Dissertação composta por 2 artigos. / INTRODUÇÃO E OBJETIVOS ─ Nos produtos cárneos industrializados cozidos a maior parte da microbiota é inativada pela aplicação de calor em processo que simula uma pasteurização. Esses produtos também recebem aditivos em sua composição que geram efeito inibidor no crescimento de determinados grupos de microrganismos. Com a microbiota patogênica reduzida a níveis aceitáveis, em amostras embaladas a vácuo ou em atmosfera modificada, criam-se condições para o crescimento de bactérias ácido láticas (BAL), descritas como microrganismos Gram positivos, não formadores de esporos, catalase negativa, desprovidos de citocromos, anaeróbios facultativos, fastidiosos, mesofílicos, ácidos tolerante, com metabolismo estritamente fermentativo, que produzem ácido láctico como o principal produto final da fermentação de carboidratos. O desenvolvimento desses microrganismos gera aroma, sabor e aspecto indesejável nos produtos cárneos. Tendo em vista estas considerações, o objetivo geral desse trabalho foi avaliar a possibilidade da inibição do crescimento de BAL em amostras de presunto fatiado embalado a vácuo, pela aplicação de bacteriocinas. Os objetivos específicos incluíram: a quantificação e identificação das espécies de BAL predominantes nas amostras de presunto cozido fatiado embalado a vácuo; o estudo dos ingredientes que compõem a formulação do presunto cozido combinados com nisina e pediocina, realizados em caldo Man Rogosa e Sharp (MRS) pela aplicação da estratégia de planejamento experimental aliada à microbiologia preditiva na avaliação do crescimento do Lactobacillus sakei; a validação dos resultados obtidos nos caldos MRS na matriz alimentar a partir da elaboração de três formulações envolvendo a utilização da nisina e uma formulação controle, submetidas a análises físico-químicas e microbiológicas durante a vida de prateleira do produto. MÉTODOS ─ Inicialmente foi realizada a quantificação das BAL totais presentes, para 4 lotes semanais de presunto cozido fatiado embalado a vácuo, nos tempos de 1 e 45 dias de armazenamento a 4 C e 8 C. Paralelamente foi realizada a identificação fenotípica dos gêneros presentes; adicionalmente, no tempo de 45 dias foram realizadas análises moleculares do gene 16S rRNA para a identificação das espécies predominantes, por meio da qual selecionou-se a espécie Lactobacillus sakei. Posteriormente, utilizou-se uma estratégia sequencial de planejamento experimental, que objetivou estudar a inibição do crescimento celular da espécie Lactobacillus sakei em caldo MRS, aplicando-se um Planejamento Fatorial Fracionário 26-2 com as variáveis cloreto de sódio, tripolifosfato de sódio, lactato de sódio, eritorbato de sódio e as bacteriocinas nisina e pediocina. Adicionalmente foi realizado um Delineamento Composto Central Rotacional (DCCR) 22 objetivando otimizar a aplicação de nisina e pediocina por meio da redução das concentrações aplicadas que apresentassem eficiência semelhante, considerando os altos custos desses ingredientes. As respostas estudadas em ambos delineamentos experimentais foram os parâmetros preditos pelos ajuste das curvas de crescimento ao Modelo de Gompertz Modificado, a citar a população máxima atingida (A), velocidade específica máxima de crescimento () e a duração da fase lag (). Para a validação dos resultados obtidos na etapa de estudo em caldo MRS, foram elaboradas três formulações teste (com diferentes concentrações de nisina) e uma controle (sem adição de nisina) para o acompanhamento da vida de prateleira do produto por meio de análises físico-químicas e microbiológicas. PRINCIPAIS RESULTADOS ─ Foram observadas contagens médias de 1,98 log UFC.g-1 no tempo 1 dia; 7,59 log UFC.g-1 a 4 C e 8,25 log UFC.g-1 a 8 C no tempo 45 dias para a contagem total de BAL. As espécies predominantemente identificadas no tempo de 45 dias foram o Lactobacillus curvatus e o Lactobacillus sakei. Na etapa de estudo em caldo MRS, os resultados do Planejamento Fatorial Fracionário evidenciaram que o tripolifosfato de sódio, a nisina e a pediocina, foram as variáveis com efeito estatisticamente significativo (p ≤ 0,05) na inibição do crescimento do Lactobacillus sakei. No Delineamento Composto Central Rotacional, a nisina foi a variável que apresentou efeito significativo na inibição do crescimento do Lactobacillus sakei, indicando a possibilidade de redução na concentração aplicada para níveis inferiores ao indicado pela legislação brasileira (12,5 mg/Kg), com efeito semelhante na inibição. A validação dos estudos em caldo MRS pela aplicação das quantidades reduzidas de nisina (indicadas pelo DCCR) na matriz alimentar, mostrou que a quantidade mínima aplicada (0,001%) apresentou um efeito semelhante na inibição das BAL em relação à quantidade máxima aplicada (0,013%); sendo em ambos os casos eficiente quando comparada à amostra controle. DISCUSSÃO E CONCLUSÃO ─ De acordo com os resultados obtidos, verificou-se que os principais gêneros de BAL envolvidos na deterioração das amostras de presunto cozido fatiado embalado a vácuo, armazenadas durante 45 dias a 4 C e 8 C, foram o Lactobacillus curvatus e Lactobacillus sakei. A contagem de BAL totais após 45 dias mostrou-se significativamente influenciada pela temperatura de armazenamento, pois as contagens foram maiores para as amostras armazenadas a 8 C quando comparadas às armazenadas a 4 C. Dentre os ingredientes comumente adicionados na formulação do presunto cozido, o tripolifosfato de sódio mostrou efeito significativo na inibição do Lactobacillus sakei nos estudos em caldo MRS. De forma semelhante, a nisina e a pediocina mostraram efeito significativo na redução das contagens do microrganismo estudado quando avaliados pela aplicação de um Planejamento Fatorial Fracionário. No Delineamento Composto Central Rotacional envolvendo as variáveis nisina e pediocina com faixas de estudos redefinidas, a nisina apresentou efeito significativo na inibição do Lactobacillus sakei, quando aplicada em concentrações inferiores ao estipulado pela legislação brasileira. Os resultados obtidos no Delineamento Composto Central Rotacional foram validados pela aplicação das quantidades otimizadas de nisina na matriz alimentar, que mesmo na concentração mínima (0,001%) mostrou um efeito similar ao obtido pela aplicação da concentração máxima (0,013%). A contagem de BAL e bactérias aeróbias mesófilas (BAM) totais mostrou-se inferior para as amostras contendo nisina quando comparada com a amostra controle, em torno de 2 a 3 ciclos logarítmicos para as BAL, e 3 ou 4 ciclos logarítmicos para as BAM; em relação ao pH, o mesmo manteve-se praticamente inalterado até o final da vida de prateleira (60 dias), enquanto o pH da amostra controle diminuiu de forma significativa. De um modo geral constatou-se que o presunto cozido fatiado embalado a vácuo é um produto com condições favoráveis ao desenvolvimento de BAL deteriorantes, a citar as espécies Lactobacillus curvatus e Lactobacillus sakei, e que dentre as bacteriocinas estudadas, a nisina apresentou efeito na inibição do Lactobacillus sakei nos estudos em caldo MRS e também na inibição das BAL deteriorantes totais no presunto cozido fatiado embalado a vácuo, mesmo em concentrações inferiores ao indicado pela legislação brasileira. / INTRODUCTION AND AIMS ─ In processed cooked meat products most of the microbiota is inactivated by heat application process which simulate a pasteurization. These products also receive additives in composition that generate inhibitory effect on the certain microorganisms groups growth. With the reduce of pathogenic microorganisms on vacuum packed or modified atmosphere samples, conditions are created for the lactic acid bacteria (LAB) growth, as described Gram positive, non-spore forming, catalase negative, devoid of cytochromes, facultative anaerobic, fastidious, mesophilic, acid tolerant, strictly fermentative metabolism that produce lactic acid as the major end product of fermentation of carbohydrates. The development of these microorganisms causes aroma, flavour and appearance undesirable in meat products. In view of these considerations, the aim of this work was to evaluate the LAB inhibition growth in samples of sliced vacuum-packed cooked ham, by the application of bacteriocins. Specific aims included: the quantification and identification of species of predominate LAB in the samples of sliced vacuum-packed cooked ham; the study of the ingredients of the formulation of cooked ham combined with nisin and pediocin conducted in Man Rogosa and Sharp (MRS) broth using a strategy of experimental design combined with predictive microbiology on evaluation of Lactobacillus sakei growth; and a validation of results obtained in MRS broths on food matrix by the elaboration of three formulations involving the use of nisin and a control formulation, which were submitted for microbiological and physico-chemical analysis during the shelf life of the product. METHODS ─ Initially, the quantification of total LAB present was performed for 4 weekly batches of sliced vacuum-packed cooked ham at time 1 and 45 days of shelf life storage at 4 C and 8 C. Simultaneously was performed phenotypic identification of the genera present; additionally, at the time of 45 days of storage, was carried out a molecular analyze of 16S rRNA gene for identification of the predominant species, whereby selected the specie Lactobacillus sakei. Subsequently, a strategy of sequential experimental design was performed to evaluate the Lactobacillus sakei cell growth inhibition in MRS broth, applying a Fractional Factorial Design 26-2 with the variables sodium chloride, sodium tripolyphosphate, sodium lactate, sodium erythorbate and the bacteriocins nisin and pediocin. The statistically significant variables in relation to Lactobacillus sakei growth inhibition were selected and a Central Composite Rotatable Design (CCRD) 22 was performed to optimize the application of nisin and pediocin by reducing the concentration applied to present similar efficiency, considering the high cost of these ingredients. The responses studied in both experimental design were the parameters logarithmic increase of population (A), maximum specific growth rate (µ) and lag phase duration (), obtained by the adjustment of Modified Gompertz predictive model. For validation of the results obtained in MRS broth, three test formulations were prepared (with different concentrations of nisin) and a control (without addition of nisin) for monitoring the shelf life of the product by physico-chemical and microbiological analyzes. MAIN RESULTS ─ Mean counts around log 1.98 CFU.g-1 at time 1 day, log 7.59 CFU.g-1 at 4C, and log 8.25 CFU.g-1 at 8C on time 45 days, was detected for the total LAB. The species predominantly identified on time 45 days were Lactobacillus curvatus and Lactobacillus sakei. In step of study in MRS broth, the results of the Fractional Factorial Design showed the sodium tripolyphosphate, nisin and pediocin like variables with statistically significant effects in Lactobacillus sakei growth inhibition. In the Central Composite Rotatable Design, nisin was the variable that had a significant effect in the Lactobacillus sakei growth inhibition, indicating that it may be reduced to smaller amounts than indicated by Brazilian law (12.5 mg/Kg) with similar effect on inhibition. The validation of MRS broth studies for applying the reduced amounts of nisin (indicated by the CCRD) in the food matrix, showed that the minimum amount applied (0.001%) presented a similar effect on LAB growth inhibition related to the maximum quantity applied (0.013%); being efficient in both cases when compared to the control sample. DISCUSSION AND CONCLUSION ─ From the results obtained, was possible to conclude that the LAB comprise the microbiota of sliced vacuum-packed cooked ham and the Lactobacillus curvatus and Lactobacillus sakei were the major species that cause deterioration on the samples stored at 4 C and 8 C for 45 days. Total LAB counts after 45 days was significantly affected by storage temperature, because the counts were higher for samples stored at 8 C compared to the samples stored at 4 C. Among the ingredients added in the formulation of cooked ham, sodium tripolyphosphate showed significant effect in Lactobacillus sakei inhibition in MRS broth. Similarly, nisin and pediocin showed significant effects on microorganism reducing, evaluated by applying a Fractional Factorial Design. The Central Composite Rotatable Design involving the variables nisin and pediocin with redefined ranges of concentrations showed that nisin presented significant effect on the Lactobacillus sakei growth inhibition, when applied in smaller amounts in comparison to the stipulated by Brazilian law. The results obtained in Central Composite Rotatable Design was validated by applying the optimized quantities of nisin in food matrix, that even when applying minimal concentration (0.001%) showed a similar effect to that obtained by applying the maximum concentration (0.013%). The count of LAB and mesophilic aerobic bacteria (MAB) total were lower for the samples containing nisin than control sample, approximately 2 to 3 log cycles for LAB, and 3 or 4 log cycles for MAB, while the pH was maintained until the end of shelf life (60 days); at the same time the pH of the control sample had a significant decrease. Altogether the sliced vacuum-packed cooked ham is a favorable product to the LAB spoilage development, principally to the species Lactobacillus curvatus and Lactobacillus sakei, and that among the studied bacteriocins against the inhibition of LAB, nisin showed effect on the Lactobacillus sakei growth inhibition by studies in MRS broth, and also on the LAB spoilage growth inhibition in sliced vacuum-packed cooked ham, even in smaller quantities than indicated by Brazilian law. / 5000

No description available.

No description available.

Phylogenetic hybridization and phenotypic fingerprinting were applied to the analysis of bacterial communities in wastewater treatment systems. These approaches were aimed at (i) developing monitoring tools able to foresee operational problems, and (ii) providing the rationale to optimize the operation of bioreactors. The work presented is intended to first describe the community found in two reactors treating pulp and paper mill effluent, and second evaluate the possibilities of these techniques with respect to the development of new monitoring tools. / Phylogenetic membrane hybridization showed that the bacterial communities were dominated by Alpha and Beta Proteobacteria, a structure probably linked to the low F:M ratio. Other important factors determining the community structure were the proportion of COD in the high molecular weight fraction, the sludge age, phosphate addition, and the concentration of specific compounds (alcohols, phenols, volatile fatty acids) in the influent. The community structure partly determined the sludge characteristics demonstrating its potential value in the assessment of reactor performance. The results obtained by phylogenetic membrane hybridization suggest that the probes used in a monitoring tool would not need to be targeted to the species level to provide relevant information. However, they also suggest that the technique is more sensitive to changes in population density as opposed to changes in bacterial metabolism. / Phenotypic fingerprinting measured a smaller difference between the communities of the two reactors studied than what was measured by phylogenetic membrane hybridization. However, differences in heterotrophic activities observed between the two communities were linked to differences in influent composition.

Although increased genome sequencing e orts have increased our understanding of genomic variability within many bacterial species, there has been limited application of this knowledge towards assessing current molecular typing methods and developing novel molecular typing methods. This thesis reports a novel in silico comparative genomic framework where the performance of typing methods is assessed on the basis of the discriminatory power of the method as well as the concordance of the method with a whole-genome phylogeny. Using this framework, we designed a comparative genomic ngerprinting (CGF) assay for Listeria monocytogenes through optimized molecular marker selection. In silico validation and assessment of the CGF assay against two other molecular typing methods for L. monocytogenes (multilocus sequence typing (MLST) and multiple virulence locus sequence typing (MVLST)) revealed that the CGF assay had better performance than these typing methods. Hence, optimized molecular marker selection can be used to produce highly discriminatory assays with high concordance to whole-genome phylogenies. The framework described in this thesis can be used to assess current molecular typing methods against whole-genome phylogenies and design the next generation of high-performance molecular typing methods from whole-genome sequence data. / xiii, 100 leaves : ill. ; 29 cm

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Wine production involves complex interactions between a variety of yeasts and bacteria. Conventional microbiological methods can be used to identify the different microorganisms present in wine, but prove to be time-consuming and certain microbial species may not grow on synthetic isolation media. The aim of this study was to evaluate the microbial population present in two South African red wines, Pinotage and Merlot, as well as five spoilt commercial South African wines by using a non-culturable approach, polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE). The results from the non-culturable approach were compared to conventional platings. Unique PCR-based DGGE fingerprints were obtained for the Bacteria and yeasts present in the South African Pinotage and Merlot wines. Using yeast specific primers the Pinotage wine showed the presence of non-Saccharomyces yeasts at the beginning of the alcoholic fermentation, while Saccharomyces cerevisiae was present until the completion of the malo-lactic fermentation (MLF). This yeast was also identified during both the alcoholic fermentation and MLF of the Merlot wine using PCR-based DGGE and conventional plating. Using Bacteria specific primers, Lactobacillus plantarum and Lactobacillus sp. was identified in the Pinotage wine using PCR-based DGGE, while Lactobacillus brevis were isolated from Merlot wine using conventional platings. Although the presence of S. cerevisiae is expected during wine fermentation, the presence of this microbe in bottled wine could lead to spoilage. Four of the spoilt commercial wine samples (RW1, RW2, RoW1 and WW1) were found to be spoilt by S. cerevisiae, while a fifth wine sample (RW3) was found to be spoilt by an Acetobacter sp. using PCR-based DGGE. Members of the family Enterobacteriaceae were identified from all the wines using PCR-based DGGE, while Enterobacter sakazakii was identified from RW1 using PCR-based DGGE and conventional plating. The members of the family Enterobacteriaceae could possibly have contributed to the spoilage of the wine by producing undesirable secondary metabolites. PCR-based DGGE proved to be an alternative to conventional microbiological methods for the identification of the microbial species in South African red grape must and wine. This method also proved to be useful in the identification of spoilage microbes in spoilt commercial South African wines. / AFRIKAANSE OPSOMMING: Die produksie van rooi wyn behels komplekse interaksies tussen ‘n verskeidenheid van giste en bakterieë. Konvensionele mikrobiologiese metodes kan gebruik word om die verskillende mikro-organismes wat in rooi wyn teenwoordig is te identifiseer, maar dit blyk tydrowend te wees, terwyl sekere mikro-organismes nie groei op sintetiese media nie. Die doel van hierdie studie was om die mikrobiologiese populasie wat in twee Suid-Afrikaanse rooi wyne, Pinotage en Merlot, en vyf bederfde kommersiële wyne teenwoordig is, te evalueer met die gebruik van ‘n kultuur-onafhanklike benadering, polimerase ketting-reaksie (PKR)-gebaseerde denaturerende gradiënt jel elektroforese (DGJE). Die resultaat van die kultuur-onhafhanklike benadering was vergelyk met konvensionele uitplating tegnieke. Unieke, ongeëwenaarde PKR-gebaseerde DGGE vingerafdrukke was verkry van die Bakterieë en giste aanwesig in die Pinotage en Merlot wyne. Deur gebruik te maak van gis-spesifieke inleiers het die Pinotage wyn die teenwoordigheid van nie-Saccharomyces giste getoon, terwyl Saccharomyces cerevisiae teenwoordig was tot en met die afhandeling van die appel-melksuur gisting (AMG). Hierdie gis is ook geïsoleer gedurende beide die alkoholiese gisting en AMG van die Merlot wyn deur gebruik te maak van PKR-gebaseerde DGGE en konvensionele uitplating tegnieke. Met Bakterieë-spesifieke inleiers, was Lactobacillus plantarum en Lactobacillus sp. geïdentifiseer in die Pinotage wyn deur gebruik te maak van PKR-gebaseerde DGGE, terwyl Lactobacillus brevis geïsoleer is uit Merlot wyn deur gebruik te maak van konvensionele uitplatings. Alhoewel die teenwoordigheid van S. cerevisiae verwag word gedurende wynfermentasie, kan die teenwoordigheid van hierdie mikrobe in gebottelde wyn tot bederwing lei. Vier van die bedorwe kommersiële wynmonsters (RW1, RW2, RoW1 en WW1) was bederf deur S. cerevisiae, terwyl ‘n vyfde wynmonster (RW3) bederf was deur ‘n Acetobacter sp. deur die gebruik van PKR-gebaseerde DGGE. Van al die wyne is lede van die Enterobacteriaceae familie geïdentifiseer deur gebruik gemaak te maak van PKR-gebaseerde DGGE, terwyl Enterobacter sakazakii geïsoleer is van RW1 met konvensionele uitplating. Die lede van die familie Enterobacteriaceae kon moontlik bygedra het tot die bederwing van die wyn deur ongewenste sekondêre metaboliete te produseer. PKR-gebaseerde DGGE bewys ‘n alternatief tot die konvensionele mikrobiologiese metodes vir die identifikasie van die mikrobiese spesies in Suid-Afrikaanse rooi druif mos en wyn te wees. Hierdie metode het ook die bruikbaarheid in die identifikasie van mikrobes wat kommersiële Suid-Afrikaanse wyne bederf, bewys.

O leite é uma das principais fontes de nutrientes essenciais ao crescimento, desenvolvimento e manutenção da saúde humana. No entanto, pode também ser um veículo de agentes tóxicos, causando sérios riscos à saúde em indivíduos que o consomem, em especial as crianças. Dentre os contaminantes de alimentos, as aflatoxinas se destacam, as quais são metabólitos secundários fúngicos relevantes na saúde humana e animal. Entre os análogos de aflatoxinas identificados até o momento, a aflatoxina B1 (AFB1) é a mais prevalente e a mais tóxica; quando ingerida por animais esta sofre biotransformação hepática convertendo-se parcialmente em aflatoxina M1 (AFM1), que é excretada no leite. Entretanto, estudos recentes tem demonstrado a presença de AFB1 em leite, discordando da literatura quanto à completa conversão desta em AFM1. Uma vez presente no leite, estas aflatoxinas podem resistir a maioria dos tratamentos para obtenção de derivados lácteos; portanto, podem estar presentes nos queijos e iogurtes. Neste trabalho, o objetivo foi avaliar a capacidade do Lactobacillus acidophilus isolado e em conjunto com prebióticos (inulina, oligofrutose, β-glucana e polidextrose) em reduzir a concentração de AFB1 e AFM1 e o efeito sobre a bioacessibilidade após digestibilidade em um modelo de digestão in vitro em leite contaminado artificialmente. Para tal, foi aplicado um delineamento experimental do tipo PlackettBurman para a avaliação do efeito de seis variáveis do processo (concentração de aflatoxina, tempo de incubação e concentração dos quatro prebióticos), um controle negativo (leite integral puro) e um controle positivo (leite integral fortificado com aflatoxina). Todos os ensaios com a adição do probiótico e prebióticos promoveram redução da AFB1 e AFM1 em leite, bem como da sua bioacessibilidade. Os níveis de redução variaram de 13,53 e 35,53% para AFB1 e 17,61 e 71,52% para AFM1. Quando comparada com o controle positivo a bioacessibilidade para AFB1 variou de 23,68 a 72,67% e 0% para AFM1 (100% de redução de bioacessibilidade). Para verificar a interação do probiótico e prebióticos com as aflatoxinas e possível modificação na estrutura proteica do leite, foi realizada espectroscopia na região do infravermelho por transformada de fourier (FTIR). Foi detectada alteração da região amida I (1700-1600cm-1) nos tratamentos adicionados do probiótico e prebióticos em relação ao tratamento contendo somente as aflatoxinas e leite, o que sugere a ocorrência de um deslocamento das aflatoxinas que estão ligadas a fração proteica do leite para a parede celular do probiótico. / Milk is a major source of essential nutrients for the growth, development and maintenance of human health. However, it can also be a vehicle for toxic agents, causing serious health risks in individuals who consume it, especially children. Among the food contaminants, aflatoxins stand out, which are important secondary fungal metabolites in human and animal health. Among aflatoxin analogues identified to date, aflatoxin B1 (AFB1) is the most prevalent and most toxic. When ingested by animals it undergoes hepatic biotransformation partially converting to aflatoxin M1(AFM1), which is excreted in the milk. However recent studies have demonstrated the presence of AFB1 in milk, disagreeing in the literature about the complete conversion of AFB1 to AFM1. Once present in milk, these aflatoxins can withstand most treatments to obtain dairy products; Therefore, may be present in cheeses and yogurts. The objective of this study was to evaluate the ability of Lactobacillus acidophilus alone and in conjunction with prebiotics (inulin, oligofrutose, β-glucan and polydextrose) to reduce the concentration of AFB1 and AFM1 and the effect on bioavailability after digestibility in a digestion model In vitro in artificially contaminated milk. For this, a Plackett-Burman experimental design was used to evaluate the effect of six process variables (aflatoxin concentration, incubation time and concentration of the four prebiotics),a negative control (pure whole milk) and a positive control (Whole milk fortified with aflatoxin). All probiotic and prebiotic assays promoted reduction of AFB1 and AFM1 in milk as well as its bioavailability. Reduction levels ranged from 13.53 and 35.53% for AFB1 and 17.61 and 71.52% for AFM1. When compared to the positive control the bioaccessibility for AFB1 ranged from 23.68 to 72.67% and 0% for AFM1 (100% reduction in bioaccessibility). To verify the interaction of probiotic and prebiotics with aflatoxins and possible modification in the protein structure of the milk, spectroscopy was performed in the infrared region by fourier transform (FTIR). It was detected alteration of the amide I region (1700-1600cm-1) in the probiotic and prebiotic treatments in relation to the treatment containing only aflatoxin and milk, suggesting the occurrence of aflatoxin displacement that is linked to the milk protein fraction to the cell wall of the probiotic.

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Bacteria identification plays an essential role in preventing health complications and saving patients' lives. The most widely used method to identify bacteria, the bacterial cultural method, suffers from long processing times. Hence, an effective, rapid, and non-invasive method is needed as an alternative. Raman spectroscopy is a potential candidate for bacteria identifi cation due to its effective and rapid results and the fact that, similar to the uniqueness of a human fingerprint, the Raman spectrum is unique for every material. In my lab at the University of Ottawa, we focus on the use of Raman scattering for biosensing in order to achieve high identifi cation accuracy for different types of bacteria. Based on the unique Raman fingerprint for each bacteria type, different types of bacteria can be identifi ed successfully. However, using the Raman spectrum to identify bacteria poses a few challenges. First, the Raman signal is a weak signal, and so enhancement of the signal intensity is essential, e.g., by using surface-enhanced Raman scattering (SERS). Moreover, the Raman signal can be contaminated by different noise sources. Also, the signal consists of a large number of features, and is non-linear due to the correlation between the Raman features. Using machine learning (ML) along with SERS, we can overcome such challenges in the identifi cation process and achieve high accuracy for the system identifying bacteria. In this thesis, I present a method to improve the identifi cation of different bacteria types using a support vector machine (SVM) ML algorithm based on SERS. I also present dimension reduction techniques to reduce the complexity and processing time while maintaining high identifi cation accuracy in the classifi cation process. I consider four bacteria types: Escherichia coli (EC), Cutibacterium acnes (CA, it was formerly known as Propi-onibacterium acnes), methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-sensitive Staphylococcus aureus (MSSA). Both the MRSA and MSSA are combined in a single class named MS in the classifi cation. We are focusing on using these types of bacteria as they are the most common types in the joint infection disease. Using binary classi fication, I present the simulation results for three binary models: EC vs CA, EC vs MS, and MS vs CA. Using the full data set, binary classi fication achieved a classi fication accuracy of more than 95% for the three models. When the samples data set was reduced, to decrease the complexity based on the samples' signal-to-noise ratio (SNR), a classi fication accuracy of more than 95% for the three models was achieved using less than 60% of the original data set. The recursive feature elimination (RFE) algorithm was then used to reduce the complexity in the feature dimension. Given that a small number of features were more heavily weighted than the rest of the features, the number of features used in the classifi cation could be signi ficantly reduced while maintaining high classi fication accuracy. I also present the classifi cation accuracy of using the multiclass one-versus-all (OVA) method, i.e., EC vs all, MS vs all, and CA vs all. Using the complete data set, the OVA method achieved classi cation accuracy of more than 90%. Similar to the binary classifi cation, the dimension reduction was applied to the input samples. Using the SNR reduction, the input samples were reduced by more than 60% while maintaining classifi cation accuracy higher than 80%. Furthermore, when the RFE algorithm was used to reduce the complexity on the features, and only the 5% top-weighted features of the full data set were used, a classi fication accuracy of more than 90% was achieved. Finally, by combining both reduction dimensions, the classi fication accuracy for the reduced data set was above 92% for a signifi cantly reduced data set. Both the dimension reduction and the improvement in the classi fication accuracy between different types of bacteria using the ML algorithm and SERS could have a signi ficant impact in ful lfiling the demand for accurate, fast, and non-destructive identi fication of bacteria samples in the medical fi eld, in turn potentially reducing health complications and saving patient lives.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnolgy, Durban University of Technology, 2007 xviii, 143 leaves / Activated sludge flocs are responsible for flocculation, settling and dewaterability. It is important to maintain the growth off loc-forming bacteria for efficient sludge settleability and compaction for good quality effluent. Filamentous bacteria on the other hand are believed to provide rigid support network or backbone upon which floc-forming bacteria adhere to form stable activated sludge flocs (Wilderer et al., 2002; Ramothokang et al., 2003). Filamentous bacteria can also be detrimental to the process when they outgrow floc-forming bacteria. Morphologically filamentous bacteria are at an advantage as they have higher outward growth velocity and can extend freely to bulk liquid substrate. Proliferation of filamentous bacteria causes foaming and bulking (Martins et al., 2004). Although chemical alleviation measures to circumvent bulking are present, they are symptomatic (Chang et al., 2004). Eikelboom (1975) developed the first identification keys for the classification of filamentous bacteria that is primarily based on morphological characteristics and microscopic examination. Although very useful, this type of identification has its limitations. For instance some filamentous bacteria can change morphology in response to changes in the environment and although some of them can be morphologically similar they may vary considerably in their physiology and taxonomy (Martins et al., 2004). A vast number of filamentous bacteria are still very poorly understood which could be due to the problems of cultivation due to their slow growing nature and maintenance of cultures (Rossetti et al., 2006). This limitation necessitates a molecular approach to resolve the taxonomy of filamentous bacteria as it is a culture-independent technique which is highly accurate. This project was undertaken to verify the identity of pure cultures of filamentous bacteria isolated previously through the application of molecular techniques. The 16S rDNA are conserved regions in bacterial cells and they can be extracted and specific nucleic acid fragments amplified. Denaturation gradient gel electrophoresis enabled the separation of fragments of identical length but different size and served as an indication of purity (Muyzer et al., 1993).