CAPES / A produção de carne suína no Brasil tem dado um grande salto nos últimos anos, no seu aspecto quantitativo e qualitativo. Isso permitiu posicionar o Brasil dentre os principais atuantes mundiais no setor da suinocultura. A garantia da inocuidade de alimentos depende da capacidade das indústrias em minimizar a contaminação de alimentos por microrganismos patogênicos, principalmente durante o processamento e estocagem, e controlar para que a garantia continue sendo mantida. Dentre os patógenos existentes, destaca-se Listeria monocytogenes que é um importante patógeno de origem alimentar emergente e causadora de infecções localizadas e generalizadas, levando o individuo ao óbito. Neste sentido, o objetivo deste estudo foi avaliar a qualidade sanitária do processo produtivo de um abatedouro-frigorifico de suínos, quanto à presença de L. monocytogenes. Inicialmente foram padronizados os métodos para aplicação da PCR e análise de sequenciamento posteriormente aplicado nas amostras coletadas. Duas coletas foram realizadas em um frigorífico que abate suínos localizado na região dos Campos Gerais – PR. Também verificou-se os prazos e custos apresentados por laboratórios particulares brasileiros, fazendo uma comparação com a técnica molecular. A quantidade de amostras positivas da seunda coleta foi superior em relação a primeira, 10 e 3 respectivamente. Com o sequenciamento foi possível observar que a origem da contaminação encontra-se no setor de triparia. Também foi possível verificar que poucos laboratórios possuem a PCR como método de diagnóstico, e provou-se ainda que a técnica pode ser uma ótima alternativa por se apresentar econômica e viável para indústria de carnes. / The production of pork in Brazil has taken a great leap in recent years, in quantitative and qualitative. This allowed position Brazil among the main active worldwide in the field of pig farming. The guarantee of the safety of food depends on the ability of industries to minimise the contamination of food by pathogenic micro-organisms, especially during processing and storage, and to ensure that the guarantee will continue to be maintained. Among the existing pathogens, stands out Listeria monocytogenes, which is an important pathogen of food origin emerging and causing localized infections and generalized, leading the individual to death. In this sense, the objective of this study was to evaluate the health quality of the production process from an abattoir-fridge of pigs, as the presence of L. monocytogenes. Initially were standardized methods for the application of PCR analysis and sequencing of subsequently applied in the samples collected. Two samples were collected in a fridge that slaughter pigs located in the region of Campos Gerais - PR. Also it was found that the deadlines and costs presented by private laboratories Brazilians, making a comparison with the molecular technique. The number of positive samples of seunda collection was superior to that of the first, 10 and 3 respectively. With the sequencing was possible to observe that the source of contamination is in the sector of triparia. It was also possible to observe that few laboratories have the PCR as a method of diagnosis, and it has been proved that the technique can be a great alternative to present economic and feasible for meat industry.

CAPES / A produção de carne suína no Brasil tem dado um grande salto nos últimos anos, no seu aspecto quantitativo e qualitativo. Isso permitiu posicionar o Brasil dentre os principais atuantes mundiais no setor da suinocultura. A garantia da inocuidade de alimentos depende da capacidade das indústrias em minimizar a contaminação de alimentos por microrganismos patogênicos, principalmente durante o processamento e estocagem, e controlar para que a garantia continue sendo mantida. Dentre os patógenos existentes, destaca-se Listeria monocytogenes que é um importante patógeno de origem alimentar emergente e causadora de infecções localizadas e generalizadas, levando o individuo ao óbito. Neste sentido, o objetivo deste estudo foi avaliar a qualidade sanitária do processo produtivo de um abatedouro-frigorifico de suínos, quanto à presença de L. monocytogenes. Inicialmente foram padronizados os métodos para aplicação da PCR e análise de sequenciamento posteriormente aplicado nas amostras coletadas. Duas coletas foram realizadas em um frigorífico que abate suínos localizado na região dos Campos Gerais – PR. Também verificou-se os prazos e custos apresentados por laboratórios particulares brasileiros, fazendo uma comparação com a técnica molecular. A quantidade de amostras positivas da seunda coleta foi superior em relação a primeira, 10 e 3 respectivamente. Com o sequenciamento foi possível observar que a origem da contaminação encontra-se no setor de triparia. Também foi possível verificar que poucos laboratórios possuem a PCR como método de diagnóstico, e provou-se ainda que a técnica pode ser uma ótima alternativa por se apresentar econômica e viável para indústria de carnes. / The production of pork in Brazil has taken a great leap in recent years, in quantitative and qualitative. This allowed position Brazil among the main active worldwide in the field of pig farming. The guarantee of the safety of food depends on the ability of industries to minimise the contamination of food by pathogenic micro-organisms, especially during processing and storage, and to ensure that the guarantee will continue to be maintained. Among the existing pathogens, stands out Listeria monocytogenes, which is an important pathogen of food origin emerging and causing localized infections and generalized, leading the individual to death. In this sense, the objective of this study was to evaluate the health quality of the production process from an abattoir-fridge of pigs, as the presence of L. monocytogenes. Initially were standardized methods for the application of PCR analysis and sequencing of subsequently applied in the samples collected. Two samples were collected in a fridge that slaughter pigs located in the region of Campos Gerais - PR. Also it was found that the deadlines and costs presented by private laboratories Brazilians, making a comparison with the molecular technique. The number of positive samples of seunda collection was superior to that of the first, 10 and 3 respectively. With the sequencing was possible to observe that the source of contamination is in the sector of triparia. It was also possible to observe that few laboratories have the PCR as a method of diagnosis, and it has been proved that the technique can be a great alternative to present economic and feasible for meat industry.

In recent years, advances in biotechnology has allowed the development of synthetic membranes associated with nanocomposites, which has shown promising results as dermal burns dressings. In this sense, silver nanoparticles (NPAg) has been the focus of interest because of their biological properties such as antimicrobial and antiinflammatory effect. The incorporation of NPAg in biological membranes of different natures, such as chitosan, polyester, polymethacrylate methyl and cellulose, has been successfully tested in several biological models. The association between NPAg and polymers produced by the micro-organism presents important advantages, such as water solubility and lack of toxicity. Recently we developed a technique for producing NPAg associated with xanthan (GX), a biopolymer with potential application in various sectors of the petrochemical industry, food and pharmaceutical, through fermentation by Xanthomonas sp performed in the presence of silver nitrate. Therefore, this study aimed to develop, characterize and evaluate the potential antimicrobial and healing membranes nanocomposite xanthan: silver on second-degree burns in the porcine model. Therefore, xanthan biocomposites: silver were used for fabrication of membranes (for casting process, which were subsequently characterized for thickness, mechanical properties (stress, strain, Young´s modulus) and the thermal profile (DSC, TG and DTG). Activity antimicrobial was tested against strains of Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). analysis for tissue repair were made two dermal burns on the back nine male pigs breed Yorkshire (25 ± 5 kg), treated with Xanthan biosensor membrane: silver (XNPAg) with topical application of silver sulfadiazine 1% (SDZ). After eight, 18 and 30 days the wounds were examined macroscopically determined for each lesion area, and the animals euthanized for Microscopic study of the scar area observed that XNPAg membranes showed a significant increase in the values of thickness (P <0.05), density (p <0.01) and Young´s modulus (p <0.001) and reduced strength strain (p <0.05) when compared to membranes of xanthan. Were revealed changes in the thermal profile of the two membranes suggesting the incorporation of silver nanoparticles in the polymer xanthan. XNPAg The membrane induced the formation of inhibition zones 9, 7 mm and 9.6 mm and death rate of 89% and 100% for Staphylococcus aureus and Escherichia coli respectively. Histological analysis showed quantitative and qualitative increase in the reaction granulation and best architectural arrangement of collagen fibers along the the healing process of wounds covered with membranes XNPAg. Could be concluded that the membranes nanocomposite xanthan:silver showed satisfactory mechanical properties for its handling, transportation and storage, as well as important antimicrobial activity and pro-healing in dermal burns using porcine model. / Nos últimos anos, os avanços na área da biotecnologia tem propiciado o desenvolvimento de membranas sintéticas associados à nanocompostos, que vem apresentando resultados promissores como curativos de queimaduras dérmicas. Nesse sentido, as nanopartículas de prata (NPAg) tem sido foco de interesse devido as suas propriedades biológicas como atividade antimicrobiana e efeito antiinflamatório. A incorporação de NPAg em membranas biológicas de diferentes naturezas, como quitosana, poliéster, polimetacrilato de metila e celulose, vem sendo testada com sucesso em diversos modelos biológicos. A associação entre NPAg e polímeros produzidos por microrganismo apresenta importantes vantagens, como solubilidade em água e ausência de toxicidade. Recentemente foi desenvolvida uma técnica de produção de NPAg associadas a goma xantana (GX), um biopolímero com aplicação potencial em vários setores da indústria petroquímica, alimentícia e farmacêutica, através de processo fermentativo realizado pela Xanthomonas sp na presença de nitrato de prata. Diante disso, o presente estudo teve como objetivo desenvolver, caracterizar e avaliar o potencial antimicrobiano e cicatrizante de membranas de bionanocompósito xantana:prata sobre queimaduras de segundo grau em modelo suíno. Para tanto, biocompósitos xantana:prata foram utilizadas para confecção de membranas (por casting process, que foram posteriormente caracterizadas quanto a espessura, propriedades mecânicas (tensão, deformação, módulo de Young) e perfil termoanalítico (DSC, TG e DTG). A atividade antimicrobiana foi avaliada frente a cepas de Escherichia coli (ATCC 25922) e Staphylococcus aureus (ATCC 25923). Para análise do reparo tecidual foram confeccionadas duas queimaduras dérmicas no dorso de nove suínos machos, da raça Yorkshire (25 ± 5 kg), tratadas com membrana de biocompósito xantana:prata (XNPAg) e com aplicação tópica da sulfadiazina de prata a 1% (SDZ). Após oito, 18 e 30 dias, as feridas foram analisadas macroscopicamente, determinada a área de cada lesão, e os animais eutanasiados para estudo microscópico da área cicatricial. Observou-se que as membranas XNPAg apresentaram aumento significativo nos valores de espessura (p<0,05), deformação (p<0,01) e módulo de Young (p<0,001), e redução da força de tensão (p<0,05) quando comparados a membranas de xantana. Foram evidenciadas alterações no perfil termoanalítico das duas membranas sugestivas da incorporação das nanopartículas de prata no polímero de xantana. As membrana XNPAg induziram a formação de halos de inibição de 9,7 mm e 9,6 mm e Taxa de letalidade de 89% e 100% para Staphylococcus aureus e Escherichia coli respectivamente. A análise histológica mostrou incremento quantitativo e qualitativo na reação de granulação, bem como melhor disposição arquitetural das fibras de colágeno ao longo do processo cicatricial das feridas cobertas com membranas XNPAg. Pôde-se concluir que as membranas de bionanocompóstico xantana:prata apresentaram propriedades mecânicas satisfatórias para sua manipulação, transporte e armazenamento, bem como importante atividade antimicrobiana e pró-cicatrizante em queimaduras dérmicas utilizando modelo suíno.

Submitted in partial fulfillment for the Degree of Master of Applied Sciences in Biotechnology, Durban University of Technology, Durban, South Africa, 2017. / The study aims to assess and compare the concentration of microbial contaminants, their sources and distribution in surface water and sediment, and to determine the impact of seasonal variations and corresponding risks of faecal contamination using conventional and molecular methods. Historical data analysis was conducted using E. coli values from the eThekwini Water and Sanitation (EWS) department for 66 months (2009-2014). E. coli and Enterococci were analysed in surface water and sediment samples using the mFC/ spread plate and Colilert-18 (IDEXX) methods. The impact of seasonal variations was assessed using E. coli and Enterococci data collected during rainfall and no rainfall events, using an auto-sampler and sediment trap in parallel. Conventional standard membrane filtration methods using mFC agar, Slanetz & Bartley/ Bile Esculin and Brilliance E. coli selective agar were compared to the enzymatic Colilert-18 and Enterolert (IDEXX) test methods along the Isipingo and Palmiet Rivers. In addition, comparison of the analytical performance of droplet digital PCR (ddPCR) and qPCR for the detection of Salmonella targeting ttr gene in river sediment samples collected from the four sites of the Palmiet River in Durban, South Africa was done. In order to assess the public health risk associated with exposure of men, women and children to microbial pathogens in polluted surface water during recreational activities, the QMRA tool was employed in relation to the risk exposure to pathogenic E. coli, Campylobacter, Salmonella and Shigella. Also, the risk associated with crop irrigation (on farmers) as well as the consumption of crops irrigated with surface water from the Isipingo river was determined. Analysis of the historical data gave a baseline of the two rivers of interest, thus helps understand the current situation of the rivers enabling researchers to pick up potential gaps. In this study after the analysis of the historical data it was evident that at the Palmiet river, microbial analysis must be conducted around the QRI settlements which is a major pollution source. Also, from this study it was found that sampling points situated close to wastewater treatment plants, pump stations or informal settlements were of major concern, thus were considered for the study. It was found that sediment exhibited higher microbial concentrations than surface water, which was observed in both rivers. Also, rainfall had a significant impact on microbial variability. Higher microbial concentrations (indicator organisms) were observed in surface water after a heavy rainfall as appose to when there was no rainfall. This was due to contamination that is washed off into the river and sediment resuspension. Methodology comparison revealed that Colilert-18 and Brilliance E. coli were more selective compared to mFC agar. Brilliance E. coli /Coliform agar was comparable with Colilert-18 IDEXX, which was also observed with Slanetz & Bartley and Enterolert IDEXX. However, when mFC agar was compared with Colilert-18 IDEXX, significant difference was observed. In comparison of two Molecular methods, ddPCR were found to be fully amenable for the quantification of Salmonella and offer robust, accurate, high-throughput, affordable and more sensitive quantitation than qPCR in complex environmental samples like sediments. Quantitative Microbial Risk Assessment (QMRA) relating to recreational and occupational exposure showed that children were at the highest risk of getting infected. Also, it was observed that the probability of infection upon exposure to surface water from the Isipingo and Palmiet rivers was significantly high, hence exceeded the WHO guidelines values. Risk assessment on crops revealed that pathogenic bacteria may pose a risk to the consumer, however, a 9-log reduction may be achieved according to the WHO multi-barrier approach which involves proper washing and proper cooking of the crop before ingestion. Overall the sampling points that had the highest pollution level and constantly exceeded the WHO and DWAF guidelines at the Isipingo river were the points situated and named “Next to the WWTP”, and “Downstream of QRI” at the Palmiet River. / M

The goal of presented dissertation has been complex analysis of changes in content of fatty acids, enzymatic and low-molecular antioxidants in apples and related evaluation of perception of originators of storage diseases for apples stored in various conditions. Main part of the work has been dedicated to study of the impact of storage of apples in modified atmosphere with reduced amount of oxygen and in reference “normal” atmosphere for six months. Next part of the work has been dedicated to study how apple storage in common, consumer affordable, conditions (storage in a cellar, in a refrigerator, and in room temperature) affects content of low-molecular antioxidants. For testing has been selected apple kinds Jonagored, Golden Delicious, Idared, Šampion, and Granny Smith. Within the scope of this work has been optimized the method for determination of fatty acids in plant material with higher content of wax. The measured values imply the apples are valuable source of many important nutrition substances like vitamins, provitamins and antioxidants. During the storage process these substances exhibit considerable protective function. Long-term storage, mainly in the atmosphere with reduced amount of oxygen (FAN), enables preservation of majority of these important nutrition substances depending on the kind of apple and conditions of storage. Freezing process is conservative to apples (mainly in the presence of protective substances), while in the process of drying the values of all monitored antioxidants decrease depending on temperature and conditions of drying. Upon the choice of the storage method one must consider nutrition, sensoric characteristic and consumer demands.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

O cloreto de trifeniltetrazólio (TTC) foi empregado neste trabalho visando padronização de método alternativo para o teste de limite microbiano em medicamentos e cosméticos, com redução no tempo de análise, além de possibilitar a aplicação da técnica de semeadura em profundidade em amostras hidroinsolúveis com opacidade e carga contaminante menor que 102 UFC/g (mL).Na técnica de tubos múltiplos, a aplicação do corante não possibilitou a redução do tempo de incubação, pois a sensibilidade para detecção de suspensão bacteriana por meio de coloração do trifenilformazano foi de 108 UFC/mL após 1 hora de reação e 107 UFC/mL após 4 horas, suspensões estas visivelmente turvas. A adição de glicose e ácido glutâmico no meio de caseina-sola, nas concentrações de 0,5% e 0,1%, respectivamente, não aumentou a sensibilidade da reação. O polissorbato 20, adicionado ao meio de cultura, líquido ou sólido, promoveu a difusão do trifenilformazano das células para o meio, fator que não favorece a visualização da massa celular no meio líquido e nem das colônias. A concentração mínima de TTC que resultou em coloração máxima no meio líquido foi de 500 e 1000 &#181;g/mL, dependendo da espécie bacteriana. Entretanto, suspensões bacterianas com 102, 10 e 1 UFC/mL testadas, pela técnica de tubos multiplos, com adição do corante na forma de solução após 20 horas em meio de caseina-soja, forneceram resultados equivalentes, quanto ao número de tubos positivos e negativos frente às duas concentrações. A comparação desta técnica alternativa com a da subcultura demonstrou ser o tempo de 20 horas de incubação insuficientes para detecção pelo TTC, quando inoculadas alíquotas de 1 mL de suspensão com número da ordem de 1 UFC/mL Na técnica de semeadura em profundidade, após incubação de 48 horas, a coloração das colônias bacterianas foi efetuada cobrindo-se a superfície do meio de cultura com 5 mL de ágar a 1,0% contendo TTC. A concentração de 0,025% do corante promoveu a coloração das colônias após 1 hora. Entretanto, na presença de suspensão de hidróxido de alumínio a 6% a concentração mínima eficaz de TTC foi de 0,1%. Para leveduras em meio líquido essa concentração foi de 2.500 &#181;/mL no meio neutralizado. Em meio sólido, na presença de material opalescente, a concentração eficaz foi de 2,0%, com o TTC adicionado na dispersão de ágar em tampão pH 7,5. Amostras de produtos comercializados com característica opalescente foram submetidas à análise pelo método de tubos múltiplos com sub-cultura e pela revelação com TTC. Simultaneamente as mesmas foram testadas pelo método de semeadura em profundidade, inoculando-se 2 mL das diluições 1:4 e 1:10 com revelação das colônias com o corante. O tempo para obtenção dos resultados foi de 72 horas pelo método oficial e de 50 horas pelo método com TTC, tanto em meio líquido como em sólido. A comparação dos dados obtidos pelos 3 métodos não resultou em diferença significativa para a maioria das amostras testadas. Entretanto, os coeficientes de variação foram sempre menores para o método de semeadura em profundidade com a amostra diluída de 1:4, indicando maior precisão deste sobre os outros, principalmente em relação ao número mais provável. / The conventional methods for bioburden assessment are restrictive when applied to evaluate microbiological quality of pharmaceuticals and cosmetics. Triphenyltetrazolium chloride (TTC) was applied in this standartization study as alternative method for microbial limit test of pharmaceuticals and cosmetics. This method aimed at to reduce the assay time by conciliating the pour-plate technique analysis low bioburden of samples containing opacifiers. The application of this dye in the multiple tubes technique did not reduce incubation time, because the bacterial suspension sensitivity to triphenylformazan colour was 108 CUF/mL to 1 hour reaction and 107 CUF/mL to 4 hours. Suspensions with these bioburden are clearly turbid. The addition of 0.5% glicose and 0.1% glutamic acid to soybean-casein digest medium did not increase the reaction sensitivity. The addition of polissorbate 20 to solid ar liquid media promotted the difusion of triphenylformazan trom the cells to media; it rendered difficult of cellular mass detection in liquid medium and as much as colonies. The minimum concentration of TTC that produced maximum colour in liquid medium was 500 and 1,000 &#181;g/mL, depending on the bacteria. However, bacterial suspensions with 102 CUF/mL tested by multiple tubes technique with 20 hours of incubation and by microbial growth detection after TTC addition provided equivalent numbers of positive and negative tubes with both concentrations. The comparison of this alternative method with that of sub-culture proved that 20 hours of microbial growth is not enough to be detected by TTC, when 1 mL of 1 CUF/mL suspension was planted. The dying of bacterial colonies was performed by covering the medium surface with 5 mL of 1% agar with TTC, molten at 48 C. The concentration of TTC that colored colonies in 1 hour was 0.025%. However, in presence of 6% aluminium hidroxide suspension the minimum effective concentration was 0.1%. The concentration required to detect yeasts in liquid media was 2,500 &#181;g/mL in neutral pH condition. To detect yeast colonies in opalescent solid medium effective TTC concentration was 2%; in this case the dye was added to agar dispersed in 7.5 phosfate buffer. Samples of marketed produtcts were analysed through multiple tubes method with sub-culture and by TTC addition. Same samples were tested through pour-plate technique by inoculating 2 mL of 1:4 and 1:10 dilutions. The time required for resutts was 72 hours by the official method and 50 hours by alternative proposed method in liquid or solid medium. When the 3 method results were compared no significant differences were observed in almost all of the samples. However, the coefficient of variation related to pour-plate method results from 1:4 sample dilution was always lower than the others, mainly when compared with those obtained trom multiple tubes method. Therefore, the alternative pour-plate method is the most accurate one.

O cloreto de trifeniltetrazólio (TTC) foi empregado neste trabalho visando padronização de método alternativo para o teste de limite microbiano em medicamentos e cosméticos, com redução no tempo de análise, além de possibilitar a aplicação da técnica de semeadura em profundidade em amostras hidroinsolúveis com opacidade e carga contaminante menor que 102 UFC/g (mL).Na técnica de tubos múltiplos, a aplicação do corante não possibilitou a redução do tempo de incubação, pois a sensibilidade para detecção de suspensão bacteriana por meio de coloração do trifenilformazano foi de 108 UFC/mL após 1 hora de reação e 107 UFC/mL após 4 horas, suspensões estas visivelmente turvas. A adição de glicose e ácido glutâmico no meio de caseina-sola, nas concentrações de 0,5% e 0,1%, respectivamente, não aumentou a sensibilidade da reação. O polissorbato 20, adicionado ao meio de cultura, líquido ou sólido, promoveu a difusão do trifenilformazano das células para o meio, fator que não favorece a visualização da massa celular no meio líquido e nem das colônias. A concentração mínima de TTC que resultou em coloração máxima no meio líquido foi de 500 e 1000 &#181;g/mL, dependendo da espécie bacteriana. Entretanto, suspensões bacterianas com 102, 10 e 1 UFC/mL testadas, pela técnica de tubos multiplos, com adição do corante na forma de solução após 20 horas em meio de caseina-soja, forneceram resultados equivalentes, quanto ao número de tubos positivos e negativos frente às duas concentrações. A comparação desta técnica alternativa com a da subcultura demonstrou ser o tempo de 20 horas de incubação insuficientes para detecção pelo TTC, quando inoculadas alíquotas de 1 mL de suspensão com número da ordem de 1 UFC/mL Na técnica de semeadura em profundidade, após incubação de 48 horas, a coloração das colônias bacterianas foi efetuada cobrindo-se a superfície do meio de cultura com 5 mL de ágar a 1,0% contendo TTC. A concentração de 0,025% do corante promoveu a coloração das colônias após 1 hora. Entretanto, na presença de suspensão de hidróxido de alumínio a 6% a concentração mínima eficaz de TTC foi de 0,1%. Para leveduras em meio líquido essa concentração foi de 2.500 &#181;/mL no meio neutralizado. Em meio sólido, na presença de material opalescente, a concentração eficaz foi de 2,0%, com o TTC adicionado na dispersão de ágar em tampão pH 7,5. Amostras de produtos comercializados com característica opalescente foram submetidas à análise pelo método de tubos múltiplos com sub-cultura e pela revelação com TTC. Simultaneamente as mesmas foram testadas pelo método de semeadura em profundidade, inoculando-se 2 mL das diluições 1:4 e 1:10 com revelação das colônias com o corante. O tempo para obtenção dos resultados foi de 72 horas pelo método oficial e de 50 horas pelo método com TTC, tanto em meio líquido como em sólido. A comparação dos dados obtidos pelos 3 métodos não resultou em diferença significativa para a maioria das amostras testadas. Entretanto, os coeficientes de variação foram sempre menores para o método de semeadura em profundidade com a amostra diluída de 1:4, indicando maior precisão deste sobre os outros, principalmente em relação ao número mais provável. / The conventional methods for bioburden assessment are restrictive when applied to evaluate microbiological quality of pharmaceuticals and cosmetics. Triphenyltetrazolium chloride (TTC) was applied in this standartization study as alternative method for microbial limit test of pharmaceuticals and cosmetics. This method aimed at to reduce the assay time by conciliating the pour-plate technique analysis low bioburden of samples containing opacifiers. The application of this dye in the multiple tubes technique did not reduce incubation time, because the bacterial suspension sensitivity to triphenylformazan colour was 108 CUF/mL to 1 hour reaction and 107 CUF/mL to 4 hours. Suspensions with these bioburden are clearly turbid. The addition of 0.5% glicose and 0.1% glutamic acid to soybean-casein digest medium did not increase the reaction sensitivity. The addition of polissorbate 20 to solid ar liquid media promotted the difusion of triphenylformazan trom the cells to media; it rendered difficult of cellular mass detection in liquid medium and as much as colonies. The minimum concentration of TTC that produced maximum colour in liquid medium was 500 and 1,000 &#181;g/mL, depending on the bacteria. However, bacterial suspensions with 102 CUF/mL tested by multiple tubes technique with 20 hours of incubation and by microbial growth detection after TTC addition provided equivalent numbers of positive and negative tubes with both concentrations. The comparison of this alternative method with that of sub-culture proved that 20 hours of microbial growth is not enough to be detected by TTC, when 1 mL of 1 CUF/mL suspension was planted. The dying of bacterial colonies was performed by covering the medium surface with 5 mL of 1% agar with TTC, molten at 48 C. The concentration of TTC that colored colonies in 1 hour was 0.025%. However, in presence of 6% aluminium hidroxide suspension the minimum effective concentration was 0.1%. The concentration required to detect yeasts in liquid media was 2,500 &#181;g/mL in neutral pH condition. To detect yeast colonies in opalescent solid medium effective TTC concentration was 2%; in this case the dye was added to agar dispersed in 7.5 phosfate buffer. Samples of marketed produtcts were analysed through multiple tubes method with sub-culture and by TTC addition. Same samples were tested through pour-plate technique by inoculating 2 mL of 1:4 and 1:10 dilutions. The time required for resutts was 72 hours by the official method and 50 hours by alternative proposed method in liquid or solid medium. When the 3 method results were compared no significant differences were observed in almost all of the samples. However, the coefficient of variation related to pour-plate method results from 1:4 sample dilution was always lower than the others, mainly when compared with those obtained trom multiple tubes method. Therefore, the alternative pour-plate method is the most accurate one.

Thesis (MSc Food Sc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The agricultural sector of South Africa is currently facing a serious water crisis. The decreased availability of water as a result of climate change and the constantly growing population has left many farmers increasingly dependant on surface water as primary source of irrigation. Urbanisation along with out-dated and insufficient wastewater treatment works have all contributed to polluting large volumes of these resources. Consequently, many farmers have been forced to use irrigation water, not only of poor quality, but often water which has been polluted with untreated sewage. As a result, this project aimed at investigating the link between the quality of irrigation water and the impact on the safety of fresh produce. A base-line of the microbial load at three sites along the Plankenburg and Eerste Rivers was established using standard microbial methods for the detection of indicator organisms such as total and faecal coliforms, Escherichia coli and Enterococci as well as potential pathogens that included Salmonella, Listeria, Staphylococcus, endosporeformers and aerobic colony counts. Chemical parameters such as pH, alkalinity, conductivity and chemical oxygen demand (COD) were also monitored, but were not correlated to microbial pollution levels in the rivers. High faecal coliform and E. coli concentrations, ranging from 310 to 7 x 106 cfu.100 mL-1 and 230 to 7 x 106 cfu.100 mL-1, respectively, were detected. The recommended irrigation water guidelines of ≤1 000 (WHO, 1989) and ≤4 000 cfu.100 mL-1 (DWAF, 2008) for faecal coliforms and E. coli were exceeded, indicating faecal pollution and thus a high health risk. This health risk was confirmed when potential pathogens such as Aerococcus viridans, Klebsiella, Listeria monocytogenes and Salmonella typhimurium were detected at all three sites. The carryover of organisms from rivers to produce (green beans and grapes) was investigated by comparing the microbial population of the Plankenburg and Eerste Rivers to the population recovered from irrigation water and the surface of fresh produce. Faecal coliforms, E. coli, Aerococcus viridans, Enterobacter aerogenes, Klebsiella, L. innocua, L. grayi, L. monocytogenes and Staphylococcus aureus were detected in all three sample types, indicating a similarity between the microbial populations found in the river, the irrigation water and produce. Thus, the transfer of potential pathogens from the rivers to produce is a strong possibility. The build-up of organisms on the surface of green beans as a result of multiple irrigations was also confirmed by an increase in faecal coliform concentrations from initial concentrations of none detected to 44 000 cfu.100 mL-1 over a 10 day irrigation period. Finally, microbial source-tracking techniques including multi-antibiotic resistance (MAR) profiling, and the API 20E classification system were used to determine genotypic and phenotypic characteristics of 92 faecal isolates (from irrigation water and produce) and 13 reference strains. Numerical classification systems was used to classify the 105 faecal isolates according to the degree of similarity between the genotypic and phenotypic characteristics of the 105 isolates. A high degree of similarity indicates a high probability that isolates originate from the same strain and therefore from the same source, thereby confirming the transfer of organisms Faecal isolates (93 and 98%, respectively) were found to be resistant to Vancomycin at both the 5 and 30 μg concentrations. The majority of isolates presented some resistance to Erythromycin (15 μg) and Ampicillin (25 μg), with 82% of isolates presenting an inhibition zone ≤4 mm. Isolates were sensitive towards Ciprofloxacin (1 and 5 μg), Ofloxacin (15 μg), Ceftriaxone (30 μg) and Cefotaxime (5 μg), which were able to inhibit the growth of 79.8, 93.3, 79.8, 88.5 and 71.2% of the isolates, respectively. The 13 medical reference strains all presented different genotypic and phenotypic characteristics and thereby indicated a high degree of variability between isolates from the same species. Finally, 35% of the isolates could be grouped together based on similar genotypic and phenotypic characteristics, therefore, more than a third of the faecal isolates obtained from the surface of the fresh produce was as a result of faecal contaminants in the irrigation water. It could therefore be concluded that a health risk is associated with the water from the Plankenburg and to a lesser extent, Eerste River when used as source of irrigation, thereby risking the transfer of potentially harmful organisms, present in the rivers as result of faecal pollution, to the surface of fresh produce. / AFRIKAANSE OPSOMMING: Suid-Afrika stuur tans af op 'n dreigende water krisis. Klimaatsverandering tesame met 'n spoedig groeiende bevolking het gelei tot 'n aansienlike vermindering in die land se varswaterbronne terwyl veranderende reënvalpatrone daartoe bygedra het dat talle boere al hoe meer afhanklik geword het van oppervlakvarswaterbronne as hul hoof-besproeïngsbron. Verstedeliking, armoede asook verouderde en onvoldoende infrastrukture het egter bygedra tot die besoedeling van baie van hierdie oppervlakvarswaterbronne. Gevolglik is meeste boere genoodsaak om klaar te kom met besproeïngswater van, nie net onaanvaarbare mikrobiese kwaliteit nie, maar dikwels water wat gekontamineer is met onbehandelde riool. Hierdie studie was gevolglik daarop gemik om die impak van die mikrobiologiese kwaliteit van besproeïngswater op die veiligheid van vars groente en vrugte te bepaal. Standaard mikrobiologiese metodes vir die bepaling van indikator organismes soos totale en fekale kolivorms, E. coli en enterococci asook potensiële patogene wat Salmonella, Listeria en Staphylococcus insluit, was gebruik om die mikrobiese lading by drie verskillende punte (P1, P2 en P3) in die Plankenburg en Eerste Rivier te bepaal. Chemiese parameters soos pH, alkaliniteit, konduktiwiteit en Chemiese Suurstof Behoefte was ook bepaal maar geen korrelasie kon tussen hierdie eienskappe en die mikrobiese besoedelingsvlakke getref word nie. Hoë konsentrasies fekale kolivorms en E. coli wat onderskeidelik vanaf 3.1 x 102 tot 7 x 106 kve.100 mL-1 en 2.3 x 102 tot 7 x 106 kve.100 mL-1 gestrek het en gereeld die voorgeskrewe riglyne van onderskeidelik ≤1 000 (WHO, 1989) en ≤4 000 kve.100 mL-1 (DWAF, 2008) oorskry het, was by al drie punte gevind. Hierdie resultate het gedui op fekale besoedeling wat gevolglik met 'n hoë gesondheidsrisiko geassosieer kon word. Hierdie risiko was bevestig deur die teenwoordigheid van talle potensiële patogene, soos Aerococcus viridans, Klebsiella, Listeria monocytogenes en Salmonella typhimurium, wat vanaf al drie punte geïsoleer was. Die oordrag van organismes vanaf die besoedelde riviere na vars vrugte en groente (groen bone en druiwe) was bepaal deur die mikrobiese lading in die Plankenburg en Eerste Rivier te vergelyk met dié verkry vanuit die besproeïngswater en vanaf groen bone wat besproei was met hierdie water. Fekale kolivorms, E. coli, Aerococcus viridans, Enterobacter aerogenes, Klebsiella, L. innocua, L. grayi, L. monocytogenes en Staphylococcus aureus was vanaf al drie die monster tipes geïsoleer. Hierdie resultate het gedui op eenderse mikrobiese populasies in al drie bronne en het daarom die moontlike oordrag van patogene bevestig. Die opbou van organismes as gevolg van veelvuldige besproeïngsessies aan die oppervlak van groen bone was bevestig deur die toename in fekale kolivorm konsentrasie vanaf 'n begin telling van nul tot 'n maksimum konsentrasie van 44 000 kve.100 mL-1. Laastens was mikrobiologiese bron naspeurbaarheidstegnieke soos multi-antibiotika weerstandbiedende profiele en die API 20E klassifikasie sisteem gebruik om individuele genotipe en fenotipe profiele van die 105 fekale isolate saam te stel. Numeriese klassifikasie sisteme was gebruik om die isolate op grond van ooreenkomste tussen hul individuele fenotipiese en genotipiese karaktereienskappe te groeppeer. 'n Hoë mate van ooreenkomstigheid sal dan daarop dui dat isolate van dieselfde besoedlingsbron afkomstig is en gevolglik die oordrag van organismes vanaf besproeïngswater na vrugte en groente bevestig. Onderskeidelik 93 en 98% van die fekale isolate het daarop gedui om weerstandbiedend te wees teen beide 5 en 30 μg Vancomycin. Die meerderheid isolate (82%) het ook 'n mate van weerstand teenoor Erythromycin (15 μg) en Ampicillin (25 μg) getoon met inhibisie sones ≤4 mm. Isolate was ook sensitief teenoor Ciprofloxacin (1 en 5 μg), Ofloxacin (15 μg), Ceftriaxone (30 μg) en Cefotaxime (5 μg). Hierdie antibiotikums was in staat om die groei van onderskeidelik 79.8, 93.3, 79.8, 88.5 en 71.2 % van die isolate te inhibeer. Alhoewel resultate 'n hoë mate van variasie tussen isolate van dieselfde spesie getoon het was dit nogtans moontlik om 35% van die isolate saam te groeppeer op grond van ooreenstemmende genotipe en fenotipe profiele. Meer as 'n derde van die fekale isolate wat vanaf die oppervlakte van die groente en vrugte geïsoleer was, was afkomstig vanaf fekale besmetting in die besproeïngswater. Die oordrag van potensieël patogene organismes vanaf besoedelde riviere na vars vrugte en groente tydens besproeïng was sodoende bevestig. 'n Hoë gesondheidsrisiko was gevolglik gekoppel aan die gebruik van water vanaf die Plankenburg Rivier, en in 'n minder mate die Eerste Rivier, as bron van besproeïngswater. / Water Research Commission / National Research Foundation

Norovirus (NoV) is a significant cause of gastroenteritis globally, and the consumption of oysters is frequently linked to outbreaks. Depuration is the principle means employed to reduce levels of potentially harmful agents or toxins in shellfish. The aim of this thesis is to construct mathematical models which can describe the depuration dynamics of water-borne pathogens, and specifically examine the dynamics of NoV during depuration for a population shellfish. Legislation is currently under consideration within the EU by the Directorate-General for Health and Consumers (DG SANCO) to limit the maximum level of NoV that consumers are exposed to via this route. Therefore it is important to the utility of the thesis that any models constructed should incorporate control measures which could be used to implement minimum NoV levels. Doing so allowed calculation of minimum depuration times that would be required to adhere to the control measures incorporated into the models. In addition to modelling the impact on pathogens during the depuration, we wished to gain some insight into how the variability, and not just the mean levels, of water-borne pathogens can be as important with respect to the length of depuration required to minimise any food safety risks to the consumer. This proved difficult in the absence of any data sets that can be used to calculate variability measures, as little data is currently available to inform these values for NoV. However, our modelling techniques were able to calculate an upper limit on the variability of water-borne pathogens that can be well approximated by lognormal distributions. Finally we construct a model which provided linkage between the depuration process and the accretion of pathogens by shellfish while still within farming waters. This model proposed that the pulses of untreated waste waters released by sewage treatment works due to high levels of rainfall would be transmitted into shellfish whilst filter-feeding.