Otimização das condições de cultivo para a produção de amilases pelo termofílico Bacillus sp. e hidrólise de amidos pela ação da enzima

A otimização das condições de cultivo para a produção de α-amilase por um termofílico Bacillus sp. cepa SMIA-2 foi realizada. Além disso, a hidrólise enzimática do amido, proveniente de várias fontes tais como batata, mandioca e milho, foi também investigada. A produção de α-amilase por Bacillus sp. SMIA-2, cultivado em meio líquido contendo amido (5 g.L-1) como fonte de carbono e suplementado com 0,5 g.L-1 de proteínas do soro de leite e 2 g.L-1 de peptona, alcançou o máximo em 32 horas com níveis de 37 U.mL-1. O microrganismo foi capaz de utilizar diversas fontes de carbono, porém a atividade da amilase variou com cada fonte. O amido foi a melhor fonte de carbono para a secreção da amilase, enquanto a sacarose, lactose, maltose, galactose e glicose não foram muito efetivas. Uma redução na concentração de amido de até 2,5 g.L-1 no meio de cultura melhorou o crescimento do organismo e a atividade enzimática. Em altas concentrações de amido, a produção da enzima foi comparativamente menor. Em relação às fontes de nitrogênio orgânico e inorgânico, a peptona (2 g.L-1) foi considerada a melhor. Considerando a quantidade de proteínas do soro de leite no meio de cultivo, a concentração de 0,25 g.L-1 foi considerada a mais efetiva para a secreção da α-amilase pelo microrganismo. A produção máxima da atividade enzimática foi observada a 50 °C e pH 8,5. A enzima foi capaz de degradar todos os amidos testados. A hidrólise do amido de batata resultou num alto rendimento de açúcares redutores em comparação às outras fontes de amido. Amido solúvel e amido de mandioca ocuparam, respectivamente, a segunda e terceira posição em relação à liberação dos açúcares redutores, enquanto que a amilase estudada mostrou apenas uma ligeira afinidade pelo amido de milho. Com o aumento da temperatura da reação para 70 °C, a hidrólise dos substratos, com exceção do amido solúvel, resultou em maiores quantidades de açúcares redutores.

Produção de poligalacturonase, pelo termofílico Bacillus sp. e algumas de suas propriedades

A produção de poligalacturonase pelo termofílico Bacillus sp. SMIA-2, cultivado em meio líquido contendo pectina cítrica como única fonte de carbono, alcançou a sua máxima atividade enzimática em 30 horas, com níveis de 42 IU.mL-1. Entre as várias fontes orgânicas e inorgânicas de nitrogênio testadas, o sulfato de amônio foi a que proporcionou maior atividade da poligalacturonase. O aumento da concentração da pectina cítrica, no meio de cultura, acima de 0,5% não proporcionou um aumento da atividade da enzima. O microrganismo foi capaz de utilizar uma variedade de fontes de carbono, mas a atividade da poligalacturonase variou com cada fonte. Pectina de maçã foi a melhor fonte de carbono para a secreção da poligalacturonase (56 IU.mL-1), enquanto frutose e maltose não foram muito efetivas. Galactose, rafinose e glicose inibiram a síntese da enzima. Estudos sobre a caracterização da poligalacturonase revelaram que a temperatura ótima dessa enzima foi 70 ºC e que ela manteve 62 e 58% de sua atividade máxima quando incubada por 2 horas a 40 e 90 ºC, respectivamente. O pH ótimo para atividade da enzima foi 7,0. A enzima manteve 90 e 75% de sua atividade máxima quando incubada a pH 8,0 e 8,5, respectivamente, por 24 horas, à temperatura ambiente. A atividade enzimática foi estimulada pelos íons Mg2+ e Zn2+. Por outro lado, foi inibida pelos íons Cs+2, Hg+2, Li+2 e Sr+2.

In vitro protein digestibility of enzymatically pre-treated bean (Phaseolus vulgaris L.) flour using commercial protease and Bacillus sp. protease

The common bean (Phaseolus vulgaris L.) is a staple food in the Brazilian diet and represents the major source of dietary protein and other micronutrients and minerals. Despite the considerable protein concentration in beans, the food is considered of low biological value when compared to animal proteins and other plant protein sources. To improve the availability of protein in beans, enzymatic treatments were performed in four cultivars (ON, OPNS, TAL and VC3). The approach was a completely randomized design with four replicates. We used a 4 × 3 factorial arrangement (four cultivars and three treatments: treatment 1-addition of commercial protease (Trypsin 250, Difco), treatment 2-addition of protease from Bacillus sp., and treatment 3:-control without enzyme addition). The enzyme: substrate ratio was 5% w/w (amount of enzyme per total protein in bean flour). The approach was a completely randomized design with four replicates. A 4 × 3 factorial arrangement (four cultivars and three treatments, the same as those mentioned above) was used. The concentration of total protein (g.100 g-1 of dry matter) in the samples ranged from 16.94 to 18.06%, while the concentration of total phenolics was between 0.78 and 1.12% (g Eq. tannic acid.100 g-1 dry matter). The in vitro protein digestibility of enzymatically untreated bean flour (control) ranged from 47.30 to 56.17% based on the digestibility of casein. Concentrations of P, K, Ca, Mg, and Zn observed in the four cultivars tested were within the average values available in the literature. Treatment 2 with protease from Bacillus sp. induced decreases in the levels of Cu and Mn. The average Fe content increased in all bean flour samples when treated with proteases, reaching a maximum increase of 102% in the TAL flour treated with protease from Bacillus sp. The digestibility of all beans tested was significantly increased (p < 0.05) after the enzyme treatment. The greatest change was observed in the OPNS cultivar treated with protease from Bacillus sp., which increased its digestibility from 54.4% (control treatment) to 81.6%.

Estudo prospectivo de 2151 pacientes com doença renal crônica em tratamento conservador com abordagem multidisciplinar no Vale do Paraíba, SP

INTRODUÇÃO: A doença renal crônica (DRC) é uma enfermidade grave, comum e tratável, cuja detecção envolve exames de baixo custo. OBJETIVO: Avaliar o efeito de uma intervenção multidisciplinar (nefrologista, assistente social, enfermeira, nutricionista e psicóloga) em parâmetros clínicos e laboratoriais de pacientes com DRC. MÉTODOS: Estudo prospectivo de 2.151 pacientes atendidos no Centro Estadual de Doenças Renais do Vale do Paraíba, SP, de fevereiro de 2008 a março de 2011. A função renal foi avaliada no início e ao final do seguimento por testes de albuminúria e taxa de filtração glomerular estimada (TFGe) pela fórmula do MDRD. Os desfechos clínicos foram: ocorrência de eventos cardiovasculares (ECV), episódios de hospitalização, necessidade de terapia renal substitutiva (TRS) e óbito. RESULTADOS: A idade média foi 62 anos (variação: 14 a 101), com acompanhamento médio de 546 dias (variação: 90 a 1540), havendo predomínio do estagio três da DRC (59%). Os diagnósticos de base mais comuns foram: hipertensão arterial (41,2%) e diabetes (32,4%). A média da pressão arterial antes e ao final do seguimento foi de 143 ± 26 mmHg x 87 ± 14 mmHg e 123 ± 16 mmHg x 79 ± 9 mmHg, respectivamente (p < 0,001); a TFGe reduziu de 58,5 ± 31 ml/min para 56,3 ± 23 ml/min (p < 0,01). A proteinúria caiu de 1,04 ± 1,44 g/dia para 0,61 ± 1,12 g/dia (p < 0,001); e a glicemia de jejum de 137 ± 73 mg/dl para 116 ± 42 mg/dl. Cento e vinte e dois pacientes (5,7%) apresentaram eventos cardiovasculares, a taxa geral de hospitalizações foi de 6,6% (n = 143 pacientes), foram observados 156 (7,3%) óbitos e 23 (1,1%) pacientes evoluíram para TRS. O risco de ECV, hospitalização e óbito aumentou de forma inversa à TFGe, mas são considerados baixos quando comparados à literatura internacional. CONCLUSÕES: A intervenção multidisciplinar com metas bem definidas é efetiva para preservação da função renal e redução da morbidade e mortalidade de pacientes com DRC.

Prevalência e fatores associados à doença renal crônica em pacientes internados em um hospital universitário na cidade de São Paulo, SP, Brasil

Introdução: A doença renal crônica (DRC) constitui importante problema de saúde pública mundial. Contudo, dados sobre prevalência e comorbidades são escassos no Brasil. Objetivo: Identificar a prevalência e fatores associados à DRC em pacientes internados em um hospital universitário. Métodos: Foram selecionados, aleatoriamente, 826 prontuários de pacientes internados em clínica médica. A DRC foi baseada no diagnóstico médico descrito no prontuário. Foram coletadas informações clínico-demográficas e feitas comparações entre pacientes com e sem DRC. Resultados: A prevalência de DRC foi 12,7%. Os pacientes com DRC se distinguiram daqueles sem a doença (p < 0,05) por terem companheiro (59,8% vs. 47,3%); idade mais elevada (65,8 ± 15,6 vs. 55,3 ± 18,9 anos); mais comorbidades como hipertensão arterial (75,2% vs. 46,3%), diabetes (49,5% vs. 22,4%), dislipidemia (23,8% vs. 14,9%), infarto do miocárdio (14,3% vs. 6,0%) e insuficiência cardíaca congestiva (18,1% vs. 4,3%); maior período de internação (11 (8-18) vs. 9 (6-12) dias) e; mais óbitos (12,4% vs. 1,4%). A análise de regressão logística indicou associação independente (OR, odds ratio; IC, intervalo de confiança de 95%) da DRC com idade (OR 1,019, IC 1,003-1,036), hipertensão arterial (OR 2,032, IC 1,128-3,660), diabetes (OR 2,097, IC 1,232-3,570) e insuficiência cardíaca congestiva (OR 2,665, IC 1,173-6,056). Conclusão: A prevalência de DRC em pacientes internados em clínica médica foi alta, sendo estes pacientes clinicamente mais complexos, visto apresentarem idade mais elevada e maior número de comorbidades, refletindo em maior risco de óbito durante internação hospitalar.

Sensibility of the PCR technique in the detection of Stenocarpella sp. associated with maize seeds

Maize seeds, infected by Stenocarpella species, are important sources of inoculum for the introduction and dissemination of stalk and ear rot and macrospore leaf spot diseases. The use of healthy seeds is an important strategy for the preventive control of these diseases. However, one of the difficulties in the health quality control programs for maize seeds is the availability of a reliable and quick method for detecting these fungi during routine seed analyses. Therefore, the objective of the present study was to investigate the possibility of using the PCR technique as an alternative method for accurately detecting these pathogens in maize seed samples. Maize seeds were kept in contact with S. maydis colonie developed in PDA media containing mannitol at -1.4 MPa for 72 h. The seed samples used in this study were prepared with infected seeds at incidences of 100, 20, 10, 2, 1 and zero %.The primers used were able to detect S. maydis fungi in association with seeds with a maximum of 2% , however those primers were not able to differentiate between the two species.

Characterization of the state transition in the cyanobacterium synechococcus sp. 7002 and a phycobilisome-less Mutant

ABSTRACT Photosynthetic state transitions were investigated in the cyanobacterium Synechococcus sp. PCC 7002 in both wild-type cells and mutant cells lacking phycobilisomes. Preillumination in the presence of DCMU (3(3,4 dichlorophenyl) 1,1 dimethyl urea) induced state 1 and dark adaptation induced state 2 in both wild-type and mutant cells as determined by 77K fluorescence emission spectroscopy. Light-induced transitions were observed in the wildtype after preferential excitation of phycocyanin (state 2) or preferential excitation of chlorophyll .a. (state 1). The state 1 and 2 transitions in the wild-type had half-times of approximately 10 seconds. Cytochrome f and P-700 oxidation kinetics could not be correlated with any current state transition model as cells in state 1 showed faster oxidation kinetics regardless of excitation wavelength. Light-induced transitions were also observed in the phycobilisomeless mutant after preferential excitation of short wavelength chlorophyll !l. (state 2) or carotenoids and long wavelength chlorophyll it (state 1). One-dimensional electrophoresis revealed no significant differences in phosphorylation patterns of resolved proteins between wild-type cells in state 1 and state 2. It is concluded that the mechanism of the light state transition in cyanobacteria does not require the presence of the phycobilisome. The results contradict proposed models for the state transition which require an active role for the phycobilisome.

Impact of different irrigation strategies on grapes and wine quality of four grapevine cultivars (Vitis sp.) in cool climate conditions. An investigation into the relationships among ABA, water status, grape cultivar and wine quality

Niagara Peninsula of Ontario is the largest viticultural area in Canada. Although it is considered to be a cool and wet region, in the last decade many water stress events occurred during the growing seasons with negative effects on grape and wine quality. This study was initiated to understand and develop the best strategies for water management in vineyards and those that might contribute to grape maturity advancement. The irrigation trials investigated the impact of time of initiation (fruit set, lag phase and veraison), water replacement level based on theoretical loss through crop evapotranspiration (ETc; 100,50 and 25%) and different irrigation strategies [partial root zone drying (PRD) versus regulated deficit irrigation (RD!)] on grape composition and wine sensory profiles. The irrigation experiments were conducted in a commercial vineyard (Lambert Vineyards Inc.) located in Niagara-on-the-Lake, Ontario, from 2005 through 2009. The two experiments that tested the combination of different water regimes and irrigation time initiation were set up in a randomized block design as follows: Baco noir - three replicates x 10 treatments [(25%, 50% and 100% of ETc) x (initiation at fruit set, lag phase and veraison) + control]; Chardonnay - three replicates x seven treatments [(25%, 50% and 100% of ETc) x (initiation at fruit set and veraison) + control]. The experiments that tested different irrigation strategies were set up on two cultivars as follows: Sauvignon blanc - four replicates x four treatments [control, fully irrigated (100% ETc), PRD (100% ETc) and RDI (25% ETc)]; Cabemet Sauvignon - four replicates x five treatments [control, fully irrigated (100% ETc), PRD (100% ETc), RDI (50% ETc) and RDI (25% ETc)]. The controls in each experiment were nonirrigated. The irrigation treatments were compared for many variables related to soil water status, vine physiology, berry composition, wine sensory profile, and hormone composition [(abscisic acid (ABA) and its catabolites]. Soil moisture profile was mostly affected by irrigation treatments between 20 and 60 em depth depending on the grapevine cultivar and the regime of water applied. Overall soil moisture was consistently higher throughout the season in 100 and 50% ETc compare to the control. Transpiration rates and leaf temperature as well as shoot growth rate were the most sensitive variables to soil water status. Drip irrigation associated with RDI treatments (50% ETc and 25% ETc) had the most beneficial effects on vine physiology, fruit composition and wine varietal typicity, mainly by maintaining a balance between vegetative and reproductive parts of the vine. Neither the control nor the 100 ETc had overall a positive effect on grape composition and wine sensory typicity. The time of irrigation initiation affected the vine physiology and grape quality, the most positive effect was found in treatments initiated at lag phase and veraison. RDI treatments were overall more consistent in their positive effect on grape composition and wine varietal typicity comparing to PRD treatment. The greatest difference between non-irrigated and irrigated vines in most of the variables studied was found in 2007, the driest and hottest season of the experimental period. Soil water status had a greater and more consistent effect on red grapevine cultivars rather than on white winegrape cultivars. To understand the relationships among soil and plant water status, plant physiology and the hormonal profiles associated with it, abscisic acid (ABA) and its catabolites [phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy-ABA (TOH-ABA), 8' -hydroxy-ABA, neophaseic acid and abscisic acid glucose ester (ABA-GE)] were analyzed in leaves and berries from the Baco noir and Chardonnay irrigation trials over two growing seasons. ABA and some of its catabolites accurately described the water status in the vines. Endogenous ABA and some of its catabolites were strongly affected in Baco noir and Chardonnay by both the water regime (i.e. ET level) and timing of irrigation initiation. Chardonnay grapevines produced less ABA in both leaves and berries compared to Baco noir, which indicated that ABA synthesis is also cultivar dependant. ABA-GE was the main catabolite in treatments with high water deficits, while PA and DPA were higher in treatments with high water status, suggesting that the vine produced more ABA-GE under water deficits to maintain rapid control of the stomata. These differences between irrigation treatments with respect to ABA and catabolites were particularly noticeable in the dry 2007 season. Two trials using exogenous ABA investigated the effect of different concentrations of ABA and organs targeted for spraying, on grape maturation and berry composition of Cabemet Sauvignon grapevines, in two cool and wet seasons (2008-2009). The fIrst experiment consisted of three replicates x three treatments [(150 and 300 mg/L, both applications only on clusters) + untreated control] while the second experiment consisted in three replicates x four treatments [(full canopy, only clusters, and only leaves sprayed with 300 ppm ABA) + untreated control]. Exogenous ABA was effective in hastening veraison, and improving the composition of Cabemet Sauvignon. Ability of ABA to control the timing of grape berry maturation was dependant on both solution concentration and the target organ. ABA affected not only fruit composition but also yield components. Berries treated with ABA had lower weight and higher skin dry mass, which constitutes qualitative aspects desired in the wine grapes. Temporal advancement of ripening through hormonal control can lead to earlier fruit maturation, which is a distinct advantage in cooler areas or areas with a high risk of early frost occurrence. Exogenous ABA could provide considerable benefits to wine industry in terms of grape composition, wine style and schedule activities in the winery, particularly in wet and cool years. These trials provide the ftrst comprehensive data in eastern North America on the response of important hybrid and Vitis vinifera winegrape cultivars to irrigation management. Results from this study additionally might be a forward step in understanding the ABA metabolism, and its relationship with water status. Future research should be focused on ftnding the ABA threshold required to trigger the ripening process, and how this process could be controlled in cool climates.

The analysis of Metarhizium robertsii potential as endophytic plant root coloniser, plant growth enhancer and antagonist to bean root pathogen Fusarium solani f. sp. phaseolis.

The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. M robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M robertsii were examined after inoculation with fungal conidia. Light and confocal microscopies were used to ascertain this rhizosphere association. Root lengths, root hair density and emergence of lateral roots were also measured. Initially, M robertsii conidia adhered to, germinated on, and colonized, roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M robertsii on the root (within 1-2 days). Plants inoculated with M robertsii AMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M robertsii did not stimulate root hair proliferation. Longer term (60 days) associations showed that M robertsii endophytically colonized individual cortical cells within bean roots. Metarhizium appeared as an amorphous mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. These results suggested that not only is M robertsii rhizosphere competent but displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs. The biocontrol of bean (Phaseolis vulgaris) root rot fungus Fusarium solani f. sp. phaseolis by Metarhizium robertsii was investigated in vitro and in vivo. Dual cultures on Petri dishes showed antagonism of M robertsii against F. solani. A relative inhibition of ca. 60% of F. solani growth was observed in these assays. Cell free culture filtrates of M robertsii inhibited the germination of F. solani conidia by 83% and the inhibitory metabolite was heat stable. Beans plants colonized by M robertsii then exposed to F. solani showed healthier plant profiles and lower disease indices compared to plants not colonized by M robertsii. These results suggested that the insect pathogenic/endophytic fungus M robertsii could also be utilized as a biocontrol agent against certain plant pathogens occurring in the rhizosphere.

Prevalencia de Aspergillus sp. y aflatoxinas en el maíz cultivado por labranza convencional y de conservación

Tesis (Maestría en Ciencias con Especialidad en Microbiología) UANL

Producción de a-Amilasa a partir de Bacillus sp.

Tesis (Maestría en Ciencias con especialidad en microbiología) U.A.N.L.

Impacto del clavicornio del agua Tropisternus sp. (Coleoptera: Hydrophilidae) sobre larvas de Culex pipiens Say (Diptera: Culicidae) [por] Humberto Quiroz Martínez

Tesis (Maestro en Ciencias con especialidad en Entomología Médica) U.A.N.L.

Acción del etanol y del acetaldehido sobre la contracción isométrica en el músculo sartorio del sapo (Bufo sp.)

Tesis (Maestría en Ciencias con Especialidad en Fisiología Médica) U.A.N.L.

Determinación del perfil de proteasas y de los requerimientos protéicos del caracol manzano (Pomacea sp.) como base para el desarrollo de una dieta artificial para su cultivo comercial

Tesis (Maestría en Ciencias con Especialidad en Recursos Alimenticios y Producción Acuícola) U.A.N.L. Facultad de Ciencias Biológicas.

Aislamiento de cepas de bacillus sp. productoras de proteasas con potencial uso industrial.

Tesis (Maestría en Ciencias con Orientación en Microbiología Industrial) UANL, 2011.