904 resultados para Bovino - Tratamentos hormonais
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Atualmente, a produtividade do feijoeiro comum (Phaseolus vulgaris L.) pode ser reduzida devido à ocorrência de doenças em todo o território nacional, destacando-se a murcha de fusário, causada por Fusarium oxysporum f. sp. phaseoli (Fop). No campo, o patógeno é disseminado a longas distâncias através das sementes infectadas e/ou contaminadas e a sua sobrevivência ocorre, principalmente, no solo. Os objetivos deste trabalho foram: avaliar a inibição do crescimento micelial de Fop por Trichoderma spp.; classificar a sensibilidade in vitro de Fop e Trichoderma spp., separadamente, a fungicidas e verificar a compatibilidade entre fungicidas químicos e biológicos para controle de Fop, presente nas sementes e no solo. Para avaliar a inibição do crescimento micelial de Fop, foram utilizados três isolados do patógeno, os quais foram confrontados, in vitro, com três isolados de Trichoderma spp. em testes de cultura pareada e produção de metabólitos voláteis a 20-22°C. Os experimentos foram conduzidos em delineamento inteiramente casualizado, com cinco repetições para cada isolado de Trichoderma. Para a classificação da sensibilidade in vitro de Fop e Trichoderma a fungicidas, foram avaliados os mesmos isolados anteriormente utilizados. Foram comparados dez fungicidas, em doses entre 0 a 100 mg L-1 que foram ajustadas de acordo com a CI50 de cada fungicida. Com base na percentagem de inibição do crescimento micelial, foram estimados os valores da concentração inibitória de 50% (CI50) e 100% (CI100) e selecionaram-se os fungicidas compatíveis com Trichoderma spp. A compatibilidade entre tratamentos químico e biológico foi avaliada através da inoculação artificial de sementes de feijão com um isolado de Fop (IAC 11.299-1) e infestação do mesmo no solo. As sementes foram tratadas com os fungicidas fludioxonil, flutriafol e tiofanato metílico, e com os três produtos biológicos, separadamente e em misturas. Avaliou-se o efeito dos tratamentos por meio dos testes de sanidade, germinação, comprimento de plântulas, massa da matéria seca em laboratório e índice de velocidade de emergência e porcentagem de emergência em estufa não climatizada. O efeito protetor dos tratamentos foi verificado através do teste de transmissão do patógeno solo-planta. Todos os isolados de Trichoderma apresentaram antagonismo in vitro contra Fop. No teste de cultura pareada foi observada uma redução de 15 a 20% no crescimento micelial do patógeno. No teste de produção de metabólitos voláteis, o isolado T12-1086G05 foi responsável pela maior inibição do crescimento micelial de Fop (10 a 48%). Os testes de sensibilidade in vitro mostraram que tiofanato metílico, flutriafol e fludioxonil foram compatíveis com Trichoderma (CI50 > 2 mg L-1). Com exceção do flutriafol e do GF 422 isolados e em mistura, todos os tratamentos foram eficientes na erradicação de Fop nas sementes, sem afetar a sua qualidade fisiológica. No teste de transmissão, verificou-se que a incidência de Fop foi de 5 a 40% no hipocótilo e de 5 a 30% nas raízes de feijoeiro provenientes de sementes tratadas com os produtos.
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A pesar de todos los avances realizados en las últimas décadas en el conocimiento sobre numerosos aspectos del Síndrome Respiratorio Bovino (SRB), entre los que se encuentran los factores de riesgo, la etiopatogenia, las características de los agentes causales, el diagnóstico, la terapéutica o la profilaxis y sus efectos globales, el SRB continúa siendo la enfermedad de mayor impacto económico en el ganado vacuno de cebo. Además, parece que tanto su incidencia como las pérdidas que origina siguen estables en los cebaderos de todo el mundo desde hace varias décadas. Sus principales características indican que debemos considerar que es una enfermedad de difícil control dado su carácter multifactorial y con la que, por tanto, debemos acostumbrarnos a convivir aunque aspirando a minimizar tanto su incidencia, como la gravedad de sus consecuencias. Por otro lado, aunque existen muchas técnicas diagnósticas a nuestra disposición, aún no se puede decir que exista una técnica de referencia o “gold standard” para el diagnóstico del SRB, y mucho menos una técnica estandarizada para su diagnóstico precoz. Establecer y estandarizar protocolos para la detección de grupos de animales en riesgo y su monitorización más intensa en fases posteriores del cebo para reducir el consumo de antibióticos de uso preventivo o metafiláctico es de gran interés. Hasta ahora, este tipo de estrategias se han basado en la detección de evidencias de contacto con gérmenes patógenos pulmonares (principalmente análisis de serología). Sin embargo, no debemos olvidar que la identificación de un agente patógeno en ausencia de una lesión y/o signos clínicos atribuibles al mismo, tan sólo es indicativa de contacto previo (no de padecimiento de un proceso de enfermedad) y que, por otro lado, la mayoría de los agentes microbiológicos ligados al SRB son comensales habituales de las vías respiratorias altas. De modo que la identificación de un agente sólo prueba enfermedad si se diagnostica junto con los signos clínicos compatibles de enfermedad y la lesión...
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Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014
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Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz
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A presente pesquisa teve como objetivo avaliar, por meio de modelos de gesso, a posição das coroas dos primeiros e terceiros molares superiores permanentes, após a irrupção espontânea dos terceiros molares, na posição dos segundos molares extraídos com finalidade ortodôntica, comparando-os com as coroas dos primeiros e segundos molares superiores permanentes de uma amostra de oclusão normal natural. A amostra experimental constituiu-se de 25 indivíduos, leucodermas, com idade média no início do tratamento de 15,6 anos, sendo 17 do sexo feminino e 8 do sexo masculino, tratados com extrações de segundos molares superiores. A amostra controle constituiu-se de 60 indivíduos do sexo masculino e sexo feminino, com idade média de 15,6 anos e faixa etária compreendida entre 12 e 21 anos, que apresentavam oclusão normal. Foram avaliadas as angulações, as inclinações e as distâncias mésio-distais das coroas dentárias dos terceiros molares e primeiros molares do grupo tratado e dos segundos molares e primeiros molares do grupo controle ou normal. Foram, também, verificadas as diferenças de alturas entre as coroas dentárias dos dois grupos. Verificou-se que nos casos tratados os valores médios das angulações mésio-distais e das inclinações vestíbulo-linguais das coroas dos terceiros molares superiores e dos primeiros molares superiores foram estatisticamente diferentes dos valores médios das angulações mésio-distais e das inclinações vestíbulo-linguais das coroas dos segundos molares superiores e dos primeiros molares superiores na amostra de oclusão normal natural. Observou-se que somente as distâncias mésio-distais das coroas dos terceiros molares superiores mostraram ser significativamente menores que as coroas dos segundos molares superiores da amostra de oclusão normal natural, tanto para o sexo feminino quanto para o masculino. O valor médio da diferença de altura entre as coroas dos primeiros molares e terceiros molares do grupo experimental e das coroas dos primeiros molares e segundos molares do grupo controle foi de 1,11mm, não havendo diferença entre os grupos. Ocorreu a presença de dimorfismo sexual nos valores da distância mésio-distais das coroas dentárias dos primeiros molares superiores do grupo controle e experimental, na distância mésio-distal das coroas dentárias dos segundos molares superiores do grupo controle e na inclinação vestíbulo-linguais das coroas dos terceiros molares superiores do grupo experimental. Nas demais avaliações não houve dimorfismo sexual.
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Electrochemical technologies have been proposed as a promising alternative for the treatment of effluents and contaminated soils. Therefore, the objective of this work was to study the treatment of contaminated soils and wastewaters using electrochemical technologies. Thus, the study regarding the scale-up of the electrochemical system with continuous flow treatment of wastewater of the petrochemical industry was investigated using platinum electrodes supported on titanium (Ti / Pt), and boron-doped diamond (BDD). The results clearly showed that under the operating conditions studied and electrocatalytic materials employed, the better removal efficiency was achieved with BDD electrode reducing the chemical oxygen demand (COD) from 2746 mg L-1 to 200 mg L-1 in 5 h consuming 56.2 kWh m-3 . The decontamination of soils and effluents by petrochemical products was evaluated by studying the effects of electrokinetic remediation for removal of total petroleum hydrocarbons (HTP) from contaminated soil with diesel. The efficiency of this process was dependent on the electrolyte used Na2SO4 (96.46%), citric acid (81.36%) and NaOH (68.03%) for 15 days. Furthermore, the effluent after treatment of the soil was treated by electrochemical oxidation, achieving a good elimination of the organic polluting load dissolved. Depending on the physical behavior of wastewater contaminated with oil (emulsified state); atrazine emulsified effluents were investigated. The main characteristics of the effluent produced during the washing of contaminated soil were studied, being dependent on the surfactant dosage used; which determined its electrolytic treatment with BDD. The electrochemical oxidation of emulsified effluent of atrazine was efficient, but the key to the treatment is reducing the size of micelles.
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The Benzylpenicillin (PENG) have been as the active ingredient in veterinary medicinal products, to increase productivity, due to its therapeutic properties. However, one of unfortunate quality and used indiscriminately, resulting in residues in foods exposed to human consumption, especially in milk that is essential to the diet of children and the ageing. Thus, it is indispensable to develop new methods able to detect this waste food, at levels that are toxic to human health, in order to contribute to the food security of consumers and collaborate with regulatory agencies in an efficient inspection. In this work, were developed methods for the quality control of veterinary drugs based on Benzylpenicillin (PENG) that are used in livestock production. Additionally, were validated methodologies for identifying and quantifying the antibiotic residues in milk bovine and caprine. For this, the analytical control was performed two steps. At first, the groups of samples of medicinal products I, II, III, IV and V, individually, were characterized by medium infrared spectroscopy (4000 – 600 cm-1). Besides, 37 samples, distributed in these groups, were analyzed by spectroscopy in the ultraviolet and near infrared region (UV VIS NIR) and Ultra Fast Liquid Chromatograph coupled to linear arrangement photodiodes (UFLC-DAD). The results of the characterization indicated similarities, between PENG and reference standard samples, primarily in regions of 1818 to 1724 cm-1 of ν C=O that shows primary amides features of PENG. The method by UFLC-DAD presented R on 0.9991. LOD of 7.384 × 10-4 μg mL-1. LOQ of 2.049 × 10-3 μg mL-1. The analysis shows that 62.16% the samples presented purity ≥ 81.21%. The method by spectroscopy in the UV VIS NIR presented medium error ≤ 8 – 12% between the reference and experimental criteria, indicating is a secure choice for rapid determination of PENG. In the second stage, was acquiring a method for the extraction and isolation of PENG by the addition of buffer McIlvaine, used for precipitation of proteins total, at pH 4.0. The results showed excellent recovery values PENG, being close to 92.05% of samples of bovine milk (method 1). While samples of milk goats (method 2) the recovery of PENG were 95.83%. The methods for UFLC-DAD have been validated in accordance with the maximum residue limit (LMR) of 4 μg Kg-1 standardized by CAC/GL16. Validation of the method 1 indicated R by 0.9975. LOD of 7.246 × 10-4 μg mL-1. LOQ de 2.196 × 10-3 μg mL-1. The application of the method 1 showed that 12% the samples presented concentration of residues of PENG > LMR. The method 2 indicated R by 0.9995. LOD 8.251 × 10-4 μg mL-1. LOQ de 2.5270 × 10-3 μg mL-1. The application of the method showed that 15% of the samples were above the tolerable. The comparative analysis between the methods pointed better validation for LCP samples, because the reduction of the matrix effect, on this account the tcalculs < ttable, caused by the increase of recovery of the PENG. In this mode, all the operations developed to deliver simplicity, speed, selectivity, reduced analysis time and reagent use and toxic solvents, particularly if compared to the established methodologies.
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The Benzylpenicillin (PENG) have been as the active ingredient in veterinary medicinal products, to increase productivity, due to its therapeutic properties. However, one of unfortunate quality and used indiscriminately, resulting in residues in foods exposed to human consumption, especially in milk that is essential to the diet of children and the ageing. Thus, it is indispensable to develop new methods able to detect this waste food, at levels that are toxic to human health, in order to contribute to the food security of consumers and collaborate with regulatory agencies in an efficient inspection. In this work, were developed methods for the quality control of veterinary drugs based on Benzylpenicillin (PENG) that are used in livestock production. Additionally, were validated methodologies for identifying and quantifying the antibiotic residues in milk bovine and caprine. For this, the analytical control was performed two steps. At first, the groups of samples of medicinal products I, II, III, IV and V, individually, were characterized by medium infrared spectroscopy (4000 – 600 cm-1). Besides, 37 samples, distributed in these groups, were analyzed by spectroscopy in the ultraviolet and near infrared region (UV VIS NIR) and Ultra Fast Liquid Chromatograph coupled to linear arrangement photodiodes (UFLC-DAD). The results of the characterization indicated similarities, between PENG and reference standard samples, primarily in regions of 1818 to 1724 cm-1 of ν C=O that shows primary amides features of PENG. The method by UFLC-DAD presented R on 0.9991. LOD of 7.384 × 10-4 μg mL-1. LOQ of 2.049 × 10-3 μg mL-1. The analysis shows that 62.16% the samples presented purity ≥ 81.21%. The method by spectroscopy in the UV VIS NIR presented medium error ≤ 8 – 12% between the reference and experimental criteria, indicating is a secure choice for rapid determination of PENG. In the second stage, was acquiring a method for the extraction and isolation of PENG by the addition of buffer McIlvaine, used for precipitation of proteins total, at pH 4.0. The results showed excellent recovery values PENG, being close to 92.05% of samples of bovine milk (method 1). While samples of milk goats (method 2) the recovery of PENG were 95.83%. The methods for UFLC-DAD have been validated in accordance with the maximum residue limit (LMR) of 4 μg Kg-1 standardized by CAC/GL16. Validation of the method 1 indicated R by 0.9975. LOD of 7.246 × 10-4 μg mL-1. LOQ de 2.196 × 10-3 μg mL-1. The application of the method 1 showed that 12% the samples presented concentration of residues of PENG > LMR. The method 2 indicated R by 0.9995. LOD 8.251 × 10-4 μg mL-1. LOQ de 2.5270 × 10-3 μg mL-1. The application of the method showed that 15% of the samples were above the tolerable. The comparative analysis between the methods pointed better validation for LCP samples, because the reduction of the matrix effect, on this account the tcalculs < ttable, caused by the increase of recovery of the PENG. In this mode, all the operations developed to deliver simplicity, speed, selectivity, reduced analysis time and reagent use and toxic solvents, particularly if compared to the established methodologies.
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This study aimed to evaluate different concentrations of kisspeptin, as well as the interaction of kisspeptin and FSH/LH in vitro maturation and oocyte competence in cattle. In Experiment 1 was determined the minimum concentration of Kisspeptin (Kp) to be used, and in Experiment 2 was evaluated its interection with FSH and LH. The oocytes were collected in a commercial slaughterhouse and only Grade I oocytes were utilized. The oocytes were cultured in TCM-199 medium with bicarbonate plus 10% FBS, sodium pyruvate (22μg/mL), amikacin (83mg/mL), FSH (0.5μg/mL), with different concentrations of Kp, the treatments were: FSH + 0M Kp-10; FSH + 10-7M Kp-10, FSH + 10-6M Kp-10; FSH + 10-5M Kp-10. In Experiment 2, was used better concentration of Kp found in Experiment 1, the following treatments: no hormones; FSH; FSH + Kp-10; FSH + LH; FSH, LH + Kp-10; Kp-10. The oocyte competence was determined by nuclear maturation, mitochondrial distribution, MitoTracker® Orange CMTMRos fluorescence intensity and DCF. The evaluation of nuclear maturation was made after 24 hours incubation and the oocytes were stained with DAPI to determine the nuclear stage (Germinal Vesicle-GV, Metaphase I-MI and Metaphase II-MII).The mitochondrial distribution was classified as peripheral/semiperipheral and diffuse in clusters/granules, evaluated after stained with the MitoTracker® Orange CMTMRos, and was also identified the intensity of it. To determine the intensity of ROS oocytes were stained with DCF. The statistical analysis was performed by SAS GLIMMIX PROC. In Experiment 1 oocytes matured only with the FSH reached a smaller nuclear maturation when compared to those who were matured with Kisspeptin at different concentrations (FSH:13/33; FSH + 10-7M Kp-10: 28/35; FSH + 10-6M Kp-10:30/34; FSH + 10-5M Kp-10:28/32; P=0,0001). There was no statistical difference in mitochondrial distribution between treatments (P>0.05). The fluorescence intensity of MitoTracker did not differ among treatments (P>0.05). The DCF fluorescence intensity was lower when the concentration of Kp was increased in the medium (FSH:12177726,1; FSH + 10-7M Kp-10:10945982,83; FSH + 10-6M Kp-10:9820536,53; FSH + 10-5M Kp-10:9147016,38; P<0,0001). Based in the Experiment 1 results, the concentration of Kp was determined in 10-7M. In Experiment 2 the mitochondrial distribution was different between treatments, because oocytes matured only with Kp or FSH+LH, reached a oocyte competence greater than those maturated with FSH only or without hormone addition (no hormones:66,66%; FSH:66,66%; FSH + Kp-10:75,86%; FSH + LH:91,17%; FSH, LH + Kp-10:82,85%; Kp-10:91,17%; P<0,05). The no hormones resulted in a lower nuclear maturation than the other treatments (no hormones: 5/18; FSH:18/32; FSH + Kp-10:22/29; FSH + LH:26/33; FSH, LH + Kp-10:26/34; Kp-10:25/34; P=0,0094). The fluorescence intensity of probes MitoTracker and DCF was lower when Kp was added to the maturation medium (no hormones:1228363/540069; FSH:2307984/1395751; FSH + Kp-10:1941890/1114948; FSH + LH:2502145/1722376; FSH, LH + Kp-10:2286173/1467782; Kp-10:1859411/979325 P<0,0001). So this is the first study that shows that Kisspeptin stimulates oocyte maturation without the presence of gonadotropins in the maturation medium.
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CHAPTER 1 - The gummy stem blight, caused by the fungus D. bryoniae, is a disease commonly found in watermelon cultivated in several countries. In Brazil, there are numerous studies related to the disease, but there are not uniform methods for quantifying of disease severity in the field. Thus, we developed a diagrammatic scale based on scanned photos of watermelon leaves infected with D. bryoniae. The scale developed showed levels of 0; 10; 20; 45; 65 and 90% of severity. The scale validation was divided into two parts: initially, 10 evaluators (half with experienced and other half without experience) estimated the disease severity based on the initial observation of 100 photos of watermelon leaves with symptoms of the disease at different severity levels. Before, the same evaluators estimated the disease severity with the support of the scale prepared from the Quant program. Data were analyzed using linear regression and were obtained angular, linear, and correlation coefficients. Based on these data, we determined the accuracy and precision of the evaluations. The correlation coefficients (R2) ranged from 0.88 - 0.97 for the experienced evaluators and from 0.55 - 0.95 for the inexperienced evaluators. The average angular coefficient (A) for inexperienced evaluators was 20.42 and 8.61 with and without the support of diagrammatic scale, respectively. Experienced evaluators showed values of average linear coefficient of 5.30 and 1.68 with and without the support of diagrammatic scale, respectively. The absolute errors analysis indicated that the use of diagrammatic scale contributed to minimize the flaws in the severity levels estimation. The diagrammatic scale proposed shown adequate for gummy stem blight severity evaluation in watermelon. CHAPTER 2 - The gummy stem blight (Didymella bryoniae) is a disease that affects the productivity of watermelon leading to losses over 40%. This study aimed to evaluate the efficiency of different production systems in control of gummy stem blight in watermelon for to establish efficient methods to combat the disease. There were applied the following treatments: conventional tillage (T1), integrated management (T2) and organic management (T3). In T1 and T2 were applied mineral fertilization and T3 was used bovine manure. There was application of fungicides and insecticides in commercial dose in T1 and T2, being after soil chemical analysis in T2. Disease severity was assessed by grading scale. The experimental design was randomized blocks. The severity of gummy stem blight has increased substantially during the fruit formation. Watermelon plants grown with integrated management (T2) showed lower levels of disease severity, while plants in organic management (T3) exhibited higher levels of severity. We conclude that management based on judicious accompaniments in field represents best way to achieve the phytosanitary aspect adequate for cultivation of watermelon in Tocantins.
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NASCIMENTO, H. G. ; FERNANDES, L. C. ; SOUSA, M. B. C. . Avaliação da fidedignidade dos ensaios de esteróides fecais realizados no Laboratório de Medidas Hormonais do Departamento de Fisiologia da UFRN. Publica , v. 2, p. 39-48, 2006.
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En la Argentina la sangre obtenida del faenado del ganado bovino es considerada un subproducto de menor valor, a pesar de su alto contenido de proteínas de gran calidad. A diferencia de lo que ocurre en algunos países, en los cuales la sangre (o alguno de sus componentes) se utiliza como complemento alimenticio humano, en nuestro país sólo una pequeña fracción de la sangre recolectada es aprovechada, destinándola principalmente a consumo animal. En este trabajo de Tesis se evaluó el potencial como aditivo alimentario humano del plasma bovino deshidratado, un derivado de la sangre bovina que se utiliza únicamente como complemento nutricional animal. En la Primera Parte de este trabajo se obtuvo Plasma Desmineralizado mediante un proceso de diafiltración y se evaluaron algunas de sus propiedades funcionales (Solubilidad, Capacidad de Retención de Agua, capacidad de Retención de Aceite, Capacidad Emulsionante, Capacidad Espumante y Capacidad Antioxidante), comparándolas con las del plasma sin diafiltrar. Como resultado de las tareas llevadas a cabo en esta Primera Parte, se lograron establecer las condiciones operativas para efectuar el proceso de diafiltración de manera eficaz y se pudo evaluar el efecto de dicho proceso sobre las Propiedades Funcionales del plasma consideradas. La Solubilidad del plasma resultó alta en un amplio rango de pH, e incluso mejoró luego del proceso de diafiltración en algunos valores de pH. Los procesos realizados al plasma aumentaron su Capacidad de Retención de Agua; en cambio, su Capacidad de Retención de Aceite disminuyó. El plasma mostró una Capacidad Emulsionante acorde a lo descripto en la bibliografía; la diafiltración y la posterior deshidratación no la afectaron, pero sí aumentaron la estabilidad de las emulsiones formadas. El plasma mostró una gran capacidad para formar espumas estables, las cuales no resultaron afectadas por los procesos efectuados sobre el mismo. El plasma deshidratado original mostró una Capacidad Antioxidante similar a la descripta en la bibliografía. El efecto del proceso de diafiltración sobre la misma no pudo ser establecido. En la Segunda Parte de este trabajo se obtuvieron dos hidrolizados enzimáticos a partir del plasma, utilizando la enzima Alcalase, con diferentes grados de hidrólisis. Se evaluaron las propiedades funcionales de los hidrolizados, comparándolas entre sí y con las del plasma original. Como resultado de estas tareas se definieron las condiciones del proceso de hidrólisis, comprobándose la obtención de fragmentos proteicos y péptidos de distinto tamaño molecular. Con respecto a las Propiedades Funcionales, los hidrolizados obtenidos resultaron ligeramente más solubles que el plasma deshidratado original, y la hidrólisis con Alcalase afectó notoriamente a las propiedades surfactantes de las proteínas plasmáticas, tanto a la Capacidad Emulsionante como a la Capacidad Espumante. En cambio, la hidrólisis enzimática aumentó marcadamente la Capacidad Antioxidante del plasma deshidratado. Los hidrolizados obtenidos presentaron una actividad de secuestro de radicales libres significativamente mayor que la del plasma original, y esa mayor actividad antioxidante resultó asociada al menor tamaño de los péptidos obtenidos. En virtud de los resultados obtenidos, se concluye que el plasma bovino deshidratado, tanto en su forma original como desmineralizado, tiene potenciales aplicaciones como aditivo alimentario para mejorar algunas propiedades funcionales de los alimentos, y que los hidrolizados que se obtuvieron de él con la enzima Alcalase poseen una alta capacidad antioxidante, que los haría viables para ser utilizados como aditivos conservantes para prolongar la vida útil de los alimentos. De ser viable su uso en alimentos humanos, esto aumentaría el valor agregado de este subproducto de la industria cárnica. Por último, se discuten algunas líneas de trabajo que deberían realizarse para completar su posible implementación en la industria alimenticia humana.
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NASCIMENTO, H. G. ; FERNANDES, L. C. ; SOUSA, M. B. C. . Avaliação da fidedignidade dos ensaios de esteróides fecais realizados no Laboratório de Medidas Hormonais do Departamento de Fisiologia da UFRN. Publica , v. 2, p. 39-48, 2006.