúcar refinado, as batatas fritas congeladas, o coco ralado, o suco de caju e as frutas secas e cristalizadas. Considerando os limites máximos permitidos (LMP) pela legislação brasileira, o consumo de SO2, em mg SO2/kg pc/dia, foi de 0,12 mg SO2/kg pc/dia, sendo que este valor médio corresponde a 17% da IDA (0,7 mg SO2/kg pc/dia). Já os resultados das análises dos sucos de frutas demonstram que a análise por injeção em fluxo (Flow injection analysis - FIA) modificada obteve resultados de sulfitos nas amostras analisadas com menor variação do que aqueles obtidos pelo método Monier-Williams (M-W) otimizado, e que nenhuma amostra ultrapassou os LMP. / In Brazil there are no data on use of sulphites by the food industry and research on their consumption by population. Thus, the objective of this research was to evaluate sulphites in fruits juices and estimate intake sulphites in a group of 5th 8th schoolchildren of public schools in São Caetano do Sul-SP, Brazil. It was applied a food frequency questionnaire (FFQ) at 313 schoolchildrens (56.9% female, 43.1% male and average age 14.7 years). Sulphited products identified were fruits juices, sugar, frozen chips, grated coconut, cashew juice and dried fruits. Considering maximum permitted levels (MPL) stabilished by Brazilian legislation, none of the students exceeded the ADI of 0.70 mg SO2/kg bw/day, with average of consumption of 0.12 mg SO2/kg bw/day (17 % of the ADI). The results of the fruits juices analysis demonstrated flow injection analysis (FIA) modified obtained better results than optimized Monier-Williams method (M-W) and no sample exceeded the MPL.

The issue of Hexavalent chromium toxicity to Portland cement hydration is a very actual these days. It takes much effort to obtain a cement containing Cr(VI) less than 2 ppm. The primary objective of this diploma theses is to find appropriate reducing substances and specify their efficiency, as well as to make an attempt to optimize their dosage. As reducing substances there were plumbed sulphites and tin(II) salts. The influence of reducing substances applied to the hydration and cement properties were validated by strenght tests, the calorimetric measurement and the volume-variation measurement. And also the changes in hydration products microstructure of Portland clinker after the gypsum replacement by sulphite ions were plumbed. The study of microstructure is based on SEM and XRD methods.