Orientador: Maria Helena Andrade Santana / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-25T22:51:52Z (GMT). No. of bitstreams: 1 Oliveira_RhelvisdeCampos_M.pdf: 1444700 bytes, checksum: 22be4f55322956156176e0226207310e (MD5) Previous issue date: 2014 / Resumo: O ácido hialurônico (AH) é um polissacarídeo linear com diversas aplicações nas áreas médica e farmacêutica. A produção de ácido hialurônico por via fermentativa vem sendo muito estudada atualmente e modificações no meio de cultura tornaram - se uma das principais alternativas para se atingir um produto com altos níveis de rendimento e massa molar (MM). A primeira etapa deste trabalho teve como objetivo estudar os efeitos de duas peptonas de soja, cuja principal diferença se encontra nas concentrações dos aminoácidos aspartato glutamina e glutamato (AGG), além das proporções de aminoácidos livres e totais. O microrganismo Streptococcus zooepidemicus ATCC 39920 foi cultivado por 24h em frascos agitados contendo uma concentração inicial de 25g L-1 de glicose e razões Carbono:Nitrogênio (C:N) de 3,4:1, 4,1:1 e 5,5:1 para a peptona rica em AGG e 4,1:1, 4,9:1 e 6,3:1 para a peptona pobre em AGG. Os resultados mostraram que a maior produção de AH foi de 1,37g L-1 à razão C:N de 4,1:1 para a peptona pobre em AGG, com MM média de 8,9x10 3 Da e cerca de 50 % das frações de MM na casa de 104 Da, enquanto que para a peptona rica em AGG esses valores foram de 0,70g L-1 e 4,1:1 em termos de produção de AH e razão C:N respectivamente, com MM média de 1,19x106 Da e cerca de 67 % das frações de MM na casa de 105 Da. Na segunda parte deste trabalho foi feita uma comparação do comportamento cinético de fermentações realizadas com as duas peptonas de soja em ensaios com aeração forçada. Os modelos logísticos de Verhulst para o crescimento celular, um análogo ao modelo de Luedeking-Piret incorporando o modelo logístico de Verhulst para o consumo de glicose, Verhulst modificado e Luedeking¿Piret para a produção de AH foram utilizados para estimar os parâmetros cinéticos. Os ajustes dos modelos apresentaram coeficientes de correlação maiores que 0,93. Os resultados mostraram que crescimento celular, consumo de glicose e produção de AH ocorreram durante as 24h de cultivo em meio de cultura contendo a peptona rica em AGG, enquanto que para a peptona pobre em AGG esse tempo reduziu para 10h. A quantidade produzida de AH foi semelhante para ambas as peptonas, entretanto, utilizando a peptona pobre em AGG, 60% da concentração final de AH veio da fase anterior à etapa de fermentação propriamente dita. Os parâmetros cinéticos apresentaram um coeficiente de manutenção maior para o cultivo com a peptona pobre em AGG e maior consumo de glicose foi verificado no cultivo com a peptona rica em AGG, refletindo-se no no rendimento YX/S, enquanto que o rendimento YP/X foi semelhante nos cultivos com ambas as peptonas. Esses resultados mostram que as peptonas não são somente fontes de nitrogênio, mas desempenham papéis específicos no cultivo celular / Abstract: Hyaluronic acid (HA) is a linear polysaccharide with many applications in the medical and pharmaceutical fields. The production of hyaluronic acid by fermentation pathway has been widely studied nowadays, and modifications made in the culture medium is one of the main alternatives to achieve a product with high levels of yield and molecular weight (MW). The first step of this work was to study the effects of two soy peptones, whose main difference is the amino acids concentrations of aspartate, glutamine and glutamate (AGG), and the proportions of free and total amino acids. The microorganism Streptococcus zooepidemicus ATCC 39920 was grown over 24 hours in shake flasks containing initial glucose concentration of 25 g L-1 and the same carbon: nitrogen (C: N) ratios 3.1:1, 1.7:1 and 1.0:1 for both peptones. The results showed that the higher production of HA was 1.37g L-1 with C:N ratio of 1.0:1 for poor AGG peptone with average MW of 8.9 x 103 Da and about 50 % of the MW fraction in the range of 104 Da, while rich AGG peptone showed values of 0.70 g L-1 and 1.7:1 in terms of HA production and C:N ratio respectively, with average MW of 1.19 x 106 Da and about 67% of the fractions of MW in the range of 105 Da. In the second part of this work was made one comparison of the kinetic behavior of cultivations using both peptones in assays with forced aeration. The logistic models of Verhulst for cell growth, analogous to Luedeking-Piret model incorporating the logistic model of Verhulst for glucose consumption, Verhulst modified and Luedeking-Piret for the production of HA were used to estimate the kinetic parameters. The adjustments of the models had correlation coefficients greater than 0.93. The results showed that cell growth, glucose consumption and HA production occurred over the 24 hours of cultivation in culture medium with rich AGG peptone, while with poor AGG peptone this time reduced for 10h. The amount of HA produced was similar for both peptones, however, using poor AGG peptone 60% of the final HA concentration came from the previous stage of the fermentation. The kinetic parameters showed higher coefficient of maintenance for cultivation with poor AGG peptone and increased glucose consumption was observed in cultivation with rich AGG peptone, reflected in terms of YX/S yield, while the yield YP/X was similar in cultivations with both peptones. These results show that peptones are not only sources of nitrogen but play specific roles in cell culture / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Orientador: Gil Eduardo Serra / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-02T16:45:35Z (GMT). No. of bitstreams: 1 Ferreira_LeonelVasco_D.pdf: 43967876 bytes, checksum: 817c781eaf50451bd81530aeeec72377 (MD5) Previous issue date: 2002 / Resumo: Os experimentos de fermentação alcoólica foram conduzidos com diferentes quantidades de inóculo, pH inicial do mosto e temperatura de fermentação, estudando estas variáveis sob diferentes concentrações de substrato, em substratos de glicose, sacarose, xarope industrial e mel final da produção de açúcar. A cinética de liberação de C02 foi determinada em paralelo, em todos os experimentos. Também se determinou parâmetros cinéticos da fermentação alcoólica em substrato de glicose. As fermentações foram monitoradas com determinação de etanol, glicerol, açúcar residual, massa celular, viabilidade e contagem de leveduras, e outras variáveis; os índices de rendimento e parâmetros cinéticos também foram calculados. Os experimentos foram conduzidos em laboratório, com frascos agitados. O inóculo com 1cf células viáveis I mL resultou em fermentações rápidas (2 a 8 horas) enquanto que com 104células viáveis I mL as fermentações foram lentas (18 a 44 horas). Os pH iniciais de 3,5 e 5,0 mostram influir de forma diversa na fisiologia da levedura, resultando em alterações significativas no rendimento da fermentação. Foi observado que sempre que três fatores desfavoráveis se apresentam - alta concentração de substrato, pH 3,5 ou temperatura elevada (38°C) - o rendimento da fermentação é reduzido e há comprometimento da viabilidade do fermento. A determinação da cinética de liberação de gás carbônico mostrou ser um parâmetro importante que permite determinar com precisão o tempo de fermentação e estimar a produção de etanol e consumo de açúcar. Através da cinética da liberação de gás carbônico se monitora o tempo final, para fins de caracterizar a paralisação da fermentação e sua amostragem imediata para determinação das variáveis de controle e especialmente a viabilidade celular. O tempo de fermentação e de consumo de substrato é um fator relevante na avaliação de linhagens. Com substrato de mel final, foi observado o efeito inibidor desse substrato; o melaço com pureza de 60 fermentou significativamente pior que o de pureza 69, mostrando ambos elevada inibição pelo substrato mas especialmente o de menor pureza. Em pH 3,5, com inóculo 108 ou 104 células viáveis I mL e temperatura de 38°C, a inibição pelo substrato mostrou-se forte a partir de 160g I L de glicose; em pH 5,0 esta inibição não se manifestou / Abstract: Fermentation trials were performed with different levels of inoculum, initial mash pH and temperature, and using different substrate conoontrations of glucose, sucrose, industrial syrup and molasses from sugarcane production. The kinetics of carbon dioxide liberation was determined in parallel in ali experiments. The kinetic parameters of the alcoholic fermentation with glucose as substrate were also determined. Fermentation was monitored by determining ethanol, glyoorol, residual sugars, 0011mass, yeast viability and 0011count and other variables. The yield and kinetic parameters were also determined. The experiments were carried out in a laboratoryin shakenflasks. The inoculumof 108viable 0011sImL resulted in rapid fermentations (2 - 8 hours) whilst with 104oolls the fermentations were sluggish (18 to 44 hours). The initial pH values of 3.5 and 5.0 caused various responses in the physiology of the yeast, resulting in significant alterations in fermentation yield. It was observed that whenever three negative factors were present - high substrate conoontration, pH 3.5 or high temperature (38°C) - the fermentation yield and yeast viability were reduood. The determination of the kinetics of carbon dioxide liberation was shown to be an effective way of precisely determining the fermentation time and estimating the production of ethanol and sugar consumption. By way of the kinetics of carbon dioxide liberation, the final time could be monitored in order to characterize fermentation paralyzation and carry out an immediate sampling for the determination of the control variables and especially of 0011 viability. Fermentation time and substrate consumption are relevant factors in the evaluation of strains. With molasses as substrate, an inhibitory effect of this substrate was observed. Using molasses with a purity of 60%, fermentation was significantlyworse than withthat of 69% purity, although both showed considerable inhibition of the fermentation. At pH 3.5, with an inoculum of 108 or 104 viable cells/mL and a temperature of 38°C, substrate inhibition was shown to be significant as trom 160 g/L of glucose. At pH 5.0 this inhibitionwas not apparent / Doutorado / Doutor em Tecnologia de Alimentos

Orientador: Glaucia Maria Postore / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T17:17:03Z (GMT). No. of bitstreams: 1 Almeida_MareciMendesde_D.pdf: 1394335 bytes, checksum: aeb99b8a1fe68ffc5a8b6d83ce4b6809 (MD5) Previous issue date: 2003 / Resumo: Os galactooligossacarídeos são produzidos a partir da lactose por ação da B-galactosidase com atividade de transgalactosilação. São carboidratos não digeríveis, sendo resistentes às enzimas digestivas e fermentados por bifidobactérias. Os benefícios da ingestão de galactooligossacarídeos são de elevar a população de bifidobactérias no cólon e por efeito antagônico, suprimir a atividade de bactérias putrefativas e reduzir a formação de produtos tóxicos por fermentação. O microrganismo Scopulariopsis sp acumulou grande quantidade de B-galactosidase por fermentação semi-sólida e a enzima foi produzida constitutivamente. A enzima obtida foi fracionada com sulfato de amônio, acetona e etanol, sendo a precipitação com sulfato de amônio a que apresentou melhores resultados. O extrato enzimático foi caracterizado bioquimicamente, apresentando um pH ótimo de 4,5 e temperatura ótima de 65°C, e a estabilidade enzimática foi em pH 5,0 e 50°C. A enzima apresentou atividade de transgalactosilação quando avaliada por cromatografia de papel. A b-?galactosidase extracelular de Scopulariopsis sp foi semi-purificada e caracterizada. A enzima foi semi-purificada 13 vezes com um rendimento de aproximadamente 14%. O peso molecular da enzima foi estimado por filtração em gel em torno de 98 KDa. Algumas características da enzima foram determinadas usando o-nitrofenil-??galactopiranosideo (ONPG) como substrato. O pH e temperatura ótimos de atividade foram em torno de 4,5 e 60°C, respectivamente. A enzima foi estável em 40°C por 24 horas e pH 5,0. Os valores de Km e Vmax para ONPG foram 2,28mM e 123?moles/mL/min, respectivamente. Os cátions Fe+2, Fe+3 e Mn+2 ativaram a enzima e N-bromosuccinimida inibiu sua atividade. A atividade de transgalactosilação da ??galactosidase foi avaliada por cromatografia líquida de alta eficiência. A temperatura ótima para atividade de galactosiltransferase foi entre 35 e 45°C e o efeito do pH foi mínimo em alta concentração de lactose; a concentração de enzima para síntese de GOS foi 4-6 U/mL. Quando uma solução de lactose a 40% (p/v) em 0,1 M de tampão acetato de sódio pH 4,5 a 45°C foi incubada com 6 U/mL de ?-galactosidase, a enzima converteu 30% da lactose em galactooligossacarídeos. Foi utilizado planejamento fatorial completo de 2 níveis para estudar o efeito da enzima (4,0 e 8,0 U/mL), pH (5,0 e 7,0) e temperatura (35°C e 45°C) na formação enzimática de galactooligossacarídeos a partir da lactose. Uma diminuição da concentração de enzima e temperatura, e um aumento do valor de pH resultou uma maior produção de galactooligossacarídeos. O produto obtido em maior quantidade foi identificado com 4'galactosil-lactose por cromatografia líquida de alta eficiência / Abstract: Galactooligosaccharides are produced frem lactose by the action of B?-galactosidases which have transgalactosylation activity. They are non-digestible carbohydrates, which are resistant to gastrointestinal digestive enzymes and are fermented by bifidobacteria. The benefits of galactooligosaccharide ingestion arise from increased population of bifidobacteria in the colon which, by their antagonistic effect, suppress the activity of putrefactive bacteria and reduce the formation of toxic fermentation products. The strain of Scopulariopsis accumulated large amounts of the ??galactosidase by semi-solid fermentation and the enzyme was produced constitutively. The enzyme obtained was fractionated with ammonium sulphate, acetone and ethanol, the precipitation with ammonium sulphate showed better results. The enzymatic extract was biochemically characterized, showing an optimum pH of 4.5 and optimum temperature of 65°C, and the enzymatic stability was at pH 5.0 and 50°C. The enzyme showed transgalactosylation activity, when it was evaluated by paper chromatography. Extracellular ??galactosidase from Scopulariopsis sp was semi-purified. The enzyme was a semi-purified 13-fold with a yield of about 14%. The molecular weight of the enzyme was estimated to be about 98 KDa by gel filtration. Some enzyme characteristics were determined using o-nitrophenyl-??galactopyranoside (ONPG) as substrate. The pH and temperature optimum of the activity were about 4.5 and 60°C, respectively. The enzyme was stable at 40°C for 24 hours and pH 5.0. The Km and Vmax values for ONPG were 2,28mM and 123llmoles/mUmin, respectively. The cations Fe+2, Fe+3 and Mn+2 activated the enzyme and N- bromosuccinimide inhibited activity. Transgalactosylation activity of the ??galactosidase was evaluated by high pressure liquid chromatography. The optimum temperature for galactosyltransferase activity was in the range of 35 to 45°C and the pH effect was minimal at high lactose concentration; the enzyme concentration for GOS synthesis was 4-6 U/mL. When 40% (w/v) lactose solution (in 0.1 M sodium acetate buffer, pH 4.5, 45°C) was incubated with 6 U/mL of ??galactosidase, the enzyme converted 30% of the lactose in galactooligosaccharides. Two-Ievel full-factorial design experiments were designed to study effects of enzyme (4.0 and 8.0 U/mL), pH (5.0 and 7.0) and temperature (35°C e 45°C) on enzymatic formation of galactooligosaccharides from lactose. A decrease of enzyme concentration and temperature and an increase of pH value resulted in a higher galactooligosaccharides production. The major product was identified as 4'galactosyl-lactose by high performance liquid chromatography / Doutorado / Doutor em Ciência de Alimentos

The use of shrimp processing waste with other cheap raw materials such as cassava was studied as a potential low cost animal feed, specially in developing countries. The impact on the economies of the shrimp industry and possible effects on the environmental were taken into consideration in developing the project.

Experiments have been conducted to identify the fouling mechanism during the crossflow filtration of a model yeast fermentation broth of Vinyl Acetate particles suspended in a Bovine Serum Albumin (BSA) solution. These have been conducted with filter modules, to obtain quantitative data for the rate and the extent of flux decline due to membrane fouling, and with filter coupons, to obtain quantitative data for the build up of the fouling layer with each individual system and the mixed system. The data from the individual systems have been analysed and then used to determine their fouling mechanisms; this information has been used to predict the fouling mechanism for the mixed system. Finally, this prediction has been compared to the actual fouling mechanism determined by analysis of the mixed system data. For the model particulate suspension, the fouling was due to the build up of a cake layer, as with dead end filtration; however, fouling was limited by membrane scouring. For the model macromolecular solution, a four part fouling mechanism was identified: initially aggregates formed within the pores; the concentration at the membrane surface increased until protein came out of solution as strands; the strands disappeared causing increased aggregation in the pores; finally, a mesh formed on the membrane surface. For the mixed system, fouling was due to the formation of a particle cake on the membrane surface with protein aggregates forming in the pores. The fouling kinetics could be predicted by considering the results from the individual systems; however, the fouling mechanism could not be predicted without using visualisation experiments due to the interactions between the particles and the macromolecules.

La fermentation en milieu solide est un bioprocédé pouvant éventuellement être utilisé comme technologie de rupture pour diminuer le coût des biocatalyseurs utilisés dans la conversion de biomasse lignocellulosique comme le son de blé. La première partie de ces travaux de recherche a donc étudié le potentiel de cette technologie par rapport à celle de fermentation en milieu submergé lors d'une comparaison en application. Plusieurs tests de saccharification ont ainsi été réalisés sur différentes matières premières (cellulose, son de blé) et ont permis de montrer l'avantage différentiateur des biocatalyseurs produits par fermentation en milieu solide. Ensuite, la deuxième partie de ces travaux de recherche a porté sur l'étude des facteurs de la récalcitrance du son de blé à l'hydrolyse enzymatique. Deux principaux facteurs ont ainsi pu être démontrés : un facteur physique, lié à l'accessibilité des biocatalyseurs aux polysaccharides, et un facteur biochimique, lié à l'absence ou à la faible présence de certaines activités enzymatiques (féruloyl estérase,…) dans le complexe enzymatique de Trichoderma reesei Rut-C30. Cette étude a également permis d'identifier l'origine des différentes fractions glucidiques hydrolysées et de déterminer le potentiel glucidique actuellement hydrolysable à partir de cette biomasse. Enfin, la dernière partie de ces travaux de recherche a été consacrée à l'étude pratique d'un concept innovant de procédé permettant de favoriser la conversion des polysaccharides contenus dans le son de blé. Une levée de la barrière physique au transfert de masse et par conséquent une validation de ce concept a finalement pu être réalisée. / Solid-state fermentation is a bioprocess that can optionally be used as disruptive technology to reduce the cost of biocatalysts used in the lignocellulosic biomass conversion like wheat bran. The first part of this research has explored the potential of this technology compared to submerged fermentation in an application comparison. Several saccharification tests have thus been carried on different feedstocks (cellulose, wheat bran) and have shown the differentiator advantage of biocatalysts produced by solid state fermentation. Then, the second part of this research has investigated the recalcitrance factors of wheat bran to enzymatic hydrolysis. Two main factors have thus been demonstrated: a physical factor, related to the accessibility of biocatalysts to the polysaccharides, and a biochemical factor, related to the absence or the low presence of some enzymatic activities (feruloyl esterase, ...) in the enzymatic complex of Trichoderma reesei Rut-C30. This study has also identified the origin of the various carbohydrate moieties hydrolyzed and has determined the carbohydrate potential currently releasable from this biomass. Finally, the last part of this research has been devoted to the practical study of an innovative concept of process to promote the conversion of polysaccharides in wheat bran. A removal of the physical barrier to mass transfer and therefore a validation of this concept has finally been achieved.

Aflatoxins are highly carcinogenic secondary metabolites that can contaminate approximately 25% of crops and that cause or exacerbate multiple adverse health conditions, especially in Sub-Saharan Africa and South and Southeast Asia. Regulation and decontamination of aflatoxins in high exposure areas is lacking. Biological detoxification methods are promising because they are assumed to be cheaper and more environmentally friendly compared to chemical alternatives. White-rot fungi produce non-specific enzymes that are known to degrade aflatoxin in in situ and ex situ experiments. The aims of this study were to (1) decontaminate aflatoxin-B-1-(AFB(1)) in naturally contaminated maize with the edible, white-rot fungus Pleurotus ostreatus (oyster mushroom) using a solid-state fermentation system that followed standard cultivation techniques, and to (2) and to assess the risk of mutagenicity in the resulting breakdown products and mushrooms. Vegetative growth and yield characteristics of P. ostreatus were not inhibited by the presence of-AFB(1).-AFB(1) was degraded by up to 94% by the Blue strain. No aflatoxin could be detected in P. ostreatus mushrooms produced from-AFB(1)-contaminated maize. Moreover, the mutagenicity of breakdown products from the maize substrate, and reversion of breakdown products to the parent compound, were minimal. These results suggest that P. ostreatus significantly degrades-AFB(1) in naturally contaminated maize under standard cultivation techniques to levels that are acceptable for some livestock fodder, and that using P. ostreatus to bioconvert crops into mushrooms can reduce-AFB(1)-related losses.

The brewing industry constantly experiences problems in trying to maintain the quality of beer produced. Unfavourable conditions during fermentation may alter the performance of the yeast strain Saccharomyces cerevisiae, resulting in a "poor" end-product. It has been established that high concentrations of extracellular ethanol, when added to the fermentation medium inhibit yeast activity. It has been recently suggested that increased carbon dioxide pressure could inactivate the yeast activity adding to further brewing problems. The aim of this study was to investigate the effect of extracellular carbon dioxide pressure and ethanol addition, on yeast performance when added to a fermentation medium, and to establish whether an inhibitory relationship existed between ethanol and carbon dioxide pressure, when combined and added to the fermentation medium. Dissolved C0₂ in the medium, medium pH and substrate utilisation were analysed daily during a fermentation, as were membrane fatty acid composition. These parameters were used to assess the effect of ethanol and carbon dioxide on the yeast performance and consequently the final end-product. Supplementing the medium with extracellular ethanol, even as low as 5%, was shown to inhibit yeast performance during fermentation. This effect was even more marked as the ethanol concentration was increased, with almost total inhibition of yeast activity occuring after the addition of 15% ethanol (v/v). A similar effect was observed when elevated C0₂ pressures were applied to the medium, and although low C0₂ pressures initially induced the synthesis of saturated yeast membrane fatty acids, elevated C0₂ pressures (greater than 1,0 atm.) was shown to follow a similar inhibitory trend, if not as dramatic, as ethanol. A combination of both ethanol and C0₂ pressure showed a further increase in the level of yeast inhibition, although the low C0₂ pressure appeared to initially inhibit the toxicity of ethanol on the yeast. Increasing the levels of the C0₂/ethanol treatment (1,0 atm.), showed a synergistic effect on yeast performance. The results of this study indicate that both extracellular ethanol and carbon dioxide do appear to inhibit yeast performance and affect membrane fatty acid composition of the cells by inhibiting the synthesis of the respective fatty acid. This affect has a significant bearing on the general metabolism of the yeast cell.

Linalool is a key intermediate for the production of important fragrance chemicals such as geraniol, nerol, geranial, and neral. Linalool can be produced via a two-step process from α-pinene which is a major component of crude sulphated turpentine (CST) a foul-smelling, volatile waste product of the pulp and paper industry. The key step in this process is the pyrolysis step which involves the isomerisation of cis-2-pinanol to linalool and requires high temperatures (600-650°C) and is not very selective due to the decomposition of the product itself under these conditions. A client of the CSIR, Teubes Pty. Ltd., is a manufacturer of flavour and fragrance compounds for the local and international fragrance market and expressed an interest in producing linalool since the company would then gain access to other valuable fragrance chemicals via relatively simple processes. Earlier work conducted by AECI, R & D did not meet with much success since the selectivity to linalool was very poor and the process could hardly be deemed as scalable. The main objective of this project was therefore to develop a process for the selective isomerisation of cis-2-pinanol to linalool with minimum by-product formation and using process equipment that could be scaled to full-scale production. Since cis-2- pinanol could not be purchased in sufficient quantities for process development, a process had to be developed for the bench-scale preparation of kilogram quantities of cis-2-pinanol from α-pinene obtained from the client. Although this synthesis formed a minor part of this investigation, several process improvements and innovations were introduced to produce high quality cis-2-pinanol, in very good yields at kilogram scale. A major part of this investigation was the design and set up of a pyrolyis rig capable of operating at elevated temperatures (400 - 750°C) for the evaluation of various process parameters. Various vaporizer, reactor, and condensation systems were evaluated for their ability to cope with the demanding conditions on a consistent basis. The initial part of the investigation was a screening exercise to evaluate various process parameters as well as solvents, materials of construction, catalysts, etc. A comprehensive statistical design was also conducted to determine the critical process parameters and the model obtained was used to predict the optimum conditions required for the preparation of in-specification product on a consistent basis. These conditions were used in the preparation of a 1kg sample which was required by theclient for market evaluation purposes. The use of a novel microreactor system was also evaluated for the pinanol pyrolysis reaction. To our knowledge, this is the first time that a microreactor has been successfully used for this type of reaction in the Fragrance industry and a patent application is being filed by the CSIR. The kinetics of the reaction in both the tubular reactor system and the microreactor system was investigated. Computer modelling studies on both the systems were also conducted. The raw material cost to produce a kilogram of linalool is $1.40. There is a significant margin of 60.8 percent between the raw material cost of linalool and the current selling price ($3.57/kg). This clearly indicates that the project is potentially feasible from an economic point of view and we can now proceed with confidence to the next stage which is the engineering design, building and commissioning of the large scale pyrolysis rig. The rest of the process steps will be conducted on existing equipment currently present at the CSIR’s large scale facility (Imbiza in Isando, Gauteng).

Orientador: Adilma Regina Pippa Scamparini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T16:03:30Z (GMT). No. of bitstreams: 1 Maldonade_IrianiRodrigues_D.pdf: 4259808 bytes, checksum: a80eab15ad28ff36edff81d33fd716be (MD5) Previous issue date: 2003 / Resumo: Este trabalho teve como objetivo isolar e selecionar leveduras produtoras de carotenóides de ecossistemas brasileiros. As leveduras pigmentadas foram isoladas de amostras de solos, flores, folhas, frutos da região de Campinas-SP e de alimentos processados. As amostras foram colocadas em frascos de erlenmeyer de 50 mL, contendo 20 mL de meio de Extrato de Malte e Levedura (YM), e incubadas a 30° C por 48 horas. Após 48 horas, as amostras foram inoculadas em placas de petri contendo meio ágar-YM e incubadas a 30°C, por 120 horas. As colônias de leveduras que apresentaram coloração entre amarelo e vermelho, foram transferidas para tubos de ensaio contendo meio ágar YM inclinado e incubadas a 30°C até crescimento satisfatório. Estas leveduras foram reisoladas, pelo método de estrias de esgotamento, em placas de petri contendo meio ágar YM (30°C por 72 horas) e, posteriormente, transferidas para tubos de ensaios contendo ágar GYMP inclinado. As culturas pigmentadas foram codificadas do seguinte modo: L12, isolada como contaminante em massa de tomate; L108, isolada de solo da região da Universidade Estadual de Campinas; L125, isolada a partir de folhas da cana-de-açúcar; L135 e L137 isoladas de solo em Holambra-SP. Através das características morfológicas, de reprodução, testes fisiológicos e bioquímicos as leveduras foram identificadas como: L12, L108, L135 e L137 como Rhodotorula mucilaginosa, e L125 como Rhodotorula graminis. A composição de carotenóides, das leveduras isoladas no Brasil, foi estudada. As culturas de leveduras foram cultivadas em 200 mL de meio YM a 200 rpm em shaker, a 25°C por 5 dias. Cromatografia de coluna aberta, cromatografia de camada delgada e cromatografia líquida de alta eficiência foram utilizadas para separar os carotenóides obtidos das leveduras, a fim de identificá-los e quantificá-los. A linhagem de Rhodotorula glutinis foi a que apresentou maior concentração total de carotenóide (881 mg/L), seguido por Rhodotorula graminis (594 mg/L), Rhodotorula mucilaginosa-137 (590 mg/L) e Rhodotorula mucilaginosa-135 (545 mg/L). Rhodotorula minuta e Sporobolomyces tiveram a menor concentração de carotenóides (168 mg/L and 237 mg/L, respectivamente). Os principais pigmentos encontrados nestas linhagens foram toruleno e b-caroteno. b-Caroteno foi o carotenóide predominante em Rhodotorula grarninis-125, Rhodotorula glutinis e Sporobolornyces, enquanto que o toruleno foi o carotenóide principal nas leveduras de Rhodotorula rnucilaginosa. Em termos de produção específica de cartenóides (_g/g de células secas), Rhodotorula glutinis foi a que obteve maior concentração de carotenóides 132 mg/g. Duas linhagens foram selecionadas para otimização da produção de carotenóides, R. mucilaginosa-137 e R. glutinis. Estas duas culturas foram cultivadas em shaker a 200 rpm, a 25°C por 5 dias, sem iluminação. Utilizou-se planejamento experimental e análise de superfície de resposta para estudar o efeito do pH inicial, concentração de glicose, extrato de levedura, sais de fosfato e sulfato de magnésio na produção de carotenóides, de biomassa e proteína celular. Para cada linhagem, foram realizados 2 planejamentos fatoriais, sendo 1 fracionário e 1 completo.Para a linhagem de R. mucilaginosa-137, o extrato de levedura foi a variável de maior influência na produção de carotenóides, enquanto que os sais de sulfato e fosfato tiveram efeito negativo. O pH inicial não teve efeito significativo tanto na produção de carotenóides como na biomassa. Através dos resultados obtidos pelo planejamento completo, observou-se que a máxima concentração de carotenóides foi de 745 mg/L com 15 g/L de extrato de levedura e 20 g/L de glicose. Em relação a produção específica de carotenóides, a máxima concentração foi de 152 mg/g com 5 g/L de extrato de levedura e 15 g/L de glicose. A concentração de extrato de leveduras e glicose também foram importantes na produção da biomassa, que atingiu o valor máximo de 8 g/L, na faixa de concentração de 15 a 17,1 g /L de extrato de levedura e de 15 a 20 g/L de glicose. Para a linhagem de Rhodotorula glutinis as variáveis de maior influência na produção de carotenóides foram pH inicial, extrato de levedura e glicose. Os sais de sulfato e fosfato não tiveram efeito significativo. Através do planejamento fatorial completo 23 com três pontos centrais, observou-se que na produção de carotenóides, apenas a glicose teve efeito positivo significativo. Na produção específica de carotenóides, o pH inicial, glicose e extrato de levedura tiveram efeito positivo. A máxima concentração de carotenóides obtida foi de 1.269 mg/L com pH inicial 4, 4 g/L de extrato de levedura e 17 g/L de glicose. Na produção específica de carotenóides, a máxima concentração foi de 337 mg/g com pH inicial 4, a 4 g/L de extrato de levedura e 7 g/L de glicose. O crescimento celular foi afetado pelo pH inicial, concentração de extrato de levedura e glicose. Entretanto, o modelo matemático referente a biomassa não apresentou uma regressão satisfatória, devendo ser utilizado apenas para estabelecer tendência da resposta / Abstract: Pigmented yeasts were collected from soils, flowers, leaves, fruits from Campinas-SP region and industrialized foods. The samples were put in 50 mL erlenmeyers flasks, containing YM broth, and they were incubated at 30°C for 48 hours. After 48 hours, these samples were inoculated in Petri plates with YM agar, and incubated at 30°C for 120 hours. The yeasts colonies that had color between red and yellow were transferred to tubes, containing YM agar, and incubated at 30°C. These yeasts were reisolated by screening in Petri plates with YM agar (30°C for 72 hours) and then, transferred into tubes containing GYMP agar. After the selection, the pigmented yeasts were identified by a code: L12, was isolated from tomato sauce; L108, from soils of State University of Campinas; L125, from leaves of sugar cane; L135 e L137, from soils of Holambra-SP. The yeasts were identified by their morphology characteristics, reproduction characteristics, physiology and biochemical tests. The yeasts L12, L108, L135 and L137 were identified as Rhodotorula mucUaginosa and L125 as Rhodotorula graminis. The carotenoid composition of yeasts isolated in Brazil was studied. The yeasts were cultured in 200 mL broth yeast malt at 200 rpm in rotary shaker, 25°C for 5 days without illumination. Open column, thin layer chromatography and high performance liquid chromatography were used to separate, identify and determine carotenoid concentrations. The yeast Rhodotorula glutinis had the highest total carotenoid concentration (881 mg/L), followed by Rhodotorula graminis (594 mg/L), Rhodotorula mucUaginosa-137 (590 mg/L) and Rhodotorula mucilaginosa-135 (545 mg/L). Rhodotorula minuta and Sporobolomyces had the lowest carotenoid contents (168 mg/L and 237 mg/L, respectively). The principal pigments found in these yeasts were torulene and b-carotene. b-Carotene predominated in Rhodotorula graminis-125, Rhodotorula glutinis and Sporobolomyces, while torulene was the major carotenoid in Rhodotorula mucilaginosa. In specific carotenoid production (mg/g of dried cells), Rhodotorula glutinis had a total carotenoid concentration of 132 mg/g. Two of these strains were selected to optimize the carotenoid production, Rhodotorula mucilaginosa-137 and Rhodotorula glutinis. The cultures were cultivated into 200 mL broth yeast malt at 200 rpm in rotary shaker, 25°C for 5 days without illumination. Response surface design was used to study the effects of initial pH and concentrations of glucose, yeast extract, magnesium sulfate and potassium phosphate. Two statistical designs were used for each strain. For the strain of Rhodotorula mucilaginosa-137, the yeast extract the most important variable in terms of enhancing carotenoid formation; magnesium sulfate and potassium phosphate had a negative influence. The initial pH had no significant effect on carotenoid formation or on cell production. Analysis of the quadratic surfaces showed that after 5 days of cultivation at 25°C, the maximum carotenoid concentration of 745 mg/L appeared at 15 g/L of yeast extract and 20 g/L of glucose. The maximum concentration of specific carotenoid production was 152 mg/g at 5 g/L of yeast extract and 10 g/L of glucose. The concentrations of yeast extract and glucose were also important on biomass production, which reached maximum value of 8.0 g/L at a range of 15 to 17.1 glL of yeast extract and 15 to 20 g/L of glucose. The variables that had most influence on carotenoid production by Rhodotorula glutinis were initial pH, yeast extract and glucose. Magnesium sulfate and potassium phosphate had no influence. The carotenoid production was described by second order p01ynomial equation. Analysis of the 23 factorial design surfaces showed that after 5 days of cultivation at 25°C, the maximum carotenoid concentration of 1,269 mg/L with initial pH 4, 4 g/L of yeast extract and 17 g/L de glucose. The maximum specific carotenoid production was 337 j..tglg with initial pH 4, 4 g/L of yeast extract and 7 g/L of glucose. Moreover, carotenoid production in mg/g per liter was more sensitive to changes in yeast extract than to changes in glucose concentrations, in the vicinity of the optimum point of carotenoid production. The growth of the microorganism was affected by initial pH and concentration of yeast extract and glucose. However, the model obtained for biomass from the experimental designs had not a good correlation and because of that it should be used only to study the tendency of response / Doutorado / Doutor em Ciência de Alimentos