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).

O projeto visou aumentar a produção de celulases em fungos T. harzianum IPT 821 e T. reesei QM 9414. Esporos foram submetidos à radiação UV-C. Células das colônias mais capacitadas ao crescimento foram sucessivamente cultivadas em meio solidificado, contendo concentrações progressivamente reduzidas fonte de carbono, de modo a resultar pressão ambiental seletiva e crescente. Após esta etapa as linhagens isoladas foram cultivadas em meio composto por bagaço de cana/farelo de trigo na proporção 80/20, 60% de umidade, 30°C por 72h. Uma linhagem, originária da cepa IPT 821, apresentou atividade de 3,7 FP U/gms, 50% superior em relação à parental: 2,6 U/gms (p=0,001; p<0,05). Análises das frações enzimáticas indicaram uma diferença significativa (p=0,001; p<0,05) na atividade de xilanase: 4,7 U/gms (mutante) e 4 U/gms (parental). Ensaios de hidrólise, Avicel como substrato (1% de sólidos; w/v) indicaram um aumento de quase 70% na hidrólise em 48h, da mutante em comparação à parental (8,7% (mutante) e 4,8% (parental), concentração enzimática de 2,5 FP U/gms). / This project aimed to increase cellulase production in fungi T. harzianum IPT 821 and T. reesei QM 9414. Spores were exposed to UV-C radiation. Colonies were plated and those showing best growth were successively cultivated in plates containing increasingly stress conditions reduced concentrations of the carbon source thus creating a progressive selective pressure. After this pre-selection step isolated strains were cultivated in medium comprising sugar cane bagasse and wheat straw, in the proportion of 80/20 respectively, 60% of moisture, 30°C for 72h. One strain, originated from IPT 821 strain, showed a cellulolytic activity of 3,7 U/gdw; 50% superior to the parental strain: 2,6 U/gdw (p=0,001; p<0,05). Analysis of the enzymatic cocktail showed significant difference (p=0,001; for p <0,05) on xylanase activity: 4,7 U/gdw (mutant strain) and 4 U/gdw (parental strain). Hydrolysis assays, using Avicel as substrate (1% w/v) showed an increase on hydrolysis of about 70%, in 48h (8,7% (mutant strain) and 4,8% (parental strain), enzyme load of about 2,5 U/gdw).

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&#176;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&#176;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&#176;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&#176;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.

Orientador: Vanildo Luiz Del Bianchi / Coorientador: Claudia Dorta / Banca: Ellen Silva Lago Vanzela / Banca: Crispin Humberto Garcia-Cruz / Banca: Gisele Ferreira Bueno / Banca: Elias de Souza Monteiro Filho / Resumo: O Brasil tem recebido grande destaque no meio econômico por conta da alta produção do setor agroindustrial, como consequência torna-se um dos maiores produtores de resíduos. A reutilização destes compostos é importante para melhorar sua disposição no meio ambiente contribuindo para a redução da poluição ambiental, além disso, é uma forma de agregar valor aos subprodutos. A indústria vinícola tem grande interesse nestas tecnologias de reutilização, já que seus principais resíduos (casca e semente de uva) são de lenta decomposição prejudicando o meio ambiente. A casca da uva pode ser hidrolisada através do emprego de ácidos ou enzimas disponibilizando açúcares fermentescíveis, que podem ser transformados em uma série de bioprodutos. Este trabalho buscou estabelecer o melhor método de pré-tratamento e hidrólise, da casca de uva para disponibilizar os açúcares que podem ser fermentados pelas leveduras Saccharomyces cerevisiae, Pichia stipitis e Pachysolen tannophilus. Para tanto foi realizado o pré-tratamento químico ácido ou alcalino junto ao pré-tratamento físico em autoclave, micro-ondas ou ultrassom na casca da uva Cabernet Sauvignon. Em etapa posterior, para seleção da melhor condição de pré-tratamento e hidrólise foi aplicado o Delineamento Composto Central Rotacional (DCCR). Também foi analisada a capacidade de consumo de açúcares do mosto hidrolisado de casca de uva pelas leveduras Saccharomyces cerevisiae, Pichia stipitis e Pachysolen tannophilus. A concentração dos açúcares foi medida através dos métodos de ADNS, xilose e glicose. Levando em consideração que ainda não existem na literatura muitos trabalhos envolvendo a casca de uva, com base nos resultados deste estudo o pré-tratamento alcalino em micro-ondas com 0,72% de NaOH por 13,5 minutos e a hidrólise ácida com 2,6% de H2SO4 por 13,5 minutos em autoclave foram os melhores métodos para a liberação de açúcares... / Abstract: Brazil has been highlighted on the economic environment due to the high production of its agroindustrial sector; as a result, it becomes one of the major residue producers. The reutilization of these compounds is relevant to improve the environment disposal, contributing towards for the environmental pollution reduction. Moreover, it is a way of adding value for the by-products.The wine industry has a great interest on reutilization techniques, once its main residues (grape seeds and skin) has a slow decomposition, which damages the environment. The grape skin can be hydrolyzed with acids or enzymes, making available fermentable sugars that can be transformed in a number of byproducts. This work sought to establish the best pre-treatment and hydrolysis method for the grape skin to provide the sugars that can be fermented for the Saccharomyces cerevisiae, Pichia stipitis and Pachysolen tannophilus yeasts. For this purpose, it was applied the acid or alkaline chemical pre-treatment along with the physic pre-treatment in autoclave, microwave or ultrasound on the Cabernet Sauvignon grape skin. Afterwards, for the pre-treatment and hydrolysis best condition selection, it was applied the Rotatable Central Composite Design (RCCD). It was also analyzed the yeasts Saccharomyces cerevisiae, Pichia stipitis and Pachysolen tannophilus consumption capacity of the grape skin hydrolysed must sugars. The sugars concentration was measured through the ADNS methods, xylose and glucose. Taking into account the lack of grape skin works on the literature, based on the results of this work the alkaline pretreatment on microwave with 0,72% of NaOH for 13,5 minutes and the acid hydrolysis with 2,6% of H2SO4 for 13,5 minutes in autoclave were the best methods for the fermentable sugars release with 10 g L-1 of reducing sugars, 3,3 g L-1 of glucose and 6,7 g L-1 of xylose.With regard to sugar consumption by the yeasts in the must of the grape skin hydrolyzate, ... / Doutor

Thesis (MScAgric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The production of quality wine is influenced by numerous factors of which grape quality is one of the most important factors. The production of quality wine, however, is not possible without good winemaking techniques and effective quality control. Critical control points (CCP) during the winemaking process must be identified to ensure optimum wine quality. Grape must is a complex medium that contains different micro-organisms which can be either beneficial or negative to wine quality, depending on the physical and chemical conditions that prevail in the must. Yeasts are responsible for alcoholic fermentation, lactic acid bacteria (LAB) for malolactic fermentation (MLF) and acetic acid bacteria (AAB) for the production acetic acid from ethanol. Yeasts and certain LAB can also produce acetic acid and thereby increasing the volatile acidity (VA) of wine. These micro-organisms can influence each other in complex fashions by competing for growth nutrients and by producing inhibitory substances. Most winemakers nowadays use commercial yeast strains to inoculate wine fermentations. This, however, does not assure a problem-free fermentation and cases of stuck and sluggish fermentations are annually reported worldwide. In these or most cases fermentation takes longer than 21 days to complete and the wine contains a residual sugar concentration of more than 4 g/L, which can be utilised by wine spoilage micro-organisms such as certain bacteria and other wild yeasts. Stuck and sluggish fermentations also increase the chances of oxidation due to the absence of the protective CO2 layer on the surface of the wine, which is formed during alcoholic fermentation. Another effect of stuck and sluggish fermentations is that valuable tank space is wasted due to the unexpected time consumption of these fermentation problems. Many factors during the winemaking process can be responsible for stuck and sluggish fermentations. In this thesis the different factors is discussed with the emphasis on the effect of the yeast strain. The way that certain yeast strains influence AAB and LAB numbers during fermentation and MLF through the production of inhibiting by-products such as medium chain fatty acids has not been investigated in detail in the past. Certain fungicides and pesticides that are used in vineyards to control pests (e.g. mildew) contain copper which can be inhibiting to yeast growth and alcoholic fermentation. Legal limits and withholding periods on these sprays are not always strictly obeyed and can lead to stuck and sluggish fermentations. This motivated us to evaluate the growth and fermentation activities of a selection of commercial wine yeasts in the presence of copper levels in the range of maximum legal limits. The effect of these commercial strains on the LAB and AAB numbers during alcoholic fermentation and MLF were also investigated. Our results showed that there was no significant difference on numbers of the AAB obtained from fermentations inoculated with different commercial wine yeast strains. However, with regards to the LAB numbers, one of the strains produced significantly more sulphur dioxide (SO2), which led to the inhibition of MLF in that wine. Our results further indicated which commercial yeast strains were capable of effectively fermenting high sugar musts and which strains were less effective. From the strains tested VIN13, N96 & L2056 were able to utilize fructose more effectively than NT50, RJ11 & D80. We could further distinguish between yeast strains that produced the lowest (VIN13 & RJ11) and the highest (WE372, NT50 & L2056) VA concentrations in must containing high sugar levels. Strains that were more tolerant against high copper levels were also identified. We tested six yeast strains in must with added copper (0.25 mM cu2+) in the form of CuSO4 .H2O. Three Cu2+-tolerant (D80, Collection Cepage Cabernet & NT50) yeast strains were distinguished from three less Cu2+-tolerant yeast strains (VIN13, NT112 & RJ11). This study made a valuable contribution in knowledge gained about commercially available wine yeast strains that can ferment effectively under certain stress conditions. Research such as this, where wine yeasts are evaluated to ferment more effectively during strenuous winemaking conditions, will be very beneficial to winemakers. / AFRIKAANSE OPSOMMING: Die produksie van gehalte wyn word deur verskillende faktore beïnvloed waarvan druifkwaliteit seker die belangrikste is. Die produksie van gehalte wyn is egter nie moontlik sonder goeie wynmaaktegnieke en effektiewe kwaliteitsbeheer nie. Kritieke kontrole punte (KKP) tydens die wynmaakproses moet dus geïdentifiseer word om sodoende ‘n verlaging in wynkwaliteit te vermy. Druiwemos het ‘n komplekse mikrobiologiese samestelling en bestaan uit verskillende mikroörganismes wat vooren nadelig vir wynkwaliteit kan wees, afhangende van die fisiese en chemiese toestande wat in die mos bestaan. Giste is verantwoordelik vir alkoholiese fermentasie, melksuurbakterieë (MSB) vir appelmelksuurgisting (AMG) en asynsuurbakterieë (ASB) vir die produksie van asynsuur vanaf etanol. Asynsuur word egter ook deur giste en MSB geproduseer en dra so by tot die vlugtige suurheid (VS) van ‘n wyn. Hierdie mikroörganismes kan mekaar op komplekse wyses beïnvloed deur o.a. te kompeteer vir voedingstowwe asook deur die produksie van inhiberende verbindings. Die meeste wynmakers maak gebruik van kommersiële gisrasse om alkoholiese fermentasies mee uit te voer. Gevalle van sogenaamde slepende en gestaakte alkoholiese fermentasies, waar suiker nie volledig na etanol en CO2 omgeskakel word nie, kom egter nog gereeld in die wynbedryf voor. In sulke gevalle neem die fermentasie gewoonlik langer as 21 dae om te voltooi met ‘n suiker konsentrasie van meer as 4 g/L wat in die wyn oorbly. Dit is nadelig vir wynkwaliteit aangesien dit nie net die kanse vir bederf deur bakterieë en giste verhoog nie, maar ook die kanse vir oksidasie verhoog a.g.v. die verlies van die beskermende CO2 lagie bo-oor die wyn. Hoe sekere gisrasse, ASB en MSB getalle gedurende fermentasie en AMG beïnvloed deur die produksie van inhiberende verbindings soos medium ketting vetsure en SO2, is ook nie baie in die verlede ondersoek nie. Sommige spuitstowwe wat gebruik word in die bekamping van swamsiektes bevat koper wat inhiberend kan wees vir gisgroei en alkoholiese fermentasie. Wetlike maksimum limiete en onthoudingsperiodes op spuitstofresidue word egter nie altyd gehoorsaam nie en kan lei tot slepende en gestaakte fermentasies. Dit het ons gemotiveer om ‘n seleksie van kommersiële gisrasse te evalueer in terme van gisgroei en fermentasie in die teenwoordigheid van kopervlakke naby die maksimum limiet. Ons resultate het gewys dat daar nie noemenswaardige verskille in AAB getalle tydens alkoholiese fermentasie tussen behandelings met verskillende kommersiële gisrasse was nie. Een van die gisrasse het wel noemenswaardig meer SO2 geproduseer wat gelei het tot inhibering van AMG in hierdie wyn. Ons het verder uitgewys watter kommersiële gisrasse instaat is om meer effektief in hoër suiker mos te fermenteer en watter van die rasse minder suksesvol was. Ons het ook rasse geïdentifiseer wat meer weerstandbiedend is teen hoë kopervlakke in mos en sodoende groter kans op ‘n suksesvolle fermentasie sal hê in mos wat koperresidue bevat wat afkomstig is van sekere spuitstowwe. Die effek van die ASB en MSB getalle gedurende fermentasie en AMG is ook ondersoek. Ons resultate het verder gewys watter kommersiële gisrasse instaat was om mos met hoë suikervlakke meer effektief te fermenteer. Vam die gisrasse wat getoets was het VIN13, N96 & L2056 fruktose meer effektief benut as NT50, RJ11 & D80. Ons kon verder onderskei tussen gisrasse wat die laagste (VIN13 & RJ11) en die hoogste (WE372, NT50 & L2056) vlakke van VS produseer in mos met hoë inisiële suikervlakke. Gisrasse wat meer tolerant was teen koperresidue in mos is ook geidentifiseer. Ons het ses gisrasse getoets in mos met bygevoegde koper (0.25 mM Cu2+) in die vorm van CuSO4 .5H2O. Daar is onderskei tussen drie Cu2+-tolerante (D80, Collection Cepage Cabernet & NT50) en drie minder Cu2+-tolerante gisrasse (VIN13, NT112 & RJ11). Hierdie studie lewer ‘n waardevolle bydrae in die invordering van kennis oor kommersieel beskikbare wyngisrasse wat meer effektief sal fermenteer onder sekere streskondisies wat in mos voorkom. Inligting soos hierdie is belangrik om die wynmaker se keuse uit die reeks bestaande kommersiële gisrasse te vergemaklik.

O objetivo deste trabalho foi à purificação da toxina alfa produzida por Costridium perfringens tipo A em sistema de duas fases aquosas (SDFA) PEG/fosfato nos modos contínuo e descontínuo. Dois planejamentos fracionários sequenciais foram aplicados para estudar a partição da toxina alfa em SDFA, em função de 4 variáveis: massa molar e concentração do PEG, concentração do fosfato e pH. Os melhores resultados para o fator de purificação, rendimento em atividade e coeficiente de partição foram obtidos com PEG 8000 g/mol (15%, w/w), fosfato 20% (w/w) e pH 8.0. Este sistema permitiu um fator de purificação de 4,6 com rendimento em atividade de 230% e coeficiente de partição de 113,9 na fase PEG. Após isto, novos experimentos foram realizados para aperfeiçoar a purificação da toxina alfa com o emprego de um planejamento experimental completo 22. Nesta pesquisa a concentração do PEG 8000 g/mol e sais de fosfato pH 8,0 foram variados. O coeficiente de partição, fator de purificação e rendimento em atividade foram fortemente influenciados por estas variáveis. Aumentando ambos os fatores estudados, foi observado um aumento nas respostas, com exceção para o fator de purificação. O melhor fator de purificação (5,7) foi obtido com 17,5% e 15% das concentrações de PEG e fosfato, respectivamente. A coluna de discos perfurados rotativos (PRDC) foi usada para extrair toxina alfa a partir do sobrenadante com (PEG) (MM = 8,000 g/mol) e solução de sais de fosfato de sódio e de potássio (pH 8.0). Os resultados obtidos demonstraram que a velocidade de fluxo da fase dispersa (VD) de 3 Ml/min foi a melhor condição para as respostas hold up (ED = 0,8), eficiência de separação (ES = 100 %) e fator de purificação (Pf = 2,37), pois os melhores resultados foram obtidos nesta taxa de fluxo usando uma velocidade de rotação dos discos baixa (35 rpm), enquanto o melhor coeficiente de transferência de massa (KDa = 2,8 x10-3 min-1) foi encontrado na maior velocidade de rotação usada (140 rpm). / The aim this work was purification alpha-toxin (phospholipase C) produced by Costridium perfringens type A by aqueous two-phase systems (ATPS) PEG/phosphate in discontinue and continue mode. Two sequential half-fraction designs were applied to studying the &#8733;-toxin partition in ATPS, as a function of four factors: PEG molar mass and concentration, phosphate concentration and pH. The highest purification factor, yield and partition coefficient results were obtained with PEG 8000 (15%, w/w), phosphate at 20% (w/w) and pH 8.0. This system allows an &#8733;-toxin purification of 4.6 fold with final activity yield of 230% and partition coefficient of 113.9 in the PEG rich phase. After this, new experiments were realized for the optimization of &#8733;-toxin purification with employed of full experimental design. In this research the concentration of PEG 8000 g/mol and phosphate salts pH 8.0 were varied. The partition coefficient (K), purification factor (Pf) and activity yield (Y%) were strongly influenced for these variables. Increasing both factors was observed an increase of these responses, except for P Pf. The higher purification factor (5.7) was obtained with 17.5% and 15% of the PEG and phosphate concentration, respectively. A continuous perforated rotating disc contactor (PRDC) was used for extraction of &#8733;-toxin from the supernatant of Clostridium perfringens type A cultivations with polyethylene glycol (PEG) (MW = 8,000 g/mol) and dipotassium and sodium phosphate salt solution (pH 8.0). The results obtained demonstrated that VD = 3 L/min was by far the optimum dispersed phase flowrate for all these response variables. Besides, maximum values of ED (0.8), Es (100 %) and Pf (2.37) were obtained at this flowrate using the lowest rotational speed (35 rpm), while optimum KDa (2.8 x 10-3 min-1 ) was achieved at the highest agitation level (140 rpm).

O objetivo deste trabalho foi à purificação da toxina alfa produzida por Costridium perfringens tipo A em sistema de duas fases aquosas (SDFA) PEG/fosfato nos modos contínuo e descontínuo. Dois planejamentos fracionários sequenciais foram aplicados para estudar a partição da toxina alfa em SDFA, em função de 4 variáveis: massa molar e concentração do PEG, concentração do fosfato e pH. Os melhores resultados para o fator de purificação, rendimento em atividade e coeficiente de partição foram obtidos com PEG 8000 g/mol (15%, w/w), fosfato 20% (w/w) e pH 8.0. Este sistema permitiu um fator de purificação de 4,6 com rendimento em atividade de 230% e coeficiente de partição de 113,9 na fase PEG. Após isto, novos experimentos foram realizados para aperfeiçoar a purificação da toxina alfa com o emprego de um planejamento experimental completo 22. Nesta pesquisa a concentração do PEG 8000 g/mol e sais de fosfato pH 8,0 foram variados. O coeficiente de partição, fator de purificação e rendimento em atividade foram fortemente influenciados por estas variáveis. Aumentando ambos os fatores estudados, foi observado um aumento nas respostas, com exceção para o fator de purificação. O melhor fator de purificação (5,7) foi obtido com 17,5% e 15% das concentrações de PEG e fosfato, respectivamente. A coluna de discos perfurados rotativos (PRDC) foi usada para extrair toxina alfa a partir do sobrenadante com (PEG) (MM = 8,000 g/mol) e solução de sais de fosfato de sódio e de potássio (pH 8.0). Os resultados obtidos demonstraram que a velocidade de fluxo da fase dispersa (VD) de 3 Ml/min foi a melhor condição para as respostas hold up (ED = 0,8), eficiência de separação (ES = 100 %) e fator de purificação (Pf = 2,37), pois os melhores resultados foram obtidos nesta taxa de fluxo usando uma velocidade de rotação dos discos baixa (35 rpm), enquanto o melhor coeficiente de transferência de massa (KDa = 2,8 x10-3 min-1) foi encontrado na maior velocidade de rotação usada (140 rpm). / The aim this work was purification alpha-toxin (phospholipase C) produced by Costridium perfringens type A by aqueous two-phase systems (ATPS) PEG/phosphate in discontinue and continue mode. Two sequential half-fraction designs were applied to studying the &#8733;-toxin partition in ATPS, as a function of four factors: PEG molar mass and concentration, phosphate concentration and pH. The highest purification factor, yield and partition coefficient results were obtained with PEG 8000 (15%, w/w), phosphate at 20% (w/w) and pH 8.0. This system allows an &#8733;-toxin purification of 4.6 fold with final activity yield of 230% and partition coefficient of 113.9 in the PEG rich phase. After this, new experiments were realized for the optimization of &#8733;-toxin purification with employed of full experimental design. In this research the concentration of PEG 8000 g/mol and phosphate salts pH 8.0 were varied. The partition coefficient (K), purification factor (Pf) and activity yield (Y%) were strongly influenced for these variables. Increasing both factors was observed an increase of these responses, except for P Pf. The higher purification factor (5.7) was obtained with 17.5% and 15% of the PEG and phosphate concentration, respectively. A continuous perforated rotating disc contactor (PRDC) was used for extraction of &#8733;-toxin from the supernatant of Clostridium perfringens type A cultivations with polyethylene glycol (PEG) (MW = 8,000 g/mol) and dipotassium and sodium phosphate salt solution (pH 8.0). The results obtained demonstrated that VD = 3 L/min was by far the optimum dispersed phase flowrate for all these response variables. Besides, maximum values of ED (0.8), Es (100 %) and Pf (2.37) were obtained at this flowrate using the lowest rotational speed (35 rpm), while optimum KDa (2.8 x 10-3 min-1 ) was achieved at the highest agitation level (140 rpm).

Poultry meat consumption in New Zealand has been increasing since 1975 with the highest peak reported in 2006. The total poultry meat consumption was 36.5 kg per capita in the year ending September 2006. Consumption of contaminated food with raw poultry can lead to campylobacteriosis, which is a food-borne disease that causes gastroenteritis in humans and it is a major problem in New Zealand. There were 12,776 reported cases of campylobacteriosis in 2007, which accounts for 65.9% of the overall notified diseases. Campylobacteriosis can lead to Guillain-Barré syndrome in some patients, an autoimmune disorder of the peripheral nervous system. Campylobacteriosis is caused by consumption of either Campylobacter jejuni or Campylobacter coli. Campylobacter spp. have been found in commercially raised poultry being infected predominantly by C. jejuni. C. jejuni has been found associated with biofilms of other bacterial species in the watering supplies and plumbing systems of animal husbandry facilities and animalprocessing plants. A biofilm is an assemblage of microbial cells that is associated with a surface and the cells are enclosed in a matrix of polysaccharides, which provides a survival advantage to the bacteria in the film. In this study, the ability to form biofilm was measured in a laboratory assay using microtitre plates. C. jejuni strains in monoculture were shown to attach to the abiotic surface and form biofilms to various degrees, thus potentially enhancing their survivability in the poultry environment. C. jejuni was also shown to have the ability to attach and survive in mixed-microbial populations. Biofilm formation may play a role in the epidemiology of C. jejuni infections. Enterococcus faecalis and Staphylococcus simulans may play a role in the biofilm formation in the poultry environment as both of these microorganisms were able to form, and harbour C. jejuni in their biofilms. Pseudomonas aeruginosa seemed to inhibit biofilm formation and C. jejuni in the mixed-microbial population. Further studies are required to establish control measures against the formation of biofilms containing C. jejuni in poultry processing plants and farms in New Zealand to reduce the reservoir of contamination and thus reduce the incidence of campylobacteriosis.

Ethanol from lignocellulosic biomass is a promising alternative to rapidly depleting oil reserves. However, natural recalcitrance of lignocelluloses to biological and chemical treatments presents major engineering challenges in designing an ethanol conversion process. Current methods for pretreatment and hydrolysis of lignocelluloses generate a mixture of pentose (C5) and hexose (C6) sugars, and several microbial growth inhibitors such as acetic acid and phenolic compounds. Hence, an efficient ethanol production process requires a fermenting microorganism not only capable of converting mixed sugars to ethanol with high yield and productivity, but also having high tolerance to inhibitors. Although recombinant bacteria and yeast strains have been developed, ethanol yield and productivity from C5 sugars in the presence of inhibitors remain low and need to be further improved for a commercial ethanol production. The overarching objective of this work is to transform Zymomonas mobilis into an efficient whole-cell biocatalyst for ethanol production from lignocelluloses. Z. mobilis, a natural ethanologen, is ideal for this application but xylose (a C5 sugar) is not its 'natural' substrate. Back in 1995, researches at National Renewable Energy Laboratory (NREL) had managed to overcome this obstacle by metabolically engineering Z. mobilis to utilize xylose. However, even after more than a decade of research, xylose fermentation by Z. mobilis is still inefficient compared to that of glucose. For example, volumetric productivity of ethanol from xylose fermentation is 3- to 4- fold lower than that from glucose fermentation. Further reduction or complete inhibition of xylose fermentation occurs under adverse conditions. Also, high concentrations of xylose do not get metabolized completely. Thus, improvement in xylose fermentation by Z. mobilis is required. In this work, xylose fermentation in a metabolically engineered Z. mobilis was markedly improved by applying the technique of adaptive mutation. The adapted strain was able to grow on 10% (w/v) xylose and rapidly ferment xylose to ethanol within 2 days and retained high ethanol yield. Similarly, in mixed glucose-xylose fermentation, the strain produced a total of 9% (w/v) ethanol from two doses of 5% glucose and 5% xylose (or a total of 10% glucose and 10% xylose). Investigation was done to identify the molecular basis for efficient biocatalysis. An altered xylitol metabolism with reduced xylitol formation, increased xylitol tolerance and higher xylose isomerase activity were found to contribute towards improvement in xylose fermentation. Lower xylitol production in adapted strain was due to a single mutation in ZMO0976 gene, which drastically lowered the reductase activity of ZMO0976 protein. ZMO0976 was characterized as a novel aldo-keto reductase capable of reducing xylose, xylulose, benzaldehyde, furfural, 5-hydroxymethyl furfural, and acetaldehyde, but not glucose or fructose. It exhibited nearly 150-times higher affinity with benzaldehyde than xylose. Knockout of ZMO0976 was found to facilitate the establishment of xylose fermentation in Z. mobilis ZM4. Equipped with molecular level understanding of the biocatalytic process and insight into Z. mobilis central carbon metabolism, further genetic engineering of Z. mobilis was undertaken to improve the fermentation of sugars and lignocellulosic hydrolysates. These efforts culminated in construction of a strain capable of fermenting glucose-xylose mixture in presence of high concentration of acetic acid and another strain with a partially operational EMP pathway.

Video files: Some images may require stereoscopic glasses / Fat in milk and cream is present as tiny droplets, which are each enveloped in a thin membrane, called the milk fat globule membrane (MFGM). The MFGM can easily be damaged by factors such as pumping the milk and applying other forms of agitation. MFGM damage is believed to reduce processing efficiency and compromise the quality of manufactured products. A comprehensive review of the literature showed that our understanding of changes occurring in the MFGM post secretion of the fat globule by the mammary secretory cell is still rudimentary. Furthermore, it was found that a fundamental understanding of MFGM damage in raw milk is lacking. Hence, this study sought to develop analytical techniques for studying the MFGM. Fluorescent probes were identified that associated with the MFGM (bovine, ovine, human) in one of two ways: either by embedding in the phospholipid bilayer (lipophilic probe) or by binding to carbohydrate moieties of glycosylated chains in the glycocalyx (lectin probes). The use of these probes, in combination with either conventional fluorescence microscopy or confocal laser scanning microscopy, allowed 2-D images and 3-D images of fat globules to be made. Application of water-soluble lipophilic probes and the lectin wheat germ agglutinin (WGA) directly to milk allowed the staining of the MFGM in its native environment. Variable distribution patterns of the probes in the MFGM were observed, which suggests that the MFGM of fat globules in harvested milk is structurally and chemically heterogeneous both within and among globules from the same species and between species, and even among fat globules within the milk of an individual animal. Furthermore, the binding behaviour of WGA to the MFGM of native fat globules (in bovine milk) and washed fat globules (in model systems) following heat treatment implicated β-lactoglobulin, α-lactalbumin, immunoglobulin M and/or the glycosylated proteins Periodic acid Schiff 6/7 in the disappearance of fat globule aggregation upon elevated heat treatment of milk. The results of the current study showed that the use of membrane-specific fluorescent probes, particularly in combination with confocal laser scanning microscopy, has significant potential for providing real time structural and chemical information about the MFGM in matrices such as harvested milk and milk products. In addition to the fluorescence microscopy techniques, development of other techniques was also conducted. Flow cytometry was shown to have significant potential for the quantitative determination of various properties of fat globules and their membranes. Although no suitable sample preparation technique could be developed in this study, atomic force microscopy is believed to have significant potential for studying structural and physical properties of the MFGM. Selective harvesting of individual fat globules was shown to be possible by using a micromanipulator. In future work, this technique is expected to be used in combination with fluorescence microscopy, or atomic force microscopy. The present study has shown that the development and application of novel analytical techniques has advanced, and in the future will further advance, understanding of the MFGM.