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Acoplamento das técnicas HPLC-ICP OES para separação e identificação de metaloproteínas em castanhas de caju / Coupling of LC-ICP OES techniques for separation and identification of metalloproteins in cashew nutsSiqueira, Bruno Menezes 02 September 2016 (has links)
O objetivo desse trabalho foi demonstrar a viabilidade do acoplamento entre as técnicas de cromatografia líquida de alta eficiência com a espectrometria de emissão óptica com plasma indutivamente acoplado (HPLC - ICP OES) em um equipamento da Thermo Fisher Scientific, visando análise de especiação elementar de espécies com maiores concentrações na amostra. A identificação de espécies moleculares associadas aos elementos Cu, Fe, Mg, e Zn em amostras de castanha de caju foi adotada como modelo para comprovar tal acoplamento. Para que o ICP OES pudesse adquirir sinais de emissão ao longo de um tempo determinado, ou seja, para que o equipamento pudesse coletar um sinal transiente, fundamental para o acoplamento HPLC - ICP OES, foi necessário executar algumas modificações. Para isso, foram feitas alterações na placa eletrônica principal do equipamento, e trocada sua caixa controladora de gases. Os parâmetros instrumentais do ICP OES foram otimizados em uma série de ensaios com o intuito de estabelecer e avaliar desempenho do instrumento e a robustez do método empregado nas análises de castanha de caju. Determinações das concentrações totais de Ca, Cu, Fe, Mg, Mn, Ni, e Zn foram feitas em amostras de castanha de caju e nos materiais de referência NIST 1515 e NIST 1547, apresentando uma exatidão do método aceitável, levando-se em consideração o preparo de amostra e a medida no ICP OES. As determinações totais elementares nas amostras de castanha de caju indicaram que os teores de Ca foram de (0,04 ± 0,01) % ; Cu (26,3 ± 5,0) mg kg-1; Fe (47,9 ± 8,8) mg kg-1; Mg (0,31 ± 0,04) %; Mn (25,6 ± 3,9) mg kg-1; Ni (24,8 ± 1,9) mg kg-1; Zn (66,3 ± 2,4) mg kg-1. Para identificação de espécies moleculares associadas aos elementos Cu, Fe, Mg, e Zn, foi feita nas amostras de castanha de caju uma extração de metais e proteínas utilizando-se de uma solução extrato de NaOH 1 mol L-1. O extrato ainda foi submetido à uma etapa de clean-up, com precipitação das proteínas com acetona e ressupenção das mesmas com tampão Tris-HCl, pH 7,4. As proteínas e metais ressupensos em tampão foram analisados no sistema HPLC ICP OES, em que a separação cromatográfica ocorreu através de uma coluna de exclusão por tamanho (SEC), a detecção molecular através do detector UV-Vis do HPLC, e a detecção elementar através do ICP OES. As espécies moleculares identificadas estão no intervalo de massa molecular compreendido entre 2 e 177 kDa, sendo que Cu, Fe e Zn apresentam associação com essas espécies moleculares. Foram monitorados ainda, no ICP OES, os sinais de intensidade de emissão de P e S. Esses elementos também apresentaram associação com espécies moleculares com peso molecular entre 2 e 177 kDa, sugerindo que essas espécies se tratam de metaloproteínas. / The aim of this study was to demonstrate the capability of coupling between liquid chromatography techniques of high efficiency with the optical emission spectrometry with inductively coupled plasma (HPLC - ICP OES) in an equipment of Thermo Fisher Scientific, aiming elemental speciation analysis of species with higher concentrations in the sample. The identification of molecular species associated with elements Cu, Fe, Mg and Zn in cashew nuts samples was adopted as a model to prove such coupling. For ICP OES could get emission signal through a certain time, so that the equipment could collect a transient signal, critical to the HPLC coupling - ICP OES, it was necessary to perform some modifications in the instrument. For this, changes were made to the main electronic board of the equipment and also changed their gas controller box. The instrumental parameters of the ICP OES were optimized in a series of tests in order to establish and evaluate performance of the instrument and the robustness of the method employed in cashew nut analysis. Determination of the total concentration of Ca, Cu, Fe, Mg, Mn, Ni and Zn were made in cashew sample and reference materials NIST 1515 and NIST in 1547, with an accuracy acceptable method, taking into account the sample preparation and measurement by ICP OES. The elemental total determinations in cashew nut samples indicated that Ca were (0.04 ± 0.01)%; Cu (26.3 ± 5.0) mg kg-1; Fe (47.9 ± 8.8) mg kg-1; Mg (0.31 ± 0.04)%; Mn (25.6 ± 3.9) mg kg-1; Ni (24.8 ± 1.9) mg kg-1; Zn (66.3 ± 2.4) mg kg-1. For the identification of molecular species associated with the elements Cu, Fe, Mg, and Zn was performed on samples of cashew nuts an extract of metals and proteins using an extract solution of NaOH 1 mol L-1. The extract was further subjected to one-step of clean-up with protein precipitation with acetone following by a resuspension with Tris-HCl buffer, pH 7.4. Proteins and metals resuspended in buffer, were analyzed in the HPLC - ICP OES system, wherein the chromatographic separation took place by a size exclusion column (SEC), the molecular detection by UV-Vis detector of HPLC, and elemental detection by ICP OES. The molecular species were identified in the molecular weight range between 2 and 177 kDa, and Cu, Fe and Zn are associated with these molecular species. Were also monitored in the ICP OES, P and S emission intensity signals. These elements also associated with molecular species having a molecular weight between 2 and 177 kDa, suggesting that this species are metalloproteins.
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Acoplamento das técnicas HPLC-ICP OES para separação e identificação de metaloproteínas em castanhas de caju / Coupling of LC-ICP OES techniques for separation and identification of metalloproteins in cashew nutsBruno Menezes Siqueira 02 September 2016 (has links)
O objetivo desse trabalho foi demonstrar a viabilidade do acoplamento entre as técnicas de cromatografia líquida de alta eficiência com a espectrometria de emissão óptica com plasma indutivamente acoplado (HPLC - ICP OES) em um equipamento da Thermo Fisher Scientific, visando análise de especiação elementar de espécies com maiores concentrações na amostra. A identificação de espécies moleculares associadas aos elementos Cu, Fe, Mg, e Zn em amostras de castanha de caju foi adotada como modelo para comprovar tal acoplamento. Para que o ICP OES pudesse adquirir sinais de emissão ao longo de um tempo determinado, ou seja, para que o equipamento pudesse coletar um sinal transiente, fundamental para o acoplamento HPLC - ICP OES, foi necessário executar algumas modificações. Para isso, foram feitas alterações na placa eletrônica principal do equipamento, e trocada sua caixa controladora de gases. Os parâmetros instrumentais do ICP OES foram otimizados em uma série de ensaios com o intuito de estabelecer e avaliar desempenho do instrumento e a robustez do método empregado nas análises de castanha de caju. Determinações das concentrações totais de Ca, Cu, Fe, Mg, Mn, Ni, e Zn foram feitas em amostras de castanha de caju e nos materiais de referência NIST 1515 e NIST 1547, apresentando uma exatidão do método aceitável, levando-se em consideração o preparo de amostra e a medida no ICP OES. As determinações totais elementares nas amostras de castanha de caju indicaram que os teores de Ca foram de (0,04 ± 0,01) % ; Cu (26,3 ± 5,0) mg kg-1; Fe (47,9 ± 8,8) mg kg-1; Mg (0,31 ± 0,04) %; Mn (25,6 ± 3,9) mg kg-1; Ni (24,8 ± 1,9) mg kg-1; Zn (66,3 ± 2,4) mg kg-1. Para identificação de espécies moleculares associadas aos elementos Cu, Fe, Mg, e Zn, foi feita nas amostras de castanha de caju uma extração de metais e proteínas utilizando-se de uma solução extrato de NaOH 1 mol L-1. O extrato ainda foi submetido à uma etapa de clean-up, com precipitação das proteínas com acetona e ressupenção das mesmas com tampão Tris-HCl, pH 7,4. As proteínas e metais ressupensos em tampão foram analisados no sistema HPLC ICP OES, em que a separação cromatográfica ocorreu através de uma coluna de exclusão por tamanho (SEC), a detecção molecular através do detector UV-Vis do HPLC, e a detecção elementar através do ICP OES. As espécies moleculares identificadas estão no intervalo de massa molecular compreendido entre 2 e 177 kDa, sendo que Cu, Fe e Zn apresentam associação com essas espécies moleculares. Foram monitorados ainda, no ICP OES, os sinais de intensidade de emissão de P e S. Esses elementos também apresentaram associação com espécies moleculares com peso molecular entre 2 e 177 kDa, sugerindo que essas espécies se tratam de metaloproteínas. / The aim of this study was to demonstrate the capability of coupling between liquid chromatography techniques of high efficiency with the optical emission spectrometry with inductively coupled plasma (HPLC - ICP OES) in an equipment of Thermo Fisher Scientific, aiming elemental speciation analysis of species with higher concentrations in the sample. The identification of molecular species associated with elements Cu, Fe, Mg and Zn in cashew nuts samples was adopted as a model to prove such coupling. For ICP OES could get emission signal through a certain time, so that the equipment could collect a transient signal, critical to the HPLC coupling - ICP OES, it was necessary to perform some modifications in the instrument. For this, changes were made to the main electronic board of the equipment and also changed their gas controller box. The instrumental parameters of the ICP OES were optimized in a series of tests in order to establish and evaluate performance of the instrument and the robustness of the method employed in cashew nut analysis. Determination of the total concentration of Ca, Cu, Fe, Mg, Mn, Ni and Zn were made in cashew sample and reference materials NIST 1515 and NIST in 1547, with an accuracy acceptable method, taking into account the sample preparation and measurement by ICP OES. The elemental total determinations in cashew nut samples indicated that Ca were (0.04 ± 0.01)%; Cu (26.3 ± 5.0) mg kg-1; Fe (47.9 ± 8.8) mg kg-1; Mg (0.31 ± 0.04)%; Mn (25.6 ± 3.9) mg kg-1; Ni (24.8 ± 1.9) mg kg-1; Zn (66.3 ± 2.4) mg kg-1. For the identification of molecular species associated with the elements Cu, Fe, Mg, and Zn was performed on samples of cashew nuts an extract of metals and proteins using an extract solution of NaOH 1 mol L-1. The extract was further subjected to one-step of clean-up with protein precipitation with acetone following by a resuspension with Tris-HCl buffer, pH 7.4. Proteins and metals resuspended in buffer, were analyzed in the HPLC - ICP OES system, wherein the chromatographic separation took place by a size exclusion column (SEC), the molecular detection by UV-Vis detector of HPLC, and elemental detection by ICP OES. The molecular species were identified in the molecular weight range between 2 and 177 kDa, and Cu, Fe and Zn are associated with these molecular species. Were also monitored in the ICP OES, P and S emission intensity signals. These elements also associated with molecular species having a molecular weight between 2 and 177 kDa, suggesting that this species are metalloproteins.
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Inactivation Of Salmonella And Surrogate Bacteria On Cashews And Macadamia Nuts Exposed To Saturated Steam And Propylene Oxide TreatmentsSaunders, Thomas Philip 30 May 2017 (has links)
Saturated steam (SS) and propylene oxide (PPO) fumigation are two common methods to improve microbiological quality and safety of tree nuts. Validation of these processes is needed to ensure adequate control of bacterial pathogens. Since pathogens cannot be studied in food processing environments, surrogates with resistance comparable to the pathogens needed to be identified. The objective was to investigate the suitability of Enterococcus faecium, Pediococcus acidilactici, and Staphylococcus carnosus as surrogate bacteria for Salmonella spp. on whole cashews and macadamia nuts, processed with SS or PPO.
Whole cashews and macadamia nuts were co-inoculated with a cocktail of Salmonella enterica and one of the three potential surrogates. Nuts were dried to original aw, packaged in poly-woven bags (2.3 kg) and commercially processed using vacuum assisted steam at 80 ͦ C or PPO fumigation. Salmonella and the potential surrogates were enumerated by serial dilution, and plated onto TSA with overlay of XLT-4 (Salmonella) or media selective for the potential surrogates.
Mean log reductions (CFU/g) of Salmonella and each potential surrogate were compared using a paired T-test. SS results: reduction of Salmonella (6.0 ± 0.14) was significantly larger than E. faecium (4.3± 0.12), or P. acidilactici (3.7± 0.14) on whole cashews. Salmonella (5.9 ± 0.18) was significantly larger than P. acidilactici (4.4± 0.18) on whole macadamia nuts.
PPO results: reduction of Salmonella (7.3 ± 0.19) was significantly greater than E. faecium (6.4± 0.31), or P. acidilactici (6.3± 0.33) on whole macadamia nuts. Reduction of Salmonella was significantly greater than E. faecium and P. acidilactici reduction on cashews.
P. acidilactici may be considered a surrogate for Salmonella reduction on whole macadamia nuts and whole cashews processed using SS at 80 ͦ C. E. faecium and P. acidilactici may be considered surrogates for Salmonella reduction on whole macadamia nuts and whole cashews processed using PPO. Reduction of St. carnosus exceeded that of Salmonella indicating it is not a suitable surrogate for Salmonella using either processing intervention. / Master of Science in Life Sciences / Tree nuts are produced and consumed worldwide, playing a role as a snack or ingredient in foods in several cultures. Traditionally, tree nuts have been believed to be microbiologically safe due to their composition and lack of water available to harbor pathogenic bacterial growth. However, recent years have proven to be problematic for many tree nuts and nut products, numerous recalls have occurred in the United States for verified contamination of <i>Salmonella</i>. Since <i>Salmonella</i> can be found in food systems worldwide, and is a leading cause of foodborne illness due to bacterial contamination of food, steps must be taken to improve the safety of nuts grown locally and imported products. With several processing options for tree nuts, two that are commonly practiced to reduce microorganisms include a fumigation of product using propylene oxide (PPO) and a thermal inactivation treatment through use of saturated steam. The comparison of <i>Salmonella</i> inactivation and non-pathogenic surrogate bacteria on cashews and macadamia nuts, being processed in these two manners, was investigated. Possible bacteria strains that were investigated for surrogacy were <i>Enterococcus faecium</i> NRRL B2354 (ATCC #8459), <i>Pediococcus acidilactici</i> (ATCC #8042), and <i>Staphylococcus carnosus</i> (ATCC #51365). Surrogates are bacteria that have similar inactivation characteristics to <i>Salmonella</i> when processed, that can be purposely introduced before processing to ensure inactivation of <i>Salmonella</i> and is harmless if consumption occurs. Studies continue to ensure safety of tree nuts as well as complying with pending and future regulations.
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Competitividade na pequena produ??o agroindustrial: estudo na agroind?stria da castanha de cajuAndrade Neto, J?lio C?sar de 28 July 2006 (has links)
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Previous issue date: 2006-07-28 / This paper looks into the cashew nut industrial process as a factor in adding value to it and involves its productions, industrialization and marketing. Its competitiveness fundamentally depends in its ability to surpass technological and non technological difficulties that contributes to increase costs and diminishes attributes to better qualities which means values to the market. The methodology applied in this paper was application of a questionnaire with a Likert model scale with closed questions and constituted by variables that composed nominated groups: exporting obstacles, market strategy, competitive advantages, broken nuts index, productive potential and social-economic profile of members of the Cooperative. A descriptive analysis was the method employed for the data analysis. After identifying some of the quantitative gains in the industrial process of the cashew nuts, recommendations are presented to COOPERCAJU to promote courses to improve the members productivity as well as technical assistance, in order to get more efficacy in the cashew nuts industrial processing / O presente estudo trata do processo industrial da castanha de caju como um fator de agrega??o de valor, envolvendo atividades de produ??o, industrializa??o e comercializa??o. Sua competitividade depende fundamentalmente da habilidade em superar gargalos tecnol?gicos e n?o tecnol?gicos que elevem os custos e limitem a implementa??o de melhorar os atributos de qualidade que o mercado valoriza. A metodologia empregada neste trabalho foi a aplica??o de um question?rio com escala do tipo Likert com quest?es fechadas, constitu?do de vari?veis que compuseram os grupos denominados: obst?culos ? exporta??o, estrat?gia de mercado, n?vel de maturidade, vantagens competitivas, ?ndice de quebra da castanha do caju, potencial produtivo e perfil s?cio-econ?mico dos cooperados. Como forma de an?lise de dados, foi utilizada an?lise descritiva. Ap?s a identifica??o de alguns fatores de ganhos quantitativos no processo de beneficiamento da castanha de caju, s?o apresentadas recomenda??es ? COOPERCAJU para promover cursos de capacita??o e consultorias t?cnicas aos seus cooperados, de modo que haja uma maior efic?cia no processamento industrial da castanha de caju
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