Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The development of the work plan proposed for this dissertation refers to the production of xanthan gum from sugarcane juice using the bacterium Xanthomonas campestris pv. campestris NRRL B-1459. Tests were conducted in 500 mL erlenmeyers flasks and in a 2.0-L reactor, in sucrose concentration range from 15 to 40 g/L, judiciously following the pre-established experimental designs, when the process variables and its levels were defined. Firstly, two varieties of sugarcane, RB 835486 and SP 791011 were tested in different production media, aiming at selecting the variety and the fermentative media whose K values (rate of consistence) and n (behavior rate) for the fermented must represented in a better way the condition of pseudoplastic fluid. In the sequence, two factorial designs in two levels and three variables defined the appropriate composition of the medium for later investigations related to the gum biosynthesis. However, the third design appears with two variables and three levels, rendering favorable an evaluation of the optimum point through surfaces generated for each response analyzed. Above all, an increase in the scale researched until then guided the execution of tests in reactor for checking, under controlled conditions, the growth of microorganisms, decrease of sucrose concentration and the product formation, the viscosity of fermented must, and the 1% solution of gum after recovery and purification. Tests in erlenmeyer flasks were carried out in stirring table at 120 rpm and 28ºC while in the reactor, the agitation, aeration, and pH maintained their values in 800 rpm, 0.5 vvm and 7.3, respectively. In that way, results found in this study indicate that the variety of sugarcane SP 791011 has remarkable potential for the production of xanthan gum and after execution of two designs was defined too the medium of production constituted by dilute sugarcane, fortified with four components for effecting the 3rd and 4th designs. Then, the feasibility of the production process of xanthan gum was attested in reactor, reaching 15.1 g/L of gum concentration, 0.629 g/Lh of productivity, 0.579 g.g-1 of conversion from substrate to product, 21509.9 cP of viscosity at 0.75 s-1 for 1% solution of xanthan gum. Gums produced were compared to a commercial sample and presented a similar spectroscopy near infrared region. / O desenvolvimento do plano de trabalho proposto para esta dissertação refere-se à produção de goma xantana a partir de caldo de cana utilizando a bactéria Xanthomonas campestris pv. campestris NRRL B-1459. Os ensaios foram conduzidos em erlenmeyers de 500 mL e em reator de 2,0 L, numa faixa de concentração de sacarose de 15 a 40 g/L, seguindo criteriosamente planejamentos experimentais pré-estabelecidos, uma vez definidas as variáveis de processo e seus níveis. Primeiramente, duas variedades de cana-de-açúcar, RB 835486 e SP 791011, foram testadas em diferentes meios de produção visando selecionar a variedade e os meios fermentativos cujos valores de K (índice de consistência) e n (índice de comportamento) para o mosto fermentado representaram melhor a condição de fluido pseudoplástico. Na seqüência, dois planejamentos fatoriais a dois níveis e três variáveis definiram a composição adequada do meio para posteriores investigações relacionadas à biossíntese da goma. Contudo, surge o 3o planejamento com duas variáveis a três níveis, propiciando uma avaliação do ponto ótimo através das superfícies geradas para cada resposta analisada. Sobretudo, um aumento na escala pesquisada até então direcionou a execução dos ensaios em reator para verificar, sob condições controladas, o crescimento de microrganismos, o decréscimo da concentração de sacarose, a formação de produto, a viscosidade do mosto fermentado e a da solução 1% da goma após recuperação e purificação. Os testes em erlenmeyers foram realizados em mesa agitadora a 120 rpm e a 28o C enquanto no reator a agitação, a aeração e o pH mantiveram seus valores em 800 rpm, 0,5 vvm e 7,3 respectivamente. Dessa forma, os resultados encontrados nesse estudo apontam a variedade de cana SP 791011 com potencialidade notável para a produção de goma xantana e definiu-se também após execução de dois planejamentos o meio de produção constituído por caldo de cana diluído fortificado com quatro componentes para efetuar o 3o e 4o planejamentos. Logo, a viabilidade do processo de produção da goma xantana foi atestada em reator, atingindo 15,1 g/L de concentração de goma, 0,629 g/Lh de produtividade, 0,579 g.g-1 de conversão de substrato a produto, 21509,9 cP de viscosidade a 0,75 s-1 para solução 1% de goma. As gomas produzidas foram comparadas a uma amostra comercial e apresentaram espectroscopia na região do infravermelho similares. / Mestre em Engenharia Química

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O Ãcido lÃtico à um composto com diversas aplicaÃÃes industriais, dos quais as indÃstrias quÃmica, farmacÃutica, de alimentos, de polÃmeros, tÃxtil e de curtume podem ser citadas alÃm de ser reconhecido como seguro pela Food and Drug Administration (FDA). Tem se tornado um importante monÃmero na indÃstria de plÃsticos, sendo polimerizado em plÃstico biodegradÃvel. Pode ser obtido industrialmente atravÃs de sÃntese quÃmica ou processo fermentativo. No entanto, à mais comumente produzido via processo fermentativo atravÃs de matÃrias-primas renovÃveis e resÃduos da agroindÃstria. O pedÃnculo do caju possui um alto valor nutricional em termos de vitaminas, sais minerais e aÃÃcares e estima-se que 88% de sua produÃÃo seja perdida devido sua alta perecibilidade sendo, portanto, um substrato em potencial para processos fermentativos. O objetivo deste trabalho foi a otimizaÃÃo da produÃÃo de Ãcido lÃtico via fermentaÃÃo submersa em meio contendo suco de caju clarificado como substrato utilizando o Lactobacillus casei NRRL B-442. A partir de dados experimentais sobre temperatura, pH, concentraÃÃes de substrato e sulfato de amÃnia inicial, foi realizado um estudo cinÃtico variando concentraÃÃes de substrato inicial de 20 a 60 g/L e mantendo a proporÃÃo ideal de 12% de sulfato de amÃnio em relaÃÃo aos aÃÃcares redutores totais iniciais. Os ensaios foram realizados em reator batelada CSTR de 1,0 L com 0,5 L de meio reacional. A temperatura foi de 37ÂC e o pH foi ajustado para 6,5 sendo controlado durante o processo. A partir dos dados experimentais, um modelo fenomenolÃgico foi desenvolvido e um programa computacional foi criado utilizando Linguagem Fortran 90. O modelo foi validado estatisticamente segundo o valor de F de Fisher. Com o modelo representativo do sistema, foi possÃvel realizar otimizaÃÃes em batelada e batelada alimentada para a fermentaÃÃo lÃtica. Os processos em batelada e batelada alimentada foram comparados entre si levando em consideraÃÃo a eficiÃncia final, a produÃÃo de Ãcido lÃtico e os custos com matÃria-prima e reagentes utilizados comparando com os custos de venda final do Ãcido lÃtico no mercado. Para a otimizaÃÃo em batelada, pÃde-se verificar que a fermentaÃÃo que apresentou melhores resultados foi a que partiu de um inÃculo com 0,3 g/L e concentraÃÃo de aÃÃcares redutores totais iniciais de 50 g/L finalizando o processo com 39,31 g/L de Ãcido lÃtico e apresentando uma eficiÃncia de 72,2%. Em relaÃÃo ao processo em batelada alimentada, conclui-se que a simulaÃÃo que apresentou melhores resultados foi a que partiu de um inÃculo de 0,3 g/L com 40 g/L de aÃÃcares redutores totais iniciais, uma vazÃo de 3 L/h e uma concentraÃÃo de suco de caju clarificado concentrado alimentado de 200 g/L obtendo 38,0 g/L de Ãcido lÃtico e uma eficiÃncia de 63,8%. Portanto, para a fermentaÃÃo lÃtica utilizando o Lactobacillus casei NRRL B-442 tendo o suco e caju clarificado como substrato, o processo em batelada foi o mais vantajoso / Lactic acid is a compound that has several industrial applications in the chemical, pharmaceutical, food, polymer, textile and tanning industries. In addition, lactic acid has being recognized as safe by the Food and Drug Administration (FDA). Lactic acid has become an important monomer in the plastic industry where it has been polymerized into biodegradable plastics. It can be obtained industrially by chemical synthesis or fermentation process. It is most commonly produced by fermentation process using raw materials and waste materials of agricultural source. Cashew apple has a high nutritional value in terms of vitamins, minerals and sugars and it is estimated that 88% of its production is lost due the high spoilage, thus it has great potential as substrate in fermentative processes. This work aimed to optimize lactic acid production by submerged fermentation in a medium containing clarified cashew apple juice as substrate using the Lactobacillus casei NRRL B-442. From available information in the literature regarding temperature, pH, initial concentrations of substrate and initial ammonium sulfate, a kinetic study was carried out changing the initial concentration of substrate from 20 to 60 g/L and maintaining the ideal ratio of 12% of ammonium sulfate in relation of the initial total reducing sugars. The experiments were carried out in a batch reactor of 1.0 L with 0.5 L of reaction medium. The temperature was set at 37 ÂC and the pH was adjusted to 6.5 and controlled during the process. From the experimental data, a phenomenological model was developed and a computer program was built in Fortran 90. The mathematical model was statistically validated according to the F value of the Fisher test. With the representative model of the reaction system, it was possible to accomplish optimizations in batch and fed-batch fermentation for lactic acid production. The results in batch and fed-batch were compared, in relation to the final efficiency of the system, lactic acid production and costs of raw materials and reagents, with the costs of the final price of the lactic acid in the market. For the optimization in batch reaction, it was found that the fermentation which had the best results was obtained from an inoculum of 0.3 g/L and a initial concentration of total reducing sugars of 50 g/L, resulting in the production of 39.31 g/L of lactic acid and an efficiency of 72.2%. Regarding the fed-batch process, the simulations showed that the best results was obtained from an inoculum of 0.3 g/L with 40 g/L of initial total reducing sugars, with a feed of clarified cashew apple juice at flow rate of 3 L/h and a concentration of 200 g/L, resulting in the production of 38.0 g/L of lactic acid concentration and an efficiency of 63.8%. Therefore, for lactic fermentation using Lactobacillus casei NRRL B-442 and the clarified cashew juice as substrate and the conditions studied in this work, the process in batch was the most advantageous

Production of xylanase and itaconic acid (IA) from Aspergillus terreus NRRL 1960 from agricultural residues was investigated in this study. Two different media were tested and the medium having itaconic acid inducing capacity was chosen for further studies due to its high xylanase and IA production capacity. The best xylan concentration was found as 2% (w/v). Addition of commercial xylanase to production culture resulted in higher initial simple sugar concentration which increased IA production slightly but decreased xylanase production. Among tested agricultural residues / corn cob, cotton stalk and sunflower stalk, the highest xylanase production was obtained on corn cob. Increasing the corn cob concentration and applying wet heat pretreatment increased the xylanase production level. In a two-step fermentation process, 70000 IU/L xylanase production was achieved in a medium containing 7% wet heat treated corn cob followed by 17 g/L IA production in a medium containing 10% glucose. Molecular weight and isoelectric point of xylanase were found as 19 kDa and pH 9.0, respectively. The enzyme was optimally active at 50&deg / C and pH 6.5-7.0. Kinetic experiments at 50&deg / C and pH 7.0 resulted in apparent Km and Vmax values of 2.5&plusmn / 0.05 mg xylan/mL and 50.2&plusmn / 0.4 IU/&micro / g protein, respectively. The major products of birchwood xylan hydrolysis were determined by thin layer chromatography as xylobiose and xylotriose. These findings indicate that the enzyme could be advantageous for use in different industrial applications due to its low molecular weight and its potential use for xylooligosaccharide production.

Au cours de cette thèse, un isolat de sol de désert, Saccharothrix algeriensis NRRL B-24137, a été évalué pour ses propriétés bioactives contre le champignon phytopathogène Botrytis cinerea, pour sa colonization sur Vitis vinifera L., et Arabidopsis thaliana ainsi qu’en vue d’étudier les méchanismes de résistance systémique induite (ISR) contre B. cinerea. Les résultats obtenus nous ont permis premièrement de montrer que Sa. algeriensis NRRL B-24137 peut présenter des activités antifongiques contre B. cinerea et que des métabolites peuvent être responsables de cette activité antifongique. Bien que ces métabolites soient encore en cours d’étude et que cette étude mérite d’être approfondie, nous avons démontré ensuite les propriétés de colonisation de l’isolat du sol du désert chez la vigne. Les résultats ont permis de montrer que la souche peut former des populations rhizosphèriques ainsi que des sous-populations endophytiques chez des plants de vigne (Cabernet Sauvignon sur porte-greffe 44-53 M) à des étapes précoces de colonisation. Puis nous avons démontré que la souche bénéfique peut induire une résistance systémique contre B. cinerea. Bien que les mécanismes impliqués ne soient pas encore compris, des parties préliminaires de ces travaux démontrent que les expressions de gènes responsables de la production de glucanase, chitinase ainsi qu’un inhibiteur de polygalacturonase ne semblent pas potentialisés pendant le phénomène de résistance systémique. Enfin nous avons démontré l’interaction entre Sa. algeriensis NRRL B-24137 et Arabidopsis thaliana qui résulte dans une association intime dûe également à colonisation rhizosphèrique et endophytique de la plante modèle. La souche bénéfique peut églement induire un phénomène de résistance systémique sur A. thaliana contre B. cinerea et les analyses de plantes mutées ont permis de determiner des parties des mécanismes impliqués dans l’ISR aini que des nouveaux mécanismes impliqués qui peuvent être induits par des microbes bénéfiques / In this thesis, the desert soil isolate, Saccharothrix algeriensis NRRL B-24137, has been evaluated for its bioactive properties towards the phytopathogenic fungus Botrytis cinerea, for its colonization of Vitis vinifera L., and Arabidopsis thaliana as well as to study the mechanisms of induced systemic resistance (ISR) towards B. cinerea. The results obtained allowed us firstly to show that Sa. algeriensis NRRL B-24137 can exhibit strong antifungal properties towards B. cinerea and that some metabolites can be responsible of this antifungal activity. Although these metabolites are still under consideration and that this study needs further works, we have demonstrated then the colonization properties of the desert soil isolate with grapevine plants. The results showed that the strain can form rhizospheric as well as endophytic subpopulations with grapevine plants (Cabernet Sauvignon cultivar graffed on 44-53 M rootstock) at early step of colonization. Then we have demonstrated that the beneficial strain could induce a systemic resistance towards B. cinerea. Although the mechanisms are not yet well understood, preliminary parts of this work demonstrated that the genes responsible of glucanase production, chitinase as well as inhibitor of polygalacturonase activity do not seems to be primed during the systemic resistance phenomenon. Finally we demonstrated that the interaction between Sa. algeriensis NRRL B-24137 and Arabidopsis thaliana plants results in a close association due also to a rhizo- and endophytic colonization of the model plant. The beneficial strain can also induce a systemic resistance in A. thaliana towards B. cinerea and analyzes of plant mutants have allowed to determine parts of the mechanisms involved in ISR as well as new mechanisms that could be trigerred by beneficial microbes

Pathogens in low-moisture foods are an emerging food safety concern due to increased survival and thermotolerance in matrices with low water activity. However, limited data is publicly available for the thermotolerance of Listeria monocytogenes, Salmonella spp., and Enterococcus faecium NRRL B-2354 (a Salmonella surrogate). The aims of this study were to identify differences in thermal inactivation rates between these organisms in three different low-moisture foods. Three model low-moisture foods (peanut butter, powder infant formula, and wheat flour) were inoculated with either E. faecium, a Salmonella spp. cocktail, or a L. monocytogenes cocktail using a dry inoculation method for a total of 9 treatments. Samples were heat treated in a hot water bath at predetermined temperatures, and bacterial survival was detected via direct plating on tryptic soy agar with 0.6% yeast extract. In peanut butter and most of the powder infant formula treatments, Salmonella spp. had significantly higher D-values than L. monocytogenes using comparable temperatures (p < 0.05). However, D-values between Salmonella spp. and L. monocytogenes were comparable in wheat flour and one of the treatment temperatures in powder infant formula (p > 0.05). For all but one of the treatments at the same temperature, E. faecium had significantly higher D-values than L. monocytogenes and Salmonella spp. in each food matrix (p < 0.05). The observed matrix effect on thermotolerance for each of the bacteria was reported in descending order as powder infant formula > peanut butter > wheat flour in the majority of the comparable D-values. While Salmonella continues to be the pathogen of concern in low-moisture foods due to survival and outbreaks, these results indicate L. monocytogenes can exhibit similar thermotolerances in relevant model low-moisture foods matrices.

Vegetative, non-sporeforming foodborne pathogens show notable survival and uncanny thermotolerance in low water activity (aw) foods. Controlled studies on Listeria monocytogenes, Salmonella spp., and Enterococcus faecium NRRL B-2354 (a Salmonella surrogate) in a variety of food matrices support thermal process validation studies required to achieve global food safety objectives. In this study, we determined and compared thermal inactivation rates using independent six-strain cocktails of pathogens in three plant-based butters. Direct determinations of decimal reduction times (D-values) for L. monocytogenes, Salmonella, and E. faecium, in corresponding butters were inoculated using peanut oil, almond oil, or sunflower oil. Thermal Death Time (TDT) studies for the organisms were conducted in triplicate. Uniform bagged plant- based butter samples of Salmonella spp. or L. monocytogenes, or E. faecium alone were sandwiched in copper plates immobilized with recessed magnets. Samples underwent rapid heat treatments via water immersion under isothermal conditions ranging from 70°C to 85°C. Bacterial destruction in peanut butter (46% fat, 0.20 aw @ 25°C), almond butter, (50% fat, 0.32 aw @ 25°C), or sunflower butter (56% fat, 0.15 aw @ 25°C) was determined by direct plating. The TDT studies showed Salmonella spp. had consistently higher D-values than L. monocytogenes in all treatments, but pair-wise comparisons found no statistical difference when assessing the thermotolerance of the two pathogens in the individual plant-based butters tested (p > 0.005). These data support Salmonella as the primary pathogen of concern in low water activity foods and show the heat resistance of L. monocytogenes can approximate destruction kinetics observed for Salmonella spp. in low aw matrices. E. faecium exhibited the highest thermotolerance. This further supports the utility of this surrogate for Salmonella spp. and L. monocytogenes in high fat, low-moisture foods similar to the plant-based butters tested. Thermotolerance differences between a dry talc vs. peanut oil-based inoculation procedures in peanut butter were also evaluated. Surprisingly, the oil-based inoculations resulted in lower D- values (p > 0.01) for Salmonella spp. and the surrogate when compared to the dry inoculum.

Limited data are currently available characterizing the thermal resistance of foodborne pathogens in semolina flour and intermediate pasta matrices representative of commercial conditions during mixing, extrusion, and drying. These data are essential to pasta producers seeking to be compliant with federal regulations since Salmonella spp. and Escherichia coli demonstrate survival in wheat flour and dried pasta products. This study investigated the heat resistance of Salmonella, pathogenic E. coli, and E. faecium NRRL B-2354 in raw semolina flour and partially dried pasta intermediates via thermal death time (TDT) studies. This study also assessed the appropriateness of E. faecium NRRL B-2354 as a surrogate in semolina flour and pasta matrices. Inoculated pasta matrices equilibrated to target water activities of 0.85, 0.88, and 0.91 (measured at 25°C) underwent isothermal inactivation treatments at 65°C, 70°C, and 75°C. Serial dilution and direct plating methods allowed for estimation of bacterial survival at each treatment. In representative pasta matrices, the D-values for each microorganism increased as water activity decreased from 0.91 to 0.85. Surprisingly, Salmonella and E. coli did not exhibit significantly different thermal resistance in pasta. The greatest heat resistance was seen in semolina flour (aw 0.45). E. faecium was significantly more thermal resistant than both pathogens in all treatments when the temperature was ≤ 70°C. The results show that E. faecium strain NRRL B-2354 is not an ideal proxy for Salmonella and E. coli in semolina and pasta matrices. Analysis of the TDT data also found that a long-goods pasta drying process can achieve ≥7-log reductions of Salmonella and E. coli when following Good Manufacturing Practices.