Orientador: Pilar Rodriguez de Massaguer / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-21T03:39:39Z (GMT). No. of bitstreams: 1 CarvalhoFilho_CelsoDuarte_M.pdf: 7345180 bytes, checksum: 62066bb1a2a00eeaa3997a883f32b44b (MD5) Previous issue date: 1996 / Resumo: Este trabalho teve por finalidade avaliar a integridade asséptica da selagem de topo das bolsas esterilizáveis, composta de um laminado de polipropileno (70µm), folha de alumínio (9µm), náilon (15µm) e poliéster (12µm) através de bioteste com esporos de Bacillus subtilis e, também, estabelecer o processo térmico adequado para produzir purê de banana comercialmente estéril nestas embalagens, processadas em autoclave por imersão total em água quente com sobrepressão. O produto selecionado foi purê de banana natural, variedade "nanica" {Musa cavendish, Lamb.) com pH 4,6 -4,7 embalado (130g) em bolsas esterilizáveis de 130 x 170mm. Microfuros de diversos diâmetros foram intencionalmente formados nestas bolsas através de fios de níquel-cromo colocados na área de selagem de topo. Após autoclavagem, os fios foram retirados das embalagens e estas foram submetidas assepticamente a um contato direto com uma suspensão de esporos da referida bactéria durante 1 hora. Foi analisado o poder de penetração dos esporos através de diferentes diâmetros de microfuros formados pelos fios na área de selagem de topo das embalagens (diâmetros dos fios : 32, 48, 79 e 97µm). O bioteste foi aplicado em bolsas cheias antes e após o processo térmico do purê a 115°C. A penetração e crescimento do B. subtilis foi confirmada por ensaios bioquímicos e subcultura em meio agar nutriente, com prévia incubação das bolsas a 30°C por 7 dias. Estas embalagens foram posteriormente submetidas ao teste eletrolítico para confirmação dos microfuros detectados. O processo térmico adequado para o purê de banana, previamente inativado enzimaticamente (97°C por 5 min.), foi de 7,5 min. a 115°C equivalente a um Fo de 0,64 min., verificado pelo método geral e ensaios de esterilidade comercial. Foi notado que o processo térmico favoreceu a penetração dos esporos através de microfuros de menor diâmetro, pois enquanto que o bioteste detectou microfuros >= 79µm m com 87,5% de penetração antes do processo, o mesmo teste detectou penetração em microfuros >= 48µm de diâmetro com 53,8% de penetração em bolsas testadas após o processo. O teste eletrolítico só foi capaz de detectar microfuros formados com fios de 79um de diâmetro em 69,23% das amostras. Em ensaios realizados antes e depois do processo térmico não foi detectada penetração em bolsas com microfuros formados com fios de 32µm . / Abstract: The aim of this work was to evaluate the integrity of the top sealing area of retortable pouches formed by a laminate of polypropylene (70 µm), aluminum foil (9um), nylon (15 µm) and polyester (12 µm), using Bacillus subtilis spores as a biotest and also to establish the proper thermal process in order to produce comercially sterile banana puree, packed in pouches processed in a full water-immersion retort. The selected product was banana puree (130g), variety "nanica" (Musa cavendish, Lamb.), pH 4.6 - 4.7, packed in 130 x 170mm retortable pouches. Microholes of different diameters were intentionally made with nickel-chrome threads placed in the top seal area. After sterilization, the threads were withdrawn from the seals and the packages were tested using the spore test. A suspension of B. subtilis spores was aseptically left on the top seal for 1 hour. The capacity of the spores to penetrate through different microholes diameters (32, 48, 79 and 97 µm) was evaluated. The biotest was carried out before and after processing the banana puree at 115°C. Bacillus subtilis penetration and growth was confirmed by biochemical tests and subculture in nutrient agar after incubation of the pouches at 30°C for 7 days. The tested packages were, latter, submitted to eletrolitic test for microholes confirmation. The proper thermal process for banana puree, previously bleached (97°C for 5 min.), was 7,5 min. at 115°C equivalent to an Fo of 0,64 min., confirmed by the general method evaluation and commercial sterility tests. It was noted that heat processing favored the penetration of spores at lower microholes sizes:while the spore test detected microholes of >= 79 µm with 87,5% of penetration before processing, the same test detected penetrations through microholes >= 48 µm with 53,8% penetration after processing. Eletrolitic test was only able to detect microholes formed with 79 µm diameter threads, showing presence of microholes in 69,23% of the tested samples. No penetration was detected in pouches with microholes formed with 32um tested before or after heat processing. / Mestrado / Mestre em Ciência de Alimentos

Orientador : Antonio de Melo Serrano / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-14T02:57:04Z (GMT). No. of bitstreams: 1 Andrade_NelioJosede_D.pdf: 4078525 bytes, checksum: 9fdf380ebd9507b2adf3c45f9232fa9f (MD5) Previous issue date: 1989 / Resumo: A eficiência de soluções de hipoclorito de sódio foi avaliada usando-se como indicadores os esporos de B. subtilis ATCC 19659. Foram produzidos, em tempos diferentes, dez lotes de esporos de B. subtilis ATCC 19659 com mesma resistência ao hipoclorito, pois não houve diferença significativa ao nível de 5% de probabilidade (P < 0,05) nas reduções decimais do número desses esporos apôs serem submetidos a 105 mg/l de cloro disponível livre em pH 9,8 a 30°C. A estabilidade do número de esporos de B. subtilis ATCC 19659 e de sua resistência às soluções de hipoclorito de sódio com 105 mg/l de cloro disponível livre em pH 9,8 no tempo de contato de 145 minutos a 30°C foi avaliada aos 30, 60, 90, 120, 150 e 180 dias era três armazenamentos; refrigeração a 4°C, congelamento a -18°C, ambos em glicerol e água (15 : 85) e liofilização em tampão fosfato M/15 de pH 7 com posterior estocagem a 4°C. A refrigeração, de acordo com as análises estatísticas, foi a melhor forma de armazenamento: o número dos esporos permaneceu constante durante os 180 dias, numa faixa de 1,41 a 1,58 x 109 unidades formadoras de colônias por mililitro (UFC/ml) e a resistência ficou estável por 150 dias, mantendo-se entre 2,62 e 2,69 reduções decimais (RD). No congelamento detectou-se uma tendência de aumento do número de esporos viáveis à medida que se prolongou a armazenagem. A resistência permaneceu estável por 120 dias, entre 2.65 e 2,72 RD. Sob liofilização, verificou-se uma maior variação no número de esporos viáveis nos diversos períodos de armazenamento. A resistência ao cloro manteve-se a mesma por 90 dias, entre 2,63 e 2,98 RD. A ação esporicida das soluções de hipoclorito de sódio contendo 50, 75, 105, 150 e 200 mg/l de cloro disponível livre em pH 9,6 e 105 mg/l com pH 9,0, 8,0 e 7,0, á temperatura de 30°C foi avaliada em lotes de esporos de B. subtilis ATCC 19659 suspensos em glicerol e água (15 : 85). Os tempos de contato, para cada condição, foram escolhidos dentro da faixa de morte acentuada, em ensaios preliminares. Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital. / Abstract: The efficiency of sodium hypoclorite as a sporicide was studied using Bacillus subtilis ATCC 19659. Ten lots of spores, produced on different occasions, showed no significant difference (P < 0.05) in resistance to 105 mg/1 free vailable chlorine at pH 9.8 and 30ºC, measured as decimal reduction in the number of spores. Spores were stored under three different conditions: at 4°C and at -18°C in glicerol: H20 (15 : 85), and at 4°C lyophilized in 0.067 M phosphate buffer at pH 7.0. The stability of the spore population and its resistance to NaOCl (105 mg/1 free available chlorine) at pH 9.6 for 145 tu at 30CC was determined every 30 d for up to 180 d. At 4ºC, no change occurred in the spore population for 180 d, maintaining a range of 1.41 to 1.58 x 109 colony forming units per ml (CFU/ml ). and no change in resistance occurred for 150 d, remaining between 2.62 and 2.69 decimal reduction (DR ). At -18°C, the number of viable spores tended to increase and resistance was slabie for 120 d, between 2.65 and 2.72 DR. Under lyophi1ization the number of viable spores was variable. Resistance to chlorine remained the same for 90 d, between 2.63 and 2.98 DR. The sporicidal action of NaOCl at 50, 75, 105, 150 and 200 mg/1 of free available chlorine at pH 9.8, and 105 mg/1 at pH 7.0, 8.0 and 9.0, was studied using lots ol spores suspended in solutions of glicerol: H20 (15 : 85) at 30°C. The times of contact were chosen from a death range in preliminary studies. The decimal reductions could be expressed by the linear regression equation Y = aX + b, where Y = decimal reduction and X = time of contact of the sanitizing agent with the spores...Note: The complete abstract is available with the full electronic document. / Doutorado / Doutor em Tecnologia de Alimentos

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Members of the Bacillus genus, when faced with unfavorable environmental conditions such as depletion of nutrients, undergo an asymmetric division process ultimately leading to the formation of an endospore. In some instances, the spore serves as the infectious agent of an associated disease; such is the case with the spore of Bacillus anthracis and the disease anthrax. Spores are resistant to a variety of unfavorable environmental conditions including traditional decontamination techniques. Spore resistance is due to the formation of specialized structures that contribute to spore dormancy through several mechanisms, including maintenance of the dehydrated state of the spore core. Spore germination is a rapid process resulting in the irrevocable transformation of the non-metabolizing dehydrated spore into a vegetative outgrowing bacterium. The exact mechanism by which individual proteins function in the germination pathway remains unknown. In this study, we have focused on the roles of putative ion transporters and other germination-active proteins in affecting spore formation and germination. Metal ions can activate enzymes during the sporulation process and/or be factors in spore resistance properties. In B. subtilis, six proteins within the spore membrane proteome (ChaA, YcnL,YflS, YloB, YugS, ZnuA) are similar to components of known cation transport systems. These proteins may play roles in the accumulation of ions during sporulation and/or the release of ions during germination. Multiple mutants altered in the putative ion transporter genes were generated, and the effects of these mutations were analyzed. All strains containing a yloB deletion showed a decrease in heat resistant cfu/ml, and >40% of the spores appeared phase dark during microscopy, indicating the formation of unstable spores. Studies were conducted to quantify the amounts of individual ions in phase-bright spores using atomic emission spectroscopy and to analyze the rate at which ions are released from germinating spores. The transport of Ca2+ from mother cell to forespore during sporulation seems to be affected in the yloB deletion mutant. This Ca2+ deficit apparently renders the spores unstable, heat sensitive, and partially germination defective, suggesting that a high-affinity transporter for Ca2+ is nonfunctional. To better understand the underlying mechanisms of germination, a high-throughput genetic screening method called transposon sequencing was used. This analysis identified genes that had not been previously implicated in germination. To investigate their functions, a number of functional assays of all the Ger mutant strains were performed that indicated a delay in stage I of germination. The mutant strains showed significant reduction in germination efficiency with L-valine: about 50% of the population failed to initiate germination suggesting a defect in the GerA-mediated response. The expression of gerA was studied using a lacZ transcriptional fusion followed by quantitative western blot analyses to determine abundance of GerA in mutant strains. The mutants were classified based upon normal or decreased gerA transcription and normal or reduced GerA protein. Further work involves understanding the functions of the identified genes and their correlation to the GerA receptor. Insight into ion transporters of spore-forming bacteria and understanding the germination apparatus may lead to promising new applications, detection methods, or therapeutics for spores, and may allow the development of better spore decontamination procedures. / Doctor of Philosophy / Bacillus subtilis is an ubiquitous bacterium that is capable of forming endospores when faced with unfavorable environmental conditions. Spores are highly resistant to heat, radiation, lack of nutrients, desiccation and oxygen deprivation. They lack metabolism, which effectively keeps them in a state of suspended animation until germinated. They may remain stable and viable in this state for extremely long periods of time. Several important pathogenic bacteria are spore formers. This leads to difficulty in their environmental eradication and the treatment of patients. Germination allows spores to resume metabolism and reestablish a vegetative state. Certain key molecules activate the germination process. Each species of spore-forming bacteria has a specific set of these molecules called germinants that will enable the spore to exit its dormant state. The work presented focuses on the understanding of the germination apparatus of Bacillus subtilis, which may provide a model to understand the germination of other spore formers and help to improve methods of decontamination.

Orientador: Ranulfo Monte Alegre / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-24T00:10:15Z (GMT). No. of bitstreams: 1 Piedrahita-Aguirre_CesarAugusto_D.pdf: 6150918 bytes, checksum: bfd7a30fcb69bbbfa8cbba5856d068f5 (MD5) Previous issue date: 2013 / Resumo: Este trabalho se propôs a estudar a produção da iturina A por Bacillus subtilis em fermentação semi-sólida em biorreatores de leito empacotado. O trabalho foi desenvolvido em quatro partes. Em uma primeira parte foi feito um screening com cepas silvestres e seus mutantes obtidos a partir da exposição de luz UV e acridina laranja. A cepa Bacillus subtilis subsp. subtilis NRRL NRS-1270 foi a que apresentou maior atividade antagônica contra os fungos Aspergillus fumigatus Fresenius NRRL 164, Aspergillus fumigatus Fresenius NRRL 166 e Aspergillus flavus var. oryzae NRRL 484. O extrato metanólico obtido da fermentação semi-sólida do Bacillus subtilis subsp. subtilis NRRL NRS-1270 foi analisado através da espectrometria de massas encontrando-se lipopeptídeos com massa molecular entre m/z 1021,43 e m/z 1087,48, mas sem a presença da iturina A. Em uma segunda etapa a cepa Bacillus Iso 1 foi isolada a partir das raízes de soja, e ante a dificuldade de identificar a iturina A através da cromatografia liquida de alta eficiência (HPLC), foi desenvolvida a metodologia de purificação da iturina A utilizando a cromatografia em coluna de vidro preenchida com sílica gel 60. A iturina A foi eluída com três sistemas de solventes compostos por 20 mL de clorofórmio-metanol-água (65:25:4,v/v/v), fração 1, seguido de 20 mL de clorofórmio-metanol-água (30:50:10, v/v/v), fração 2, e a fração final composta por 10 mL de clorofórmio-metanol-água (20:60:15, v/v/v). As frações obtidas foram analisadas através da HPLC e da espectrometria de massa, identificando 5 isômeros da iturina A (C13-C16). Na terceira etapa, foi feito um delineamento composto central rotacional (DCCR) para avaliar o efeito da casca de arroz como suporte inerte e da vazão volumétrica de ar na produção de iturina A; como substratos foram utilizados o farelo de soja desengordurado e o farelo de trigo. Nenhuma variável do DCCR foi estatisticamente significativa, mas operacionalmente foram importantes, devido à redução da oxigenação do Bacillus Iso 1 pela baixa vazão de ar e menor concentração de casca de arroz, favorecendo a produção de iturina; nestas condições obteve-se 6,88 g/kg de substrato seco de iturina A.Esta é a maior quantidade de iturina A produzida em biorreatores de leito empacotado (coluna) com aeração forçada até hoje. Na quarta etapa, a partir dos resultados obtidos no DCCR foram estudados os parâmetros do processo: queda de pressão, consumo de oxigênio e perfis de temperatura, visando entender o comportamento da fermentação a 0,4 L/min e 0,8 L/min. A máxima produção de iturina obtida foi 5,58 g/kg de substrato seco com a vazão de 0,4 L/min. O incremento na queda de pressão é ocasionado não unicamente pelo incremento da vazão volumétrica, mas também pela produção do biopolímero ?-PGA o qual ocupa os espaços livres entre as partículas, dificultando o fluxo normal de ar através do leito, reduzindo o consumo de oxigênio. A baixa oxigenação favoreceu a alta produção da iturina A e gerou baixo calor metabólico (5,75 W/kg-dry substrato·min). Os resultados obtidos podem ser úteis na elaboração de estratégias para ampliação de escala do processo em fermentadores aerados de leito empacotado / Abstract: This work covers a study of the production of iturin A by Bacillus by solid-state fermentation in packed bed bioreactors. The study was conducted in four parts. At first a screening was conducted with wild strains and their mutants obtained from exposure to UV light and mutagenic agent acridine orange. The strain Bacillus subtilis subsp subtilis NRRL NRS 1270 showed the highest antagonistic activity against Aspergillus fumigatus NRRL 164, Aspergillus fumigatus NRRL 166 and Aspergillus flavus var . oryzae NRRL 484. A methanolic extract obtained by solid state fermentation of Bacillus subtilis subsp subtilis NRRL NRS 1270 was analyzed with mass spectrometry showing lipopeptides with molecular mass between m/z 1021.43 and m/z 1087.48, but without the presence of iturin A. In the second stage, the strain Bacillus Iso 1 was isolated from soybean roots. Given the difficulty of identifying iturin A by high performance liquid chromatography (HPLC), a iturin A purification methodology was developed using glass column chromatography packed with activated Silica gel 60 and alumina. This methodology involved three solvent systems for elution of the iturin A from the column. A first fraction consisted of 20 ml of chloroform-methanol-water (65:25:4 , v/v/v) and was followed by 20 ml of chloroform - methanol- water (30:50 : 10, v/v/v), that was then followed by a final fraction consisting of 10 ml of chloroform-methanol-water (20:60:15, v/v/v). The fractions obtained of fermentation were analyzed by both HPLC and mass spectrometry, identifying five iturin A isomers (C13-C16). In the third stage of the study, an experimental design was constructed in the form of a central composite rotational design (CCRD) to evaluate the effect of rice husk as an inert support and air flow rates to the iturin A production, using defatted soybean meal and wheat bran as substrate. Although none of the studied variables showed statistical significance, the operational importance of reduction of oxygenation of the Bacillus Iso 1 fermentation due to the low concentration of rice husk and air flow rate was observed to favor the production of iturin; in these conditions high productivity was obtained reaching 6.88 g/kg-dry substrate of iturin A. Concluding from available literature, this is the highest concentration of iturin A ever produced in packed bed bioreactor (column) with forced aeration to date. In the fourth stage, in order to understand the behavior of the fermentation under aeration conditions between 0.4 L/min and 0.8 L/min, the following process parameters were studied, based on the results obtained from the CCRD: pressure drop, oxygen consumption and temperature profiles. The maximum production of iturin obtained was 5.58 g/kg-dry substrate with the air flow rate at 0.4 L/. The increase of the pressure gradients is caused not only by increasing the volumetric air flow rate but also by the production of biopolymer ?-PGA by Bacillus iso 1, which occupies the free interparticle space, hindering or preventing the normal flow of air through the bed and thus leading to reduced oxygen consumption. The low oxygenation favored the high iturin A production and resulted in low metabolic heat generation (5.75 W/kg-dry substrate.min). The results of this work are expected to be conducive for designing strategies to scale up the process in aerated packed bed bioreactors / Doutorado / Engenharia de Alimentos / Doutor em Engenharia de Alimentos

Bacillus subtilis (ATCC 19659) were damaged by exposure to various concentrations of glutaraldehyde, as shown by decreased germination rates. The damage caused was repaired or otherwise obviated by the presence of sodium lactate in the holding medium. When two different salts of lactic acid were compared for ability to overcome the effect of glutaraldehyde, it was found that calcium salt of lactate was more effective than the sodium salt. The damage repair system involved l-alanine, lactate and either the sodium or calcium ions. The study involved in determining the difference in efficiency of spore repair was due to an organic or an amino acii snowed that the presence of two carboxylic functional groups did not effectively alter the reactivity.

The initiation of transcription by the Bacillus subtilis sigma-M3 RNA polymerase at two Bacillus phage ɸ29 promoters and the effect of the delta subunit on initiation have been investigated by an in vitro kinetic analysis. The templates for the analysis were plasmids which carried the ɸ29 A2 or G2 promoter. The cloning and localization of the A2 promoter are reported here. The kinetics of RNA synthesis initiation were examined using a single-round run-off transcription assay in which multiple initiation events at a single promoter were inhibited with heparin. It was observed that the formation of heparin-resistant complexes at the A2 promoter required the presence of the initiating nucleotides, while the RNA polymerase alone was able to form heparin-resistant, non-initiated complexes at the G2 promoter. The G2 promoter was also shown by a competition assay to be a stronger promoter than A2. The effect of the delta subunit on complex formation at the two promoters was investigated with the single-round transcription assay. Delta had no effect on the formation of initiation complexes at the G2 promoter but lowered the rate and extent of complex formation at the A2 promoter. The effect of delta on the kinetic parameters of complex formation at the A2 promoter was also investigated. The data suggested that delta affects the efficiency with which the enzyme/promoter complexes undergo the transition(s) to a complex from which RNA synthesis can be initiated, although other interpretations were possible. A model for the effect of delta is proposed, in which it is postulated that the release of delta from the enzyme/promoter complex is essential for initiation. Enzyme which is associated with delta can interact with both the A2 and G2 promoters but complexes at the weaker A2 promoter do not efficiently release delta, thus slowing the formation of initiation complexes. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate