Efeito da homogeneização à alta pressão na atividade e estabilidade de enzimas

Orientador: Marcelo CristianiniTexto em português e inglêsTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: A homogeneização à alta pressão (HAP) é uma operação unitária capaz de alterar a conformação e, consequentemente a funcionalidade de polissacarídeos,proteínas e enzimas. O objetivo deste trabalho foi avaliar o efeito da HAP na atividade e estabilidade de cinco enzimas comerciais com aplicação na indústria de alimentos (x-amilase de Aspergillus niger, protease neutra de Bacillus subtilis, b-galactosidase de Kluyveromyces lactis, amiloglicosidase de A. niger e glicose oxidase de A. niger). Para cada enzima, a atividade foi avaliada antes e após a HAP (até 200 MPa) em diferentes temperaturas e pH. Além disso, a reversibilidade dos efeitos do processo foi determinada indiretamente através da medida de atividade da enzima após um período de repouso. Os resultados de x-amilase demonstraram que a enzima é altamente estável ao processo de HAP (em pressões de até 150 MPa), independentemente do pH e temperatura de processo e da ausência de cálcio no tampão de diluição da enzima. Os resultados da b-galactosidase, por outro lado, mostraram que a enzima é pouco estável, apresentando redução da atividade (~30%) após HAP a 150 MPa quando processadas em pH não ótimo para atividade da enzima. Os resultados obtidos para a protease neutra, amiloglicosidase e glicose oxidase indicaram que o efeito da HAP foi dependente dos parâmetros utilizados no processo (pH, temperatura e pressão de homogeneização) e das condições utilizadas na medida de atividade (pH, temperatura e tempo de estocagem). Para estas três enzimas, significativos ganhos de atividade e/ou estabilidade foram observados para pelo menos uma das condições avaliadas, sendo que os mais importantes foram: (i) redução da temperatura ótima de atividade da protease neutra de 55 para 20ºC após HAP a 200 MPa, (ii) aumento da atividade da glicose-oxidase à 75ºC após HAP a 150 MPa, (iii) aumento da atividade residual entre 100 e 400% após armazenamento refrigerado de glicose-oxidase homogeneizada em diferentes pressões, (iv) aumento da atividade de amiloglicosidase à 80ºC após a HAP a 100 MPa. A reversibilidade das alterações observadas foi inferida pela avaliação da atividade da enzima após um período de repouso, sendo as alterações determinadas como reversíveis (protease neutra, glicose-oxidase, amiloglicosidase) ou irreversíveis (protease neutra, glicose-oxidase, b-galactosidase) em função dos parâmetros de processo. O efeito de processamentos sequenciais sobre a glicose oxidase, a protease e a amiloglicosidase também foi avaliado e os resultados demonstraram que, para a maioria das condições estudadas, a atividade da enzima se manteve igual à obtida após o primeiro ciclo de homogeneização ou apresentou uma redução. Uma exceção foram os resultados da glicose oxidase homogeneizada a 150 MPa por 3 vezes, que apresentou aumento de atividade de aproximadamente 150% em relação à enzima nativa. A partir dos resultados, conclui-se que o efeito da HAP é diferente para cada enzima e que as maiores alterações ocorrem em condições de atividade não ótima e para enzimas de estruturas mais complexas, como é o caso da glicose oxidase. Os resultados obtidos apresentam aplicação direta, para modificação e melhoria do desempenho de enzimas comerciais, e preenchem uma lacuna científica importante sobre o conhecimento dos efeitos do processo de HAP em enzimasAbstract: High pressure homogenization (HPH) is a unitary operation capable to alter the conformation and, consequently, the functionality of polyssacharides, proteins and enzymes. This work aimed to study the HPH effects on activity and stability of five commercial enzymes intensively applied in food industry (x-amylase from Aspergillus niger, b-galactosidase from Kluyveromyces lactis, neutral protease from Bacillus subtilis, amyloglucosidase from A. niger and glucose-oxidase from A. niger). The activity of each enzyme was studied before and after HPH process (up to 200 MPa) at different temperatures and pH. Moreover, the process reversibility was indirectly determined by the activity measured after a rest period under refrigeration (8ºC).The results revealed that x-amylase was highly stable to HPH up to 150 MPa,independent on the pH or temperature used in the HPH process or the presence of calcium in buffer. On the other hand, the results of b-galactosidase indicated that enzyme was partially inactivated (~ 30%) after homogenization at 150 MPa when processed at non optimum pH. The HPH effects on neutral protease, amyloglucosidase (AMG) and glucose oxidase (GO) were dependent on the process parameters (pH, temperature and homogenization conditions) and the activity measurement conditions (pH, temperature and storage time). For these enzymes, it was observed activity and/or stability improvement after some process. The main improvements were: (i) change of the optimum temperature of neutral protease from 55 to 20ºC after HPH at 200 MPa, (ii) improvement of GO activity at 75ºC after HPH at 150 MPa, (iii) enzyme activity improvement between 100 and 400% after GO refrigerated storage, (iv) improvement on amyloglucosidase activity at 80ºC after HPH at 100 MPa. The reversibility of the HPH effects was evaluated after a rest period.The reversibility was dependent on the process parameters; but, in general, neutral protease, GO and AMG were reversible, while the results of neutral protease, GO and b-galactosidase were irreversible. The effects of sequential homogenization processes (sequential passes) on GO, AMG and neutral protease were evaluated and the results showed that the enzyme activity remained equal or reduced after 2 or 3 cycles of homogenization. An exception was the result obtained for GO homogenized at 150 MPa, which showed an activity improvement of 150% after three passes. The results evaluation of this research showed that HPH effects on enzymes were different for each enzyme. The main alterations occured at non optimum condition of enzyme activity and for enzymes with complex structure as the GO. The obtained results can be directly applied for improvement of enzyme industrial production. Also, the results enriched the scientific knowledge about the HPH effects on enzymesDoutoradoTecnologia de AlimentosDoutora em Tecnologia de Alimento

“O que eu faço, o chefe não faz; o que ele faz, eu aprendo” : trabalho cooperado como alternativa à exploração no trabalho?

Trabalho de Conclusão de Curso (graduação) — Universidade de Brasília, Instituto de Ciências Sociais, Departamento de Sociologia, 2021.Este projeto objetiva compreender as experiências prévias e posteriores ao trabalho cooperado; o contexto que leva trabalhadores a transitar de vínculos tradicionais ou precarizados ao trabalho cooperado; o que impulsiona e o que permite esse movimento; identificar condições, contingências, motivações, oportunidades, desafios que surgem durante a transição; compreender como se dá a nova relação, como a organização do local de trabalho se modifica; e as consequências do novo arranjo para os trabalhadores. Para atingir esses objetivos, propomos uma revisão bibliográfica sobre as transformações recentes no mundo do trabalho e entrevistas com trabalhadores cooperados no setor de projetos arquitetônicos de Brasília.This project tries to understand the experiences before and after cooperative work; the context that drives workers to move away from traditional or precarious bonds to cooperative ones, conditions that drive and allow this movement; to identify the conditions, contingencies, motivations, opportunities, challenges that arise during this kind of transition; to understand how the new relationship takes place, how the organization of the workplace changes; and the consequences of the new arrangement for workers. In order to achieve these goals, we propose a literature review of recent transformations in the world of work and interviews with cooperative workers in the architectural design sector of Brasilia

Motivos que llevan a la práctica del baloncesto : un estudio con jóvenes atletas brasilenos

Este estudio tiene como objetivo caracterizar los motivos que llevan a adolescentes a escoger el baloncesto como una práctica deportiva sistemática y competitiva, a través de la categorización en cada sexo analizado. A través del Formulario para Identificación de Situaciones de Estrés en Baloncesto (FISSB), fueron evaluados 135 jugadores de baloncesto, siendo 74 de sexo masculino y 61 del femenino, entre 15 a 18 anos de la Región de la Gran Sao Paulo. De este instrumento fue analizada la pregunta nQué te llevó a iniciar la práctica del balonceston. Fueron identificadas 225 manifestaciones sobre motivos de la práctica del baloncesto, siendo 156 por parte de ninos y 69 por parte de las ninas. Los motivos intrínsecos representaron 41,8% de las respuestas (94) y 131 respuestas estaban vinculadas a motivos extrínsecos (58,2%). Para los motivos intrínsecos fueron determinadas 4 categorías generales (aspiraciones, necesidades cognitivas y emocionales, placer por la modalidad siendo ésta la categoría más importante para los atletas, tanto del sexo masculino como femenino. Para los motivos extrínsecos fueron determinadas 2 categorías generales (personas significativas y ambiente social), siendo que las personas significativas fueron consideradas como las de mayor importancia para el inicio de la práctica del baloncesto, como destacable para los padres y técnicos.The purposes of this study were to identify the real motives that influenced adolescents in choosing basketball as the sport they would practice and compete and to categorize those motives according to similar characteristics, showing the frequency of the motives in each category. The study was made with 135 athletes (74 boys and 61 girls) with age ranging from 15 to 18 years old, from basketball clubs at the Region of Great São Paulo. The athletes answered the Formulary for indentification of Stressful Situations in Basketball (FISSB). For this study it was considered one specific question of the FISSB: «Why did you choose to practice and play basketball?». It were found 225 different manifestations: 156 for the boys and 69 for the girls. The intrinsic motives represented 41.8% of the answers (94), while the extrinsic motives represented 58.2% (131). For the intrinsic motives it were defined four categories: aspirations, emotional necessities, cognitive necessities and pleasure in playing basketball. This category was the most important for both boys and girls. For the extrinsic motives it were defined two categories: social environment and significant people that was the most important both for boys and girls, specially parents and coaches

Effects Of High Pressure Homogenization On The Activity, Stability, Kinetics And Three-dimensional Conformation Of A Glucose Oxidase Produced By Aspergillus Niger.

High pressure homogenization (HPH) is a non-thermal method, which has been employed to change the activity and stability of biotechnologically relevant enzymes. This work investigated how HPH affects the structural and functional characteristics of a glucose oxidase (GO) from Aspergillus niger. The enzyme was homogenized at 75 and 150 MPa and the effects were evaluated with respect to the enzyme activity, stability, kinetic parameters and molecular structure. The enzyme showed a pH-dependent response to the HPH treatment, with reduction or maintenance of activity at pH 4.5-6.0 and a remarkable activity increase (30-300%) at pH 6.5 in all tested temperatures (15, 50 and 75°C). The enzyme thermal tolerance was reduced due to HPH treatment and the storage for 24 h at high temperatures (50 and 75°C) also caused a reduction of activity. Interestingly, at lower temperatures (15°C) the activity levels were slightly higher than that observed for native enzyme or at least maintained. These effects of HPH treatment on function and stability of GO were further investigated by spectroscopic methods. Both fluorescence and circular dichroism revealed conformational changes in the molecular structure of the enzyme that might be associated with the distinct functional and stability behavior of GO.9e10341

Effect of High-Pressure Technologies on Enzymes Applied in Food Processing

High isostatic pressure (HIP) and high-pressure homogenization (HPH) are considered important physical technologies that able to induce changes on enzymes. HIP and HPH are emerging food processing technologies that involve the use of ultra high pressures (up to 1200 MPa for HIP and up to 400 MPa for HPH), where the first process is based on the principle that the maintenance of a product inside vessels at high pressures induces changes in the molecules conformation and, consequently, in the functionality of polysaccharides, proteins and enzymes. To the contrary, for HPH process, the high shear and sudden pressure drop are the responsible phenomena for the changes on the processed product. This chapter aims to evaluate comparatively the effects of HIP and HPH on the activity of enzymes currently applied in food industry and to identify the main structural changes induced by each process. The overall evaluation of the results shows that mild conditions of both processes were recently highlighted as able to improve the activity and the stability of several enzymes, whereas extreme process conditions (pressure, time and temperature) induce enzyme denaturation with consequent reduction of biological activity. Considering the complexity and diversity involved in the enzyme structure and its ability to react, it is not possible to determine specific conditions that each process is able to promote increase or reduction of enzyme activity, being necessary to evaluate HIP and HPH for each enzyme. Finally, in terms of molecular structure, the effect of HIP and HPH on enzymes can be explained by the alterations in the quaternary, tertiary and secondary structures of enzymes, which directly affects its active site configuration

Effect of High Pressure Homogenization Process on Bacillus Stearothermophilus and Clostridium Sporogenes Spores in Skim Milk

AbstractHigh pressure homogenization (HPH) is an alternative food processing technique. As product heating is minimum, its characteristics are not affected in a large extend. This characteristic makes the HPH an interesting process to guarantee the safety of thermo-labile food. Inactivation of Bacillus stearothermophilus ATCC 7953 and Clostridium sporogenes PA 3679 spores in skim milk by HPH was studied. Results showed that pressures up to 300 MPa were not able to cause any reduction on spore counts or promote changes on its thermal resistance. The application of heat shock (100°C/15 min) before HPH treatment and the homogenization process realized at mild inlet temperature (45°C) – which results in homogenization temperature of around 84°C at 300 MPa - also did not cause reduction on viable spores counts. A few spores reduction (0.67 logarithmic cycles) were only observed when the milk samples were subjected to homogenization treatment 16 times (multiple passes) at 300 MPa. Therefore, although HPH be recognized as an effective method for milk pasteurization, it was conclude that the HPH process is not able to guarantee the commercial sterility of milk, being necessary the association of the homogenization with another preservative method, as refrigeration

Aspergillus niger inactivation in mango nectar by dynamic high pressure combined with mild heat treatment

Orientadores: Marcelo Cristianini, Pilar Rodriguez de MassaguerDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: O néctar de manga é apreciado por sua cor e aroma. Este produto, pela sua alta acidez, pode ser contaminado por bolores, dentre os quais são de grande relevância aqueles capazes de sobreviver ao processamento térmico. O processamento a alta pressão dinâmica (APD) é um tratamento ¿a frio¿ realizado para inativação de microrganismos visando uma melhor qualidade sensorial e nutricional do produto obtido. Neste estudo, avaliou-se o efeito do processamento a APD isoladamente ou combinado com tratamento térmico (TT) brando na inativação de Aspergillus niger em néctar de manga e, posteriormente, foi realizada a avaliação do efeito dos processos sobre a cor e a concentração de vitamina C do néctar. Paralelamente foi desenvolvida uma metodologia para limpeza e sanificação do equipamento e um processo para a redução de viscosidade do néctar de manga. Foram estudadas pressões entre 100 e 300 MPa para a inativação do Aspergillus niger. A pressão de 300 MPa inativou toda a carga inicial (> 6,24 reduções decimais), 200 MPa provocou uma inativação parcial (aproximadamente 2 ciclos) e, pressões inferiores a 150 MPa não resultaram em inativação significativa do A. niger. Quando o processo de APD foi associado à TT brando (80ºC/15minutos) foi observado um efeito sinérgico entre pré tratamento a APD e posterior TT (aproximadamente 1 ciclo logaritmo); já quando utilizado pré TT seguido de tratamento a APD, foi observado apenas um efeito aditivo. Utilizando-se aplicação de APD seguida de TT, foi realizado um planejamento experimental fixando-se a pressão em 200 MPa e variando-se temperatura e tempo do TT e ratio do néctar de manga (brix/acidez), de forma a obter um modelo que descrevesse o processo. Os resultados indicaram que tempo e temperatura afetaram positivamente a inativação e o ratio do néctar afetou negativamente. A partir do modelo foram estabelecidos que processos a 200 MPa seguido de TT a 73,5ºC/10 minutos ou 61,5ºC/20 minutos eram suficientes para obter 5 reduções decimais do bolor. A avaliação desses processos combinados, do processo térmico tradicional (100ºC/10 minutos) e do processo utilizando apenas pressão (300 MPa) sobre o conteúdo de vitamina C e cor do néctar demonstrou uma inativação de vitamina C muito similar para todos os processos (perdas de aproximadamente 45%) e uma melhor retenção de cor para os produtos processados à APD. O desenho do processo de limpeza e sanificação para o equipamento foi considerado eficaz uma vez que reduziu para 6.24 decimal reduction), 200 MPa caused a partial inactivation (2 log cycles) and pressures up to 150 MPa did not inactivate the mold. A synergistic effect was observed when pre treatment of DHP was associated to mild TT (80ºC/ 15 minutes) with increase of 1 log cycle on mold inactivation. However, only an additive effect was observed when pre TT was associated to DHP. An experimental design was carried out using fixed pressure of 200 Mpa followed by mild TT. Temperature and time of thermal treatment and ratio of mango nectar (brix/acidity) was select as experimental design variables. The results indicated that time and temperature affected positively and nectar ratio affected negatively the mold inactivation. By the mathematical model, process of 200 MPa followed by 73.5ºC/10 minutes or by 61.5ºC/20 minutes was able to promote 5 decimal reduction of A. niger. The effect of these processes, the conventional heat and DHP at 300 MPa on vitamin C concentration and nectar color retention was evaluated. The vitamin C losses were around 45% for all processes studied but better color retention was observed on the nectar processed by DHP. The CIP design was considered efficient once it was able to reduce the initial load of A. niger from ~106 CFU.mL-1 to <1 CFU.mL-1. 2.5% of a commercial detergent (active agents: sodium toluenosulfonate and didecylmethilamonium chlorine) and peracetic acid at 0.05% were used in this process. The nectar viscosity reduction was effective by using pectic enzymes and celullase, being able to reduce natural mango nectar viscosity by 50%. The results obtained indicated that DHP isolated or combined with mild thermal treatment are a viable alternative to inactivate A. niger in mango nectar. The process has a negative effect on vitamin C retention. It can be attributed to the oxygen dissolved in mango nectar and also to metals seals of equipment (Be-Cu) which can had promoted an increase in metallic ions concentration in nectar. These ions are pro oxidant of vitamin C, resulting in intense vitamin oxidationMestradoMestre em Tecnologia de Alimento

Influence of high pressure homogenization on commercial protease from Rhizomucor miehei: Effects on proteolytic and milk-clotting activities

AbstractThis work studied the influence of high pressure homogenization (HPH) on a commercial fungal protease. The enzyme solutions (2 and 20 g/100 mL) were processed up to 190 MPa and the proteolytic activity (PA), milk-clotting activity (MCA) and the rheological behavior of the milk coagulation phase were evaluated. The effects of multi-pass (three cycles) HPH at 25 and 190 MPa was evaluated for enzyme processed at concentration of 2 g/100 mL. No differences in PA and MCA were observed for the samples of 2 g/100 mL of enzyme concentration processed by HPH. On the other hand, increase in PA (∼3%) and MCA (∼10%) were observed for the enzymes processed at 190 MPa at high concentration, which consequent faster clotting and higher consistency of the milk gel. The multi-pass increased PA (≤6%) but did not alter MCA nor improved the milk coagulation phase. The results highlight that the energy supplied from HPH to enzyme at low concentration is not enough to promote positive changes in the enzyme coagulant profile; however, the HPH of solution with high enzyme concentration showed a positive effect, indicating that the collisions between enzymes during the process was important to reach the observed changes