Desenvolvimento de um bioprocesso para a produção, recuperação e formulação de fitase termoestável de Ganoderna sp. MR-56 obtida por cultivo submerso

Orientadora: Prof(a). Dr(a). Michele Rigon SpierCoorientadores: Prof. Dr. Carlos Ricardo Soccol, Prof(a). Dr(a). Luciana P.S. VandenbergheTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa : Curitiba, 12/02/2015Inclui referênciasResumo: A presente tese teve como objetivo a produção de fitase termoestável de Ganoderma sp. MR-56 empregando o extrato de farelo de trigo, um subproduto agroindustrial, como substrato para o cultivo submerso. As fitases são enzimas comumente utilizadas em processamento de ração animal e alimentos para catalisar a degradação do ácido fítico em inositol e fosfatos. O ácido fítico é considerado um fator anti-nutricional presente nos vegetais, pois diminui a biodisponibilidade de alguns nutrientes em aves e suínos. Dessa forma, a aplicação da fitase reduz a adição de fosfato inorgânico nas rações, consequentemente, um benefício econômico e a diminuição da excreção desse mineral no meio ambiente. Atualmente as fitases produzidas industrialmente têm origem microbiana e são obtidas por processos fermentativos. Porém, a produção de fitases de macromicetos ainda é limitada e por cultivos submersos nunca reportados na literatura até o presente estudo. A tese teve como objetivo identificar o micro-organismo produtor de fitase e otimizar a produção da enzima. Além disso, a recuperação, caracterização, formulação e o estudo da estabilidade da enzima também foram realizados. O Ganoderma sp. foi identificado por métodos morfológico e molecular. A otimização da produção de fitase foi constituída por 2 etapas: a primeira consistiu em um delineamento experimental do tipo DCCR (Delineamento Composto Central Rotacional) para estudar a suplementação do meio de cultivo com melaço de soja, extrato de levedura e cloreto de cálcio. A segunda etapa da otimização foi constituída de um DCCR e os fatores estudados foram temperatura de cultivo, pH inicial e taxa de inoculação. O meio de cultivo otimizado continha extrato de levedura 8% m/v, temperatura de 30°C, pH 6,0 e taxa de inoculação de 3% v/v e a produção máxima de fitase foi de 14,5 U mL-1. Nos estudos de cultivo submerso em diferentes tipos de biorreatores, o frasco tipo Dreschel com 2 vvm resultou na melhor produção da enzima de 20,94 U mL-1, porém o STR alcaçou uma melhor produtividade de fitase de 0,14 U-1mL-1h-1. A concentração por ultrafiltração em dispositivos de centrifugação em tubos com poro de 30 kDa apresentou maior concentração da enzima em 36,75 vezes. As estratégias de purificação de cromatografia de troca iônica, filtração em gel S-100 e S-400 não foram eficientes para a separação da fitase. Por outro lado, o zimograma indicou bandas com atividade de fitase as quais apresentaram elevada massa molecular. A fitase foi caracterizada e apresentou pH ótimo entre 4,5 e 5,0, e adequada termoestabilidade à 80 e 90ºC/30 min com 100 e 94,24% de atividade residual, respectivamente. A fitase foi ativada por Mn2+, Ca2+, Na+, Co2+, Fe2+, EDTA e fosfato inorgânico na concentração de 1 mM. Após seriados estudos de formulação líquida com emprego de aditivos, verificou-se que a presença do antimicrobiano A1 0,21% e antioxidante O2 0,0021% foram importantes para a manutenção da estabilidade da enzima durante o armazenamento (103,5% de atividade relativa) em 15 dias em condições aceleradas (40ºC). Os estudos de armazenamento à temperatura ambiente da fitase em pó resultaram que o polímero E2 apresentou 79% de atividade residual após 90 dias de estudo. A fitase produzida e formulada apresentou um potencial para possível aplicação em ração animal e produtos para consumo humano com o intuito de diminuir o fitato, bem como melhorar a absorção de alguns nutrientes. Palavras-chave: fitase, Ganoderma sp. MR-56, otimização, concentração, formulação, aditivos e spray-drying.Abstract: The aim of this thesis was the production of a thermostable phytase from Ganoderma sp. MR-56 by submerged culture, using wheat bran extract as agroindustrial by-product. Phytases are enzymes employed in processing feed and food to catalyze the degradation of phytic acid to inositol and phosphates. The phytic acid is considered an antinutritional factor present in vegetables and decreases the bioavailability of some nutrients in poultry and pigs. The phytase application reduces the inorganic phosphate addition on feed, therefore, an economic benefit and diminished excretion of this mineral into the environment. Currently, phytases from microbial source have been obtained by industrial fermentation processes. However, phytases production by macromycetes is still limited and by submerged culture production had never been reported before in the literature. This thesis aims to identify the microorganism producing phytase and optimize enzyme production by submerged culture. In addition, recovery, characterization, formulation and stability studies of phytase were performed. The Ganoderma sp. was identified by morphological and molecular methods. The optimization of phytase bioprocess was composed by two steps: the first consisted of an experimental design CCRD two levels and three factors to study the submerged culture medium supplementation composed of soybean molasses, yeast extract and calcium chloride. The second optimization consisted of CCRD and the factors studied were culture temperature, initial pH and inoculum rate. The optimized medium contained 8% w/v yeast extract, temperature of 30°C, pH 6.0 and 3% v/v inoculum rate and the maximum phytase production reached 14.5 U mL-1. Studies of different bioreactors for phytase synthesis, the Dreschel bottle type with 2 vvm was the best for enzyme production (20.94 U mL-1), however in STR reached the best phytase productivity of 0,14 U-1mL- 1h-1. The ultrafiltration tubes using 30 kDa fraction retained was 36.75-fold of concentrated enzyme. Strategies purification, of ion exchange chromatography, gel filtration S-100 and S-400 were not effective for the phytase separation. Phytase zymogram suggested activity bands with estimated high molecular weight. The enzyme was characterized and had optimum pH of 5.0 and a thermo stability phytase at 80 to 90ºC for 30 min showed 100 and 94.24% of residual activity, respectively. The enzyme was activated by Mn2+, Ca2+, Na+, Co2+ and Fe2 +, EDTA and inorganic phosphate at concentrations of 1 mM. After series of studies for phytase liquid formulation additives, it was found that the antimicrobial 0.21% A1 and antioxidant 0.0021% O2 were important to maintain enzyme stability during storage 103.5% phytase residual activity on 15 days at 40°C. The formulation powder studies allowed select the polymer E2 for phytase encapsulation with 79% phytase activity relative at room temperature after 90 days of storage. The produced and formulated phytase presented a potential application for feed and processed food in order to reduce the phytate as well as improving some nutrients absorption. Keywords: phytase, Ganoderma sp., optimization, concentration, formulation, additives and spray-drying

Desenvolvimento de um bioprocesso para a produção, caracterização e recuperação da Fitase de Schizophyllum Commune obtida por fermentação em estado sólido

Orientadora: Profa. Dra. Michele Rigon SpierCo-orientadores: Prof. Dr. Carlos Ricardo Soccol, Profa. Dra. Luciana P.S. VandenbergheDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduaçao em Processos Biotecnológicos. Defesa: Curitiba, 29/04/2011Bibliografia: fls. 91-107Área de concentração:Agroindústria e BiocombustíveisResumo: As fitases hidrolisam o acido fitico em inositol e fosfatos, os quais se encontram armazenados em graos, sementes e legumes. Fitases podem ser produzidas por processos de fermentacao submersa (FSm) ou fermentacao no estado solido (FES). A FES e a mais vantajosa, pois pode utilizar residuos agroindustriais como substrato/suporte, os quais apresentam baixo custo e possuem elevado rendimento de producao final. As fitases apresentam aplicacoes em racao animal e produtos para consumo humano. Estas melhoram a absorcao do fosforo, de outros nutrientes no organismo e tambem reduzem a quantidade de fosforo eliminado nos excrementos dos animais resultando em beneficios para o meio ambiente. O presente trabalho teve como objetivo otimizar a producao da enzima fitase de S. commune por FES utilizando o farelo de trigo como substrato/suporte, caracterizar, recuperar a fitase produzida, estudar a extracao liquido-liquido da fitase alem de estudar a sua estabilidade durante o armazenamento. O basidiomiceto Schizophyllum commune foi selecionado como um produtor de enzima fitase utilizando o farelo de trigo como substrato/suporte para a producao de fitase. Para a otimizacao da producao de fitase foi realizado um planejamento experimental fatorial fracionado Box-Behncken design 35 utilizando cinco variaveis independentes (temperatura, concentracao de sacarose, concentracao de extrato de levedura, pH e taxa de inoculo) em tres niveis (+1, 0, -1) e cinco pontos centrais totalizando 37 experimentos. A producao maxima de fitase (113,76 U/gbs) foi obtida com o meio suplementado com sacarose 5%, pH 7, taxa de inoculo 7,5% e temperatura de fermentacao de 33 ‹C. Esse resultado aumentou 285% a producao de fitase em 72 horas de fermentacao. A fitase apresentou uma atividade otima em pH 5 e temperatura de 50 ‹C, Km e Vmax de 0,16 mM e 1,85 ƒÊmol/mL.min respectivamente. A fitase foi ativada na presenca de K+, Ca2+, Mg2+, Mn2+, Zn2+, Cu2+, Fe2+, Fe3+, Co2+, Ni2+, Na+ acetato e citrato e completamente inibida por molibdato de amonio. A melhor condicao de armazenamento para a manutencao da estabilidade da enzima sob refrigeracao (4 ‹C) com 22% de atividade relativa em 125 dias. Estudos preliminares com estabilizantes no extrato bruto enzimatico foram realizados apontando o aditivo A (0,25% p/v) como o melhor agente estabilizante o qual mantem 109% de atividade relativa durante 90 dias de armazenamento a temperatura ambiente de 25 ‹C. A extracao liquido-liquido da enzima utilizando as condicoes de concentracao de citrato 14% (m/m), massa molar de PEG 1500, concentracao do PEG 22% (m/m) e pH 7 tambem apresentou um resultado satisfatorio, com recuperacao de 367 %, fator de purificacao de 5,43 e coeficiente de particao de 2,63.Abstract: Phytases hydrolyze phytic acid to inositol and phosphates, which are stored in grains, seeds and vegetables. Phytases can be produced by submerged fermentation (SmF) and solid-state fermentation which is most used and commercially advantageous. It can use agroindustrial residues as substrate/support, which reduces the cost of the bioprocess and the final price of the enzyme. Phytases have applications in feed and products for human consumption. Phytases improve phosphorus absorption and others nutrients and also reduce the amount of phosphorus eliminated in the animals excrements resulting in benefits to the environment. This study aimed to optimize the phytase production of S. commune by SSF, characterize, recovery the produced phytase, study the liquid-liquid extraction and the stability during storage. The basidiomycete Schizophyllum commune was selected as a major of phytase producer using wheat bran as substrate/support for the phytase production. The optimization of phytase production was carried out by a full 35 fractional factorial Box-Behnken experimental designs using five independent variables (temperature, sucrose concentration, yeast extract concentration, pH and inoculum rate) at two levels (+1, 0, -1) and five central points totalizing 37 experiments. The maximal level of phytase (113.76 U/gds) was obtained in a medium containing sucrose 5%, pH 7.0, inoculum rate 7.5% at 33ºC during 72 hours. This result increased 285% the phytase production in 72 hours fermentation. The enzyme had an optimum pH 5 and 50°C, Km and Vmax of 0.16 mM and 1.85 mmol / mL min, respectively. The enzyme was activated in the presence of K+, Ca2 +, Mg2 +, Mn2 +, Zn2 +, Cu2 +, Fe2 +, Fe3 +, Co2 +, Ni2+, Na+, acetate and citrate and completely inhibited by ammonium molybdate. The best storage condition for maintaining the enzyme stability was at 4 ° C, with 22% relative activity in 125 days. Preliminary studies with stabilizers agents in crude enzymatic extract were performed, resulting in additive A (0.25% w/v) as the best stabilizing agent with 109% relative activity during 90 days of storage at room temperature 25°C. The liquid liquid extraction of ezyme using citrate 14% (m/m), PEG 1500, PEG concentration of 22% (w/w) and pH 7 also had satisfactory results, with a partition coefficient of 2.63, yield 367% and purification factor of 5.43

Analysis of inducers of xylanase and cellulase activities production by Ganoderma applanatum LPB MR-56

n/

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Desenvolvimento de um bioprocesso para a produção, recuperação e formulação de fitase termoestável de Ganoderna sp. MR-56 obtida por cultivo submerso

Orientadora: Prof(a). Dr(a). Michele Rigon SpierCoorientadores: Prof. Dr. Carlos Ricardo Soccol, Prof(a). Dr(a). Luciana P.S. VandenbergheTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa : Curitiba, 12/02/2015Inclui referênciasResumo: A presente tese teve como objetivo a produção de fitase termoestável de Ganoderma sp. MR-56 empregando o extrato de farelo de trigo, um subproduto agroindustrial, como substrato para o cultivo submerso. As fitases são enzimas comumente utilizadas em processamento de ração animal e alimentos para catalisar a degradação do ácido fítico em inositol e fosfatos. O ácido fítico é considerado um fator anti-nutricional presente nos vegetais, pois diminui a biodisponibilidade de alguns nutrientes em aves e suínos. Dessa forma, a aplicação da fitase reduz a adição de fosfato inorgânico nas rações, consequentemente, um benefício econômico e a diminuição da excreção desse mineral no meio ambiente. Atualmente as fitases produzidas industrialmente têm origem microbiana e são obtidas por processos fermentativos. Porém, a produção de fitases de macromicetos ainda é limitada e por cultivos submersos nunca reportados na literatura até o presente estudo. A tese teve como objetivo identificar o micro-organismo produtor de fitase e otimizar a produção da enzima. Além disso, a recuperação, caracterização, formulação e o estudo da estabilidade da enzima também foram realizados. O Ganoderma sp. foi identificado por métodos morfológico e molecular. A otimização da produção de fitase foi constituída por 2 etapas: a primeira consistiu em um delineamento experimental do tipo DCCR (Delineamento Composto Central Rotacional) para estudar a suplementação do meio de cultivo com melaço de soja, extrato de levedura e cloreto de cálcio. A segunda etapa da otimização foi constituída de um DCCR e os fatores estudados foram temperatura de cultivo, pH inicial e taxa de inoculação. O meio de cultivo otimizado continha extrato de levedura 8% m/v, temperatura de 30°C, pH 6,0 e taxa de inoculação de 3% v/v e a produção máxima de fitase foi de 14,5 U mL-1. Nos estudos de cultivo submerso em diferentes tipos de biorreatores, o frasco tipo Dreschel com 2 vvm resultou na melhor produção da enzima de 20,94 U mL-1, porém o STR alcaçou uma melhor produtividade de fitase de 0,14 U-1mL-1h-1. A concentração por ultrafiltração em dispositivos de centrifugação em tubos com poro de 30 kDa apresentou maior concentração da enzima em 36,75 vezes. As estratégias de purificação de cromatografia de troca iônica, filtração em gel S-100 e S-400 não foram eficientes para a separação da fitase. Por outro lado, o zimograma indicou bandas com atividade de fitase as quais apresentaram elevada massa molecular. A fitase foi caracterizada e apresentou pH ótimo entre 4,5 e 5,0, e adequada termoestabilidade à 80 e 90ºC/30 min com 100 e 94,24% de atividade residual, respectivamente. A fitase foi ativada por Mn2+, Ca2+, Na+, Co2+, Fe2+, EDTA e fosfato inorgânico na concentração de 1 mM. Após seriados estudos de formulação líquida com emprego de aditivos, verificou-se que a presença do antimicrobiano A1 0,21% e antioxidante O2 0,0021% foram importantes para a manutenção da estabilidade da enzima durante o armazenamento (103,5% de atividade relativa) em 15 dias em condições aceleradas (40ºC). Os estudos de armazenamento à temperatura ambiente da fitase em pó resultaram que o polímero E2 apresentou 79% de atividade residual após 90 dias de estudo. A fitase produzida e formulada apresentou um potencial para possível aplicação em ração animal e produtos para consumo humano com o intuito de diminuir o fitato, bem como melhorar a absorção de alguns nutrientes. Palavras-chave: fitase, Ganoderma sp. MR-56, otimização, concentração, formulação, aditivos e spray-drying.Abstract: The aim of this thesis was the production of a thermostable phytase from Ganoderma sp. MR-56 by submerged culture, using wheat bran extract as agroindustrial by-product. Phytases are enzymes employed in processing feed and food to catalyze the degradation of phytic acid to inositol and phosphates. The phytic acid is considered an antinutritional factor present in vegetables and decreases the bioavailability of some nutrients in poultry and pigs. The phytase application reduces the inorganic phosphate addition on feed, therefore, an economic benefit and diminished excretion of this mineral into the environment. Currently, phytases from microbial source have been obtained by industrial fermentation processes. However, phytases production by macromycetes is still limited and by submerged culture production had never been reported before in the literature. This thesis aims to identify the microorganism producing phytase and optimize enzyme production by submerged culture. In addition, recovery, characterization, formulation and stability studies of phytase were performed. The Ganoderma sp. was identified by morphological and molecular methods. The optimization of phytase bioprocess was composed by two steps: the first consisted of an experimental design CCRD two levels and three factors to study the submerged culture medium supplementation composed of soybean molasses, yeast extract and calcium chloride. The second optimization consisted of CCRD and the factors studied were culture temperature, initial pH and inoculum rate. The optimized medium contained 8% w/v yeast extract, temperature of 30°C, pH 6.0 and 3% v/v inoculum rate and the maximum phytase production reached 14.5 U mL-1. Studies of different bioreactors for phytase synthesis, the Dreschel bottle type with 2 vvm was the best for enzyme production (20.94 U mL-1), however in STR reached the best phytase productivity of 0,14 U-1mL- 1h-1. The ultrafiltration tubes using 30 kDa fraction retained was 36.75-fold of concentrated enzyme. Strategies purification, of ion exchange chromatography, gel filtration S-100 and S-400 were not effective for the phytase separation. Phytase zymogram suggested activity bands with estimated high molecular weight. The enzyme was characterized and had optimum pH of 5.0 and a thermo stability phytase at 80 to 90ºC for 30 min showed 100 and 94.24% of residual activity, respectively. The enzyme was activated by Mn2+, Ca2+, Na+, Co2+ and Fe2 +, EDTA and inorganic phosphate at concentrations of 1 mM. After series of studies for phytase liquid formulation additives, it was found that the antimicrobial 0.21% A1 and antioxidant 0.0021% O2 were important to maintain enzyme stability during storage 103.5% phytase residual activity on 15 days at 40°C. The formulation powder studies allowed select the polymer E2 for phytase encapsulation with 79% phytase activity relative at room temperature after 90 days of storage. The produced and formulated phytase presented a potential application for feed and processed food in order to reduce the phytate as well as improving some nutrients absorption. Keywords: phytase, Ganoderma sp., optimization, concentration, formulation, additives and spray-drying

Desenvolvimento de um bioprocesso para a produção, caracterização e recuperação da Fitase de Schizophyllum Commune obtida por fermentação em estado sólido

Orientadora: Profa. Dra. Michele Rigon SpierCo-orientadores: Prof. Dr. Carlos Ricardo Soccol, Profa. Dra. Luciana P.S. VandenbergheDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduaçao em Processos Biotecnológicos. Defesa: Curitiba, 29/04/2011Bibliografia: fls. 91-107Área de concentração:Agroindústria e BiocombustíveisResumo: As fitases hidrolisam o acido fitico em inositol e fosfatos, os quais se encontram armazenados em graos, sementes e legumes. Fitases podem ser produzidas por processos de fermentacao submersa (FSm) ou fermentacao no estado solido (FES). A FES e a mais vantajosa, pois pode utilizar residuos agroindustriais como substrato/suporte, os quais apresentam baixo custo e possuem elevado rendimento de producao final. As fitases apresentam aplicacoes em racao animal e produtos para consumo humano. Estas melhoram a absorcao do fosforo, de outros nutrientes no organismo e tambem reduzem a quantidade de fosforo eliminado nos excrementos dos animais resultando em beneficios para o meio ambiente. O presente trabalho teve como objetivo otimizar a producao da enzima fitase de S. commune por FES utilizando o farelo de trigo como substrato/suporte, caracterizar, recuperar a fitase produzida, estudar a extracao liquido-liquido da fitase alem de estudar a sua estabilidade durante o armazenamento. O basidiomiceto Schizophyllum commune foi selecionado como um produtor de enzima fitase utilizando o farelo de trigo como substrato/suporte para a producao de fitase. Para a otimizacao da producao de fitase foi realizado um planejamento experimental fatorial fracionado Box-Behncken design 35 utilizando cinco variaveis independentes (temperatura, concentracao de sacarose, concentracao de extrato de levedura, pH e taxa de inoculo) em tres niveis (+1, 0, -1) e cinco pontos centrais totalizando 37 experimentos. A producao maxima de fitase (113,76 U/gbs) foi obtida com o meio suplementado com sacarose 5%, pH 7, taxa de inoculo 7,5% e temperatura de fermentacao de 33 ‹C. Esse resultado aumentou 285% a producao de fitase em 72 horas de fermentacao. A fitase apresentou uma atividade otima em pH 5 e temperatura de 50 ‹C, Km e Vmax de 0,16 mM e 1,85 ƒÊmol/mL.min respectivamente. A fitase foi ativada na presenca de K+, Ca2+, Mg2+, Mn2+, Zn2+, Cu2+, Fe2+, Fe3+, Co2+, Ni2+, Na+ acetato e citrato e completamente inibida por molibdato de amonio. A melhor condicao de armazenamento para a manutencao da estabilidade da enzima sob refrigeracao (4 ‹C) com 22% de atividade relativa em 125 dias. Estudos preliminares com estabilizantes no extrato bruto enzimatico foram realizados apontando o aditivo A (0,25% p/v) como o melhor agente estabilizante o qual mantem 109% de atividade relativa durante 90 dias de armazenamento a temperatura ambiente de 25 ‹C. A extracao liquido-liquido da enzima utilizando as condicoes de concentracao de citrato 14% (m/m), massa molar de PEG 1500, concentracao do PEG 22% (m/m) e pH 7 tambem apresentou um resultado satisfatorio, com recuperacao de 367 %, fator de purificacao de 5,43 e coeficiente de particao de 2,63.Abstract: Phytases hydrolyze phytic acid to inositol and phosphates, which are stored in grains, seeds and vegetables. Phytases can be produced by submerged fermentation (SmF) and solid-state fermentation which is most used and commercially advantageous. It can use agroindustrial residues as substrate/support, which reduces the cost of the bioprocess and the final price of the enzyme. Phytases have applications in feed and products for human consumption. Phytases improve phosphorus absorption and others nutrients and also reduce the amount of phosphorus eliminated in the animals excrements resulting in benefits to the environment. This study aimed to optimize the phytase production of S. commune by SSF, characterize, recovery the produced phytase, study the liquid-liquid extraction and the stability during storage. The basidiomycete Schizophyllum commune was selected as a major of phytase producer using wheat bran as substrate/support for the phytase production. The optimization of phytase production was carried out by a full 35 fractional factorial Box-Behnken experimental designs using five independent variables (temperature, sucrose concentration, yeast extract concentration, pH and inoculum rate) at two levels (+1, 0, -1) and five central points totalizing 37 experiments. The maximal level of phytase (113.76 U/gds) was obtained in a medium containing sucrose 5%, pH 7.0, inoculum rate 7.5% at 33ºC during 72 hours. This result increased 285% the phytase production in 72 hours fermentation. The enzyme had an optimum pH 5 and 50°C, Km and Vmax of 0.16 mM and 1.85 mmol / mL min, respectively. The enzyme was activated in the presence of K+, Ca2 +, Mg2 +, Mn2 +, Zn2 +, Cu2 +, Fe2 +, Fe3 +, Co2 +, Ni2+, Na+, acetate and citrate and completely inhibited by ammonium molybdate. The best storage condition for maintaining the enzyme stability was at 4 ° C, with 22% relative activity in 125 days. Preliminary studies with stabilizers agents in crude enzymatic extract were performed, resulting in additive A (0.25% w/v) as the best stabilizing agent with 109% relative activity during 90 days of storage at room temperature 25°C. The liquid liquid extraction of ezyme using citrate 14% (m/m), PEG 1500, PEG concentration of 22% (w/w) and pH 7 also had satisfactory results, with a partition coefficient of 2.63, yield 367% and purification factor of 5.43

Characterization of Hemicellulolytic Enzymes Produced by Aspergillus niger NRRL 328 under Solid State Fermentation on Soybean Husks

This manuscript describes the analysis of xylanase production by Aspergillus niger NRRL 328 in solid state fermentation (SSF) of soybean husks. A maximum value of extracellular xylanase activity of approximately 950 U g-1 was achieved after 96 h. Proteomic analyses performed on the enzymatic mixture responsible for the maximum value of xylanase activity in SSF revealed the presence of two xylanases. This xylanolytic mixture was partially purified and characterized. It followed Michaelis-Menten kinetics towards xylan, with a KM of 7.92 ±0.97 mg xylan/mL and a Vmax of 262.2 ± 27.8 g L-1 s-1. The optimum pH for the enzyme is 5.3, and the optimal temperature is 50 °C. The enzyme retains 100% of its activity at 40 °C for at least 1 month. It shows very high stability in a broad pH range, with a half-life of 40 days at pH 5.3, pH 6.0, pH 6.5, pH 7.0, and pH 8.0

Thrombotic and hemorrhagic complications of COVID-19 in adults hospitalized in high-income countries compared with those in adults hospitalized in low- and middle-income countries in an international registry

Background: COVID-19 has been associated with a broad range of thromboembolic, ischemic, and hemorrhagic complications (coagulopathy complications). Most studies have focused on patients with severe disease from high-income countries (HICs). Objectives: The main aims were to compare the frequency of coagulopathy complications in developing countries (low- and middle-income countries [LMICs]) with those in HICs, delineate the frequency across a range of treatment levels, and determine associations with in-hospital mortality. Methods: Adult patients enrolled in an observational, multinational registry, the International Severe Acute Respiratory and Emerging Infections COVID-19 study, between January 1, 2020, and September 15, 2021, met inclusion criteria, including admission to a hospital for laboratory-confirmed, acute COVID-19 and data on complications and survival. The advanced-treatment cohort received care, such as admission to the intensive care unit, mechanical ventilation, or inotropes or vasopressors; the basic-treatment cohort did not receive any of these interventions. Results: The study population included 495,682 patients from 52 countries, with 63% from LMICs and 85% in the basic treatment cohort. The frequency of coagulopathy complications was higher in HICs (0.76%-3.4%) than in LMICs (0.09%-1.22%). Complications were more frequent in the advanced-treatment cohort than in the basic-treatment cohort. Coagulopathy complications were associated with increased in-hospital mortality (odds ratio, 1.58; 95% CI, 1.52-1.64). The increased mortality associated with these complications was higher in LMICs (58.5%) than in HICs (35.4%). After controlling for coagulopathy complications, treatment intensity, and multiple other factors, the mortality was higher among patients in LMICs than among patients in HICs (odds ratio, 1.45; 95% CI, 1.39-1.51). Conclusion: In a large, international registry of patients hospitalized for COVID-19, coagulopathy complications were more frequent in HICs than in LMICs (developing countries). Increased mortality associated with coagulopathy complications was of a greater magnitude among patients in LMICs. Additional research is needed regarding timely diagnosis of and intervention for coagulation derangements associated with COVID-19, particularly for limited-resource settings

Association of Country Income Level With the Characteristics and Outcomes of Critically Ill Patients Hospitalized With Acute Kidney Injury and COVID-19

Introduction: Acute kidney injury (AKI) has been identified as one of the most common and significant problems in hospitalized patients with COVID-19. However, studies examining the relationship between COVID-19 and AKI in low- and low-middle income countries (LLMIC) are lacking. Given that AKI is known to carry a higher mortality rate in these countries, it is important to understand differences in this population. Methods: This prospective, observational study examines the AKI incidence and characteristics of 32,210 patients with COVID-19 from 49 countries across all income levels who were admitted to an intensive care unit during their hospital stay. Results: Among patients with COVID-19 admitted to the intensive care unit, AKI incidence was highest in patients in LLMIC, followed by patients in upper-middle income countries (UMIC) and high-income countries (HIC) (53%, 38%, and 30%, respectively), whereas dialysis rates were lowest among patients with AKI from LLMIC and highest among those from HIC (27% vs. 45%). Patients with AKI in LLMIC had the largest proportion of community-acquired AKI (CA-AKI) and highest rate of in-hospital death (79% vs. 54% in HIC and 66% in UMIC). The association between AKI, being from LLMIC and in-hospital death persisted even after adjusting for disease severity. Conclusions: AKI is a particularly devastating complication of COVID-19 among patients from poorer nations where the gaps in accessibility and quality of healthcare delivery have a major impact on patient outcomes

Effect of Antiplatelet Therapy on Survival and Organ Support–Free Days in Critically Ill Patients With COVID-19

International audienc