Orientador: Roberto da Silva / Banca: Adriane Maria Ferreira Milagres / Banca: Heloisa Ferreira Alves do Prado / Resumo: A ciclodextrina glicosiltransferase (CGTase) é uma enzima microbiana que converte o amido em ciclodextrinas (CDs). Estes compostos são moléculas cíclicas, formadas por monômeros de D-glicoses unidas por ligações glicosídicas do tipo a-1,4. As CDs podem ser uma alternativa para controlar o escurecimento de batatas e frutas devido a sua capacidade de formar complexo de inclusão com substratos da reação de escurecimento. Alimentos contendo amido, quando adicionados da CGTase são também, potencialmente capazes de sofrer a ação da enzima formando CD localmente. Esse trabalho teve por finalidade estudar a aplicação direta da enzima CGTase em batatas e a aplicação de ciclodextrina comercial em batatas, maçãs, bananas e pêras visando controlar as reações de escurecimento enzimático ou não enzimático, destes alimentos. A formação de complexos com ciclodextrina foi determinada por calorimetria diferencial de varredura (DSC). Este trabalho mostrou pela primeira vez que tanto a CGTase produzida por Bacillus clausii E16 quanto a CGTase comercial (Toruzyme., Novozymes), foram capazes de controlar o escurecimento químico de batata e enzimático de batata e maçã. A utilização da CD mostrou efeito similar a ação das CGTases para batata e maçã, reduzindo dessa forma o escurecimento enzimático causado pela enzima polifenoloxidase. Pode-se inferir que ocorreu a produção da CD pela ação da CGTase em presença do substrato natural (amido de batata e maçã) e por conseqüência sua ação complexante junto aos agentes responsáveis pelo escurecimento, inibindo assim a efetivação das reações. Essa possível encapsulação foi demonstrada pela análise de DSC referente à diminuição do pico da amostra de batata tratada com CGTase. / Abstract: The cyclodextrin glycosyltransferase (CGTase) is a microbial enzyme that converts starch to cyclodextrins (CDs). These compounds are cyclical molecules, formed by D-glucose monomers linked by a-1,4-glycosidic linkages type. The CDs can be an alternative to control the potato and fruits browning due its capacity to form complexes of inclusion with substrate of the browning reaction. Foods containing starch, when added of the CGTase are also potentially able to the action from the enzyme, then forming CD. This work had the purpose to study the direct application of the CGTase enzyme in potatoes and the commercial cyclodextrin application in potatoes, apples, bananas and pears aiming to control the browning enzymatic or no enzymatic reactions of these foods. The cyclodextrin complexes formation was determined by differential scanning calorimetry (DSC). This work showed for the first time that as the CGTase produced for Bacillus clausii E16 as the commercial CGTase (Toruzyme., Novozymes), were capable to control the chemical browning of potato and enzymatic browning of potato and apple. The use of the CD showed to similar effect to the action of CGTases for potato and apple, reducing in both the enzymatic browning caused by polyphenoloxidase enzyme. It can be concluded that the production of CD occurred by CGTase action in presence of the natural substrate (starch of potato and apple) and for consequence it’s action to form complexes next to the responsible agents for the browning, thus inhibiting effectively the reactions. This possible encapsulation was demonstrated by analysis of DSC referring to the reduction of the peak of the potato sample treated with CGTase. / Mestre

Orientador: Eleni Gomes / Banca: Henrique Ferreira / Banca: Crispin Humberto Garcia Cruz / Resumo: Os refrigerantes são bebidas gaseificadas, obtidas através da diluição do suco ou extrato vegetal de sua origem em água potável, adicionada de açúcares. Devido ao elevado teor de açúcar neste produto e ao baixo pH, os refrigerantes tornam-se propícios ao desenvolvimento de microrganismos, em especial as leveduras. A contaminação microbiológica é mais comum na etapa de produção, entretanto, a maioria dos contaminantes pode ser proveniente de matérias-primas como água ou equipamentos quando não higienizados de maneira adequada. De acordo com o histórico de análises microbiológicas da Indústria BEBIDAS POTY Ltda, foram detectados alguns pontos de contaminação por leveduras dentro do fluxograma de produção, as quais têm apresentado certa resistência aos sanificantes utilizados. Desta forma o presente trabalho teve como objetivo isolar, identificar leveduras de matérias primas e diversos equipamentos da produção e avaliar o efeito inibitório dos sanificantes nas leveduras isoladas, buscando conhecer a eficácia dessas preparações. Neste estudo, foram isoladas 26 leveduras apenas da água de enxágue após o processo de higienização dos equipamentos de produção em todas as linhas de produtos carbonatados. Dentre as leveduras isoladas, os gêneros mais comuns foram Candida tropicalis, C. orthopsilosis, C. viswanathii, C. boidinii, Pichia manshurica, Zygosaccharomyces bisporus, sendo a espécie C. sojae a mais frequente. Os isolados foram testados na presença de 1) detergente alcalino clorado, 2) detergente alcalino não clorado, 3) desinfetante ácido peracético e 4) base ácida com ação de detergência/desinfetante de duas marcas comerciais identificadas como Marca I e Marca II, na perspectiva de encontrar a concentração mínima inibitória. Os agentes químicos de basicidade elevada com ação de limpeza foram os mais eficientes nos testes de... / Abstract: Soft drinks are carbonated beverages, obtained by diluting the juice or vegetable extract in drinking water, added of sugars. Because of the high sugar content of this product and the low pH, the refrigerants are susceptible to development of microorganisms, in particular yeasts. Microbial contamination is most common in the production step, however, most of the contaminants can come from raw materials and equipment such as water when not cleaned properly. According to the history of microbiological analyzes of Industry DRINKS POTY Ltda, some points of contamination by yeasts were detected within the production flow chart, which have shown some resistance to the sanitizers used. Thus, the present study aims to isolate and identify yeast from raw materials and from various equipment and evaluate the inhibitory effect of sanitizers on yeasts. It was isolated 26 yeasts strains from rinse water after the process of cleaning production equipment in all lines of carbonated products. Among the yeasts, the most common genera were Candida tropicalis. C. orthopsilosis, C. viswanathii, C. boidinii, Pichia manshurica, Zygosaccharomyces bisporus, the most frequent species was C. sojae. The isolates were tested in the presence of 1) chlorinated alkaline detergent, 2) alkaline detergent non-chlorinated, 3) disinfectant peracetic acid and 4) acid based detergent with disinfectant action two trademarks identified as Marca I and Marca II with a view to find the minimum inhibitory concentration. Chemical agents with high alkalinity were the most efficient in inhibition of yeast growth the less effective was alkali detergent containing chlorine being necessary to use very high concentrations to achieve the minimum inhibitory action. The data showed that the yeasts were very sensitive, to oxidizers such as nitric acid, peracetic acid and O3 gas / Mestre

Orientadores: Lucimara Gaziola de La Torre, Reinaldo Gaspar Bastos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-25T04:50:36Z (GMT). No. of bitstreams: 1 Oliveira_AlineFurtado_M.pdf: 2165174 bytes, checksum: d3aa7acdee3edc7f222daf3f7a82f910 (MD5) Previous issue date: 2014 / Resumo: A microfluídica é uma ciência que opera em pequenos volumes de fluídos dentro de canais em dimensões de micrômetros (10-6 m). Estes sistemas permitem controlar moléculas no espaço e no tempo, gerando resultados rápidos e confiáveis num sistema precisamente controlado e capaz de mimetizar ambientes celulares. Os dispositivos microfluídicos apresentam uma diversidade de geometrias aplicáveis para diversas áreas de pesquisas, sendo que a capacidade de formar gradientes permite avaliar as condições e o desempenho celular microbiano. Assim, este trabalho teve como objetivo desenvolver dispositivos microfluídicos capazes de formar gradiente de concentração difusivo e investigar sua aplicabilidade em bioprocessos. Diante disso, foram propostos três modelos de dispositivos usando materiais biocompatíveis: (i) dispositivo em base de vidro, denominado de Vidro-vidro; (ii) em base de vidro e poli dimetilsiloxano (PDMS), chamado de Vidro-PDMS e (iii) vidro e PDMS modificado quimicamente para tornar a superfície hidrofílica, Vidro-mPDMS. Os três dispositivos foram avaliados quanto à capacidade de formação de gradiente de concentração difusivo, os quais apresentaram um perfil linear. Além disso, validou-se o estudo do comportamento de Saccharomyces cerevisiae ATCC 7754 num gradiente de concentração de glicose de 0 a 40 g/L de glicose, sendo usado o dispositivo vidro-vidro. Foi observado que houve crescimento de células ao longo das câmaras microfluídicas, e isso possibilitou na determinação de parâmetros cinéticos, os quais não apresentaram diferença estatisticamente significativa com o cultivo em batelada convencional. As condições da microfluídica possibilitaram também a determinação da cinética de Monod, usando menores intervalos de gradiente. Portanto, este dispositivo microfluídico mostrou-se uma ferramenta com potencial para investigar comportamento celular frente à diferença de concentração e contribuirá para a otimização de bioprocessos através da determinação de parâmetros cinéticos / Abstract: Microfluidic is a science that operates in small amounts of fluids inside channels in dimensions of micrometers (10-6 m). These systems allow the precise control of molecules in space and time, generating fast and reliable results and it can also be used to mimics environment cellular . Microfluidic devices can be produced in diversity of geometries, it can be applied in several scientific areas and especially the formation of concentration gradients can be used to evaluate conditions and performance of microbial cell. Therefore, this work had the objective to develop microfluidic devices that are able to generate diffusive concentration gradients and investigate their applicability in bioprocesses. In this context, we propose three models of microfluidics devices using biocompatible materials: (i) Glass-based device, named glass-glass; (ii) glass and poli dimetilsiloxane (PDMS) based device, Glass-PDMS and (iii) glass and chemically modified PDMS (hydrophilic surface), Glass-mPDMS. The three devices were evaluated by their capacity of generating difusive concentration gradient, demonstrating linear concentration profile. Furthermore, the behavior of Saccharomyces cerevisiae ATCC 7754 inside of glucose concentration gradient ranging from 0 to 40 g/L were validated, using the glass-glass device . It was observed that cell growth along the microfluidic chambers, having determined the kinetic parameters, which was considered statistically similar to conventional batch cultivation. Conditions of microfluidics also allowed determination of the Monod kinetic, using smaller intervals gradient Therefore, the use of concentration gradient in microfluidic device is a potential tool for investigate of microbial cell behavior against the concentration difference and it can contribute to the optimization of bioprocesses through the determination of kinetic parameters / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestra em Engenharia Química

Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.

No description available.

Dissertation (PhD)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Constructed wetlands have been widely used in the treatment of industrial and domestic wastewater to reduce biological and chemical oxygen demand (BOD and COD), to remove nitrate and enteric viruses as well as to generally improve water quality. Distillery wastewater has a complex character due to high concentrations of sugars, lignins, hemicellulose, dextrins, resins, polyphenols and organic acids, leading to a high COD that may exceed 100 000 mg/L. The potential application for the treatment of distillery wastewater by means of constructed wetlands is relatively unexplored. In 1999 a study was initiated at Distell Goudini distillery, Western Cape, South Africa, to explore the possibility of using constructed wetlands to treat distillery wastewater. It was found that constructed wetlands do have the ability to treat distillery wastewater providing that the influent COD does not exceed 15 000 mg/L for extended periods and the correct substrate material is used. The present study expanded on the above-mentioned study and specifically aimed to provide information on the microbiological controls in wetland systems in an applied sense that may contribute to improved treatment efficiency. Furthermore, this project aimed to contribute to our fundamental understanding of the microbial ecology of constructed wetlands used for the treatment of distillery wastewater. This study revealed that a highly dynamic microbial composition exists within wetlands. Furthermore it was found that wetlands can efficiently remove COD even though a low degree of similarity exists between microbial communities in various zones of the same wetland and those between different wetlands, as well as low similarity between communities sampled from the same zone over time. This demonstrates that it will be difficult to define the ‘ideal’ degradative community in terms of microbiological criteria and serves as a reminder that various indicators should be considered for monitoring system health. Furthermore the shifts in microbial community composition illustrate the ability of microbial communities to adapt to changes in the environment without compromising their functional efficacy. When studying the attached microbial communities within wetland systems it was found that different morphotypes are detected at certain stages of biofilm development while some organisms are present at most phases of biofilm formation. Measurement of CO2 production and dissolved organic carbon (DOC) removal in laboratory scale columns showed that grazing protists had a notable effect on overall microbial activity and that organic loading influenced these predator-prey interactions. Interestingly, increased clogging of pores occurred in the presence of protists, resulting in reduced flow through the porous matrix. Terminalrestriction fragment length polymorphism (T-RFLP) analysis of biofilms on gravel in experimental wetlands indicated that the presence of protists and algae had an effect on the microbial community composition. Scanning electron microscopy (SEM) showed that the presence of algae also had an influence on biofilm structure suggesting that the algae provided labile nutrients that were utilized by the bacterial and yeast members of the community. Finally, augmentation with a commercial mixture or microbial populations isolated from distillery effluent demonstrated that the concentration at which supplements are applied influence degradative efficiency. / AFRIKAANSE OPSOMMING: Kunsmatige vleilande word wêreldwyd gebruik in die behandeling van indusriële en huishoudelike afvalwater om biologiese en chemiese suurstof aanvraag (BSA en CSA) te verminder, om nitrate en ingewandsvirusse te verwyder asook om waterkwaliteit in die algemeen te verbeter. Distilleerafvalwater het komplekse eienskappe as gevolg van hoë konsentrasies suiker, lignien, hemisellulose, dekstrien, harpuis, polifenole en organiese sure, wat lei tot ‘n hoë CSA wat 100 000 mg/L kan oorskry. Daar is tot op hede relatief min studies gedoen oor die potensiële gebruik van kunsmatige vleilande vir die behandeling van distilleerafvalwater. In 1999 is ‘n studie by Distell Goudini distilleeraanleg in die Wes Kaap van Suid Afrika onderneem om die moontlikheid van kunsmatige vleilande vir die behandeling van distilleerafvalwater te bestudeer. Daar was bevind dat kunsmatige vleilande die vermoë het om distilleerafvalwater te behandel gegewe dat die invloeiende CSA nie 15 000 mg/L oorskry nie en dat die regte substraat materiaal gebruik word. Die huidige studie het by die bogenoemde studie aangesluit met die doel om informasie oor die mikrobiologiese kontroles in vleilandsisteme op ‘n toegepaste wyse te voorsien, wat tot verbeterde behandeling doeltreffendheid kan lei. Hierdie studie het verder beoog om by te dra tot ons fundementele kennis van die mikrobiese ekologie van kunsmatige vleilande wat gebruik word vir die behandeling van distilleerafvalwater. Dié studie het bevind dat daar ‘n hoogs dinamiese mikrobiese samestelling binne vleilande bestaan. Daar was verder bevind dat CSA steeds effektief deur vleilande verwyder kan word alhoewel daar ‘n lae graad van ooreenstemming is tussen mikrobiese gemeenskappe in verskeie sones van dieselfde vleiland en verskillende vleilande, asook ‘n lae graad van ooreenstemming tussen gemeenskappe wat in dieselfde sone oor tyd gemonster is. Dit demonstreer dat dit moeilik sal wees om die ‘ideale’ degraderende gemeenskap te vind in terme van mikrobiologiese kriteria en dien as ‘n herinnering dat verkeie indikatore in ag geneem moet word om die welstand van ‘n ekologiese sisteem te monitor. Die verskuiwings in mikrobiese gemeenskapsamestelling illustreer verder die vermoë van natuurlike sisteme om aan te pas by veranderinge in die omgewing sonder om funksionele doeltreffendheid te verminder. Die studie van aangehegte mikobiese gemeenskappe het aangedui dat veskillende morfotipes bespeur kan word tydens sekere fases van biofilm formasie terwyl sekere organismes tydens meeste van die fases teenwoordig is. Die bepaling van CO2 produksie en die verwydering van opgeloste organiese koolstof in laboratoriumskaal kolomme het geïlustreer dat voedende protiste ‘n waarneembare effek gehad op die algehele mikrobiese aktiwiteit en dat die organiese lading hierdie predator-prooi interaksie beïnvloed het. Dit was interessant om te vind dat die teenwoordigheid van protiste die verstopping van porieë aangehelp het en dus tot verlaagde vloei deur die poreuse matriks gelei het. Terminale-restriksie fragment lengte polimorfisme (T-RFLP) analiese van biolfilm op klipgruis in eksperimentele vleilande het aangedui dat die teenwoordigheid van protiste en alge ‘n effek gehad het op die mikrobiese gemeenskapsamestelling. Skandeerelektronmikroskopie (SEM) het bewys dat die teenwoordigheid van alge ook ‘n invloed op biofilm struktuur gehad het wat daarop dui dat alge maklik afbreekbare voedingstowwe aan die bakterieë en giste van die mikrobiese gemeenskap beskikbaar gestel het. Laastens was bewys dat die konsentrasie van toevoeging van ‘n kommersiële mikrobiese mengsel of mikrobiese populasies wat uit afvoer geïsoleer was, die effektiwiteit van degradering kan beïnvloed.

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: This study strives to improve two current industrial processes by making them more cost effective through the use of hydrolytic enzymes or microbial systems. The first process targeted is the industrial conversion of starch to ethanol. In the second process, hydrolytic enzymes are applied to the manufacturing of instant coffee. The engineering of microbial systems to convert starch to bio-ethanol in a one-step process may result in large cost reductions in current industrial processes. These reductions will be due to decreased heating energy requirements, as well as a decrease in money spent on the purchase of commercial enzymes for liquefaction and saccharification. In this study, a recombinant Saccharomyces cerevisiae strain was engineered to express the wild-type Aspergillus awamori glucoamylase (GA I) and α-amylase (AMYL III) as well as the Aspergillus oryzae glucoamylase (GLAA) as separately secreted polypeptides. The recombinant strain that secreted functional GA I and AMYL III was able to utilise raw corn starch as carbon source, and converted raw corn starch into bio-ethanol at a specific production rate of 0.037 grams per gram dry weight cells per hour. The ethanol yield of 0.40 gram ethanol per gram available sugar from starch translated to 71% of the theoretical maximum from starch as substrate. A promising raw starch converter was therefore generated. In the second part of this study, soluble solid yields were increased by hydrolysing spent coffee ground, which is the waste generated by the existing coffee process, with hydrolytic enzymes. Recombinant enzymes secreted from engineered Aspergillus strains (β-mannanase, β-endoglucanase 1, β-endo-glucanase 2, and β-xylanase 2), enzymes secreted from wild-type organisms (β-mannanases) and commercial enzyme cocktails displaying the necessary activities (β-mannanase, cellulase, and pectinase) were applied to coffee spent ground to hydrolyse the residual 42% mannan and 51% cellulose in the substrate. Hydrolysis experiments indicated that an enzyme cocktail containing mainly β-mannanase increased soluble solids extracted substantially, and a soluble solid yield of 23% was determined using the optimised enzyme extraction process. Soluble solid yield increases during the manufacturing of instant coffee will result in; (i) an increase in overall yield of instant coffee product, (ii) a decrease in amount of coffee beans important for the production of the product, and (iii) a reduction in the amount of waste product generated by the process. / AFRIKAANSE OPSOMMING: Hierdie studie poog om twee huidige industriële prosesse te verbeter deur die prosesse meer kosteeffektief met behulp van hidroltiese ensieme en mikrobiese sisteme te maak. Die eerste industrie wat geteiken word, is die omskakeling van rou stysel na etanol, en die tweede om hidrolities ensieme in die vervaardiging van kitskoffie te gebruik. Die skep van mikrobiese sisteme om rou-stysel in ’n ’een-stap’ proses om te skakel na bio-etanol sal groot koste besparing tot gevolg hê. Hierdie besparings sal te wyte wees aan die afname in verhittingsenergie wat tydens die omskakelingsproses benodig word, asook ’n afname in die koste verbonde aan die aankoop van duur kommersiële ensieme om die stysel na fermenteerbare suikers af te breek. In hierdie studie is ’n rekombinante Saccharomyces cerevisiae-gis gegenereer wat die glukoamilase (GA I) and α-amilase (AMYL III) van Aspergillus awamori, asook die glukoamilase van Aspergillus oryzae (GLAA) as aparte polipeptide uit te druk. Die rekombinante gis wat die funksionele GA I en AMYL III uitgeskei het, was in staat om op die rou-stysel as koolstofbron te groei, en het roustysel na bio-etanol teen ’n spesifieke tempo van 0.037 gram per gram droë gewig biomassa per uur omgeskakel. Die etanolopbrengs van 0.40 gram per gram beskikbare suiker vanaf stysel was gelykstaande aan 71% van die teoretiese maksimum vanaf stysel as substraat. ’n Belowende gis wat roustysel kan omskakel na bio-etnaol was dus geskep. In die tweede deel van hierdie studie is die opbrengs in oplosbare vastestowwe vermeerder deur die koffie-afval wat tydens die huidige industrieële proses genereer word, met hidrolitiese ensieme te behandel. Rekombinante ensieme afkomstig vanaf Aspergillus-rasse (β-mannanase, β-endoglukanase 1, β-endo-glukanase 2 en β-xilanase 2), ensieme deur wilde-tipe organismes uitgeskei (β-mannanase), asook kommersiële ensiempreparate wat die nodige ensiemaktiwiteite getoon het (β-mannanase, sellulase en pektinase) is gebruik om die oorblywende 42% mannaan en 51% sellulose in koffie-afval te hidroliseer. Hidrolise eksperimente het getoon dat ’n ensiempreparaat wat hoofsaaklik mannanase bevat, die oplosbare vastestofopbrengs grootliks kan verbeter, met ’n verhoogde opbrengs van 23% tydens geöptimiseerde ensiembehandelings. ’n Verhoogde opbrengs in oplosbare vastestowwe tydens die vervaardiging van kitskoffie sal die volgende tot gevolg hê: (i) ’n toename in totale opbrengs van kitskoffie produk, (ii) ’n afname in die hoeveelheid koffiebone wat vir die produksie ingevoer moet word, en (iii) ’n afname in die hoeveelheid afval wat tydens die vervaardigingsproses produseer word.

O objetivo desse estudo foi investigar o efeito protetor do íon magnésio em células de levedura Saccharomyces cerevisiae submetidas aos estresses térmico e etanólico. No processo industrial de fermentação as leveduras estão sujeitas ao estresse térmico, originado pelo calor produzido pela própria fermentação, e ao estresse pelo etanol, originado pelos efeitos adversos do álcool etílico produzido pelo catabolismo dos açúcares pelas leveduras. O magnésio tem a capacidade de atenuar os efeitos nocivos de estresse em leveduras S. cerevisae, principalmente através da estabilização da membrana celular. Foi cultivada a levedura Saccharomyces cerevisiae Y-904, a 30°C por 24h sob agitação de 80 rpm em meio YEPD e em meio à base de caldo de cana-de-açúcar, acrescido ou não de 20 mmol de magnésio, na forma de sulfato de magnésio hepta-hidratado. As culturas foram expostas ao estresse térmico, através da elevação da temperatura de incubação de 30 para 42°C e/ou ao estresse etanólico, em meio com 10% (v/v) de etanol. A viabilidade celular da levedura foi determinada por microscopia ótica às 0, 1, 2, 3, 4, 5, 24 e 48 horas do período de incubação sob as condições de estresse. A concentração de magnésio nas células e nos meios foi determinada por espectroscopia de absorção atômica. Os resultados foram analisados estatisticamente através de Análise de Variância, com posterior aplicação de Teste de Tukey. O enriquecimento do meio YEPD e/ou das células da levedura com magnésio proporcionou maior população e viabilidade celular da levedura. Em meio à base de caldo de cana o enriquecimento com magnésio não influenciou a população ou a viabilidade celular da levedura, provavelmente porque tal meio de cultivo já apresentava concentração suficiente de magnésio para a proteção da levedura contra os estresses térmico e etanólico. / The aim of this study was to investigate the protective effect of the ion magnesium in cells of Saccharomyces cerevisiae under thermal and ethanolic stresses. In the industrial process of fermentation the yeasts might be submitted either to thermal stress, originated from the heat produced by fermentation, or to ethanol stress, originated from the damages effect of ethylic alcohol produced by the catabolism of the sugars by the yeasts. Magnesium has the capability to attenuate harmful effects of stresses in yeast, mainly through the stabilization of the cellular membrane. The strain Y-904 of Saccharomyces cerevisiae was cultivated at 30°C during 24h under 80 rpm agitation in medium YEPD or in a broth from sugar cane, both added or not with 20 mmol of magnesium from magnesium sulphate hepta-hydrated (MgSO4 7H2O). The cultures were exposed either to heat shock, by rising of the temperature of incubation from 30 to 42°C, either/or to ethanol shock, in broth with 10% (v/v) of ethanol. The cellular viability of the yeasts was determined by optical microscopy at 0, 1, 2, 3, 4, 5, 24 and 48 hours of the period of incubation under the stress conditions. The magnesium concentration in the cells and in the mediums was determined for atomic absorption spectroscopy. The results were statistically analyzed by variance analysis and Tukey test. The yeast population and cell viability were higher in the medium YEPD enriched with magnesium (intra or extracellular) compared the same medium without magnesium supplementation. However it was not observed difference in population or viability of the yeasts in the broths from sugar cane enriched or not with magnesium. This happened probably because the broth from sugar cane already presented a concentration of magnesium enough to protect the yeast cells against the thermal and ethanolic stresses.

Nisina, um antimicrobiano produzido por cepas de Lactococcus lactis subsp. Lactis, pode ser utilizada como preservante em alimentos e, possivelmente, como agente antimicrobiano. Para aumentar sua produção por Lactococcus lactis ATCC 11454 em meio MRS (pH = 6,5) realizamos ensaios em agitador rotativo (100 rpm/ 30 °C/ 36h) a partir de 3 grupos de ensaios, designados por desenho fatorial de dois níveis (2 (4-1) no grupo 01) e 2(3) nos grupos 2 e 3. As concentrações de sacarose (2,2 a 15 g/L), asparagina (7,5 a 75 g/L), fosfato de potássio (6 a 18 g/L) e Tween 80 (1,0 a 6,6 g/L) foram adicionadas ao meio MRS (pH = 6,5). A produção de nisina foi acompanhada a partir do método de difusão em ágar a partir de utilização de Lactobacillus sake ATCC 9924 como microorganismo sensível. Os melhores níveis de nisina atingidos foram de 1,6 x 104 AU/mL (grupo 2) a 1,8 x 104 AU/mL (grupo 1) e obtidos para os níveis máximos de sacarose e asparagina. Dependendo das proporções entre as concentrações dos outros nutrientes, quando a concentração final de sacarose variou entre 0,33 g/L a 3,64 g/L, o pH oscilou entre 5,10 to 6,01, o acido lático produzido variou de 1,19 g/L a 4,44 g/L e a concentração celular variou de 0,99 a 4,184 mg/L. produção de nisina mostrou-se independente das velocidades de crescimento, as quais variaram de 0,0070 h-1 a 0,0401 h-1. A analise de regressão mostrou se adequar a um modelo polinomial quadrático: Log AU/mL = 1,98905 - 0,213558 x2 + 1,80028 x3 - 1,40776 x4 + 0,23436 x1 x2 + 0,35936 x1 x3 +1,47217 x3 x4 (equação principal). Onde os índices representam as concentrações de sacarose; asparagina; fosfato e tween 80 respectivamente. A análise de variância foi realizada para os parâmetros fixados para demonstrar a adequação do modelo proposto. (x são os índices dos nutrientes adicionados). / Nisin, an antimicrobial substance tha is produced by Lactococcus lactis, can be utilized as a preservative and therapeutic agent. To improve its production by Lactococcus lactis ATCC 11454 in MRS broth (pH =6.5), in rotatory shaker (100 rpm/ 30 °C/ 36h), three groups of assays, set up by a fractional factorial design of two-levels (2 (4-1) in group 01), and 2(3) in groups 2 and 3 were carried out. The minimum and maximum concentrations of sucrose (5 to 12.5 g/L), asparagine (7.5 to 75 g/L), potassium phosphate (6 to 18 g/L) and Tween 80 (1.0 to 6.6 g/L) were added to the MRS broth (pH = 6.5). The nisin production was accompanied through the agar diffusion assay using Lactobacillus sake ATCC 9924 as a sensitive microorganism. The best nisin activities ranged from 1.67 x 104 AU/mL (group 2) to 1.84 x 104 AU/mL (group 1) and were obtained for maximum levels of sucrose and asparagine, dependending of the proportions of the other nutrients concentrations, when the final sucrose content varied from 0.33 g/L to 3.64 g/L, the pH value oscillated beTween 5.10 to 6.01, the produced lactic acid was ranged from 1.19 g/L to 4.44 g/L and the dry-cell content was about 0.99 to 4,184 mg (DCW)/L. Nisin production was shown to be independent of growth rates, which ranged from 0.0070 h-1 to 0.0401 h-1. The regression analysis attained a quadratic polynomial model: Log AU/mL = 1.98905 - 0.213558 x2 + 1.80028 x3 - 1.40776 x4 + 0.23436 x1 x2 + 0.35936 x1 x3 +1.47217 x3 x4 (main equation). The analysis of variance performed to the fitted parameters of the independent variables was performed for checking model fitting results adequacy. (x are the index for the nutrients added).

Este trabalho consiste em um estudo sobre monitoramento ambiental de áreas limpas. Os parâmetros comparados foram partículas não viáveis de 0,5 e 5,0 µm, contaminação do ambiente (UFC do ar) e de superfícies (Rodac®). Procedeu-se ao estudo em áreas de manipulação crítica (Classe A). Nestas áreas, a amostragem foi feita em situações de repouso e dinâmica, antes e após sanitização, e em etapas de certificação e rotina. Os dados de literatura indicam que os parâmetros não são particularmente dependentes do lay out ou classificação das áreas, mas sim do seu uso e do comportamento dos operadores. As conclusões foram positivas quanto a correlação entre diferentes locais de amostragem, para partículas não viáveis de 0,5 e 5,0 µm, e ausência deste tipo de correlação em posições em fluxo laminar. Também, os valores de correlação foram quase sempre decrescentes com a maior limpeza do ambiente. Os microrganismos mais frequentemente isolados, nas áreas A2., A3 e A4 foram Bacillus sp, Staphylococcus sp e Corynebacterium sp. / This paper represents a study about environmental monitoring data for cleanrooms. The parameters that were compared are: total airborne particles of 0.,5 and 5.0 µm, airbome colony forming units (CFU\'s) and CFU\'s on surfaees (Rodac®). Most attention was paid to areas where critical handling is performed (class A areas). In these areas sampling was performed \"at rest\" and \"dynamic\" conditions, before and after sanitization and during certification and routine steps. These data indicates that the parameters are not particularly dependent upon the lay out or classification of areas, but rather on the use of the areas, and how the operators behave in them. Evaluating the data, the conclusion was about a correlation among different sampling places, for total airborne particles of 0.,5 and 5.0 µm, and absence of this kind of correlation in laminar flows positions. Also, correlation values were always increasing with the cleanless of the environment. The microorganisms most frequently isolated in areas A2, A3 and A4 were Bacillus sp, Staphylococcus sp and Corynebacterium sp.