Evaluación in Vitro De La Actividad Antimicrobiana De Una Cepa De Bacillus Subtilis Con Potencial Probiótico

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Evaluación in Vitro De La Actividad Antimicrobiana De Una Cepa De Bacillus Subtilis Con Potencial Probiótico Universidad de Matanzas “CAMILO CIENFUEGOS” FACULTAD DE AGRONOMÍA Evaluación in vitro de la actividad antimicrobiana de una cepa de Bacillus subtilis con potencial probiótico Tesis presentada en opción al Título Académico de Ingeniero Agrónomo Autor: Benoit Bayoda Hodabalo Tutoras: Dr C. Ana Julia Rondón Castillo MsC. Marta E. Laurencio Silva Ing. Marvelys Socorro Ortega Matanzas 2013 PENSAMIENTO Amargas son las raíces del estudio, pero los frutos son dulces. Albert Einstein NOTA DE ACEPTACIÓN _________________________________________________________________________________________ _________________________________________________________________________________________ _________________________________________________________________________________________ _________________________________________________________________________________________ _________________________________________________________________________________________ _________________________________________________________________________________________ _________________________________________________________________________________________ __________________________________________ ________________ Presidente del Tribunal Firma __________________________________________ ________________ Miembro del Tribunal Firma __________________________________________ ________________ Miembro del Tribunal Firma __________________________________________ ________________ Miembro del Tribunal Firma Dado en Matanzas, el día ____ del mes de ______________ del año 2013. “Año 55 de la Revolución” DECLARACIÓN DE AUTORIDAD Yo, Benoit Bayoda Hodabalo, estudiante de 5to año de la facultad de Agronomía, declaro que soy el único autor de este trabajo de diploma y autorizo a la Universidad de Matanzas “Camilo Cienfuegos” a hacer uso del mismo para lo que sea necesario. _________ Firma DEDICATORIA A mis padres Bayoda Essobyou y Belei Essodezam quienes han confiado en mí en los estudios. A mi tio Belei Estache por su apoyo y estimulo en todo mi trayectoria estudiantil. A mis hermanos, especialmente Juliette y Romeo y a mi tutor de casa Mr Innocent, quien me enseñó a ser una persona responsable. OPINIÓN DE LOS TUTORES AGRADECIMIENTOS Quiero expresar mis más sinceros agradecimientos en primer lugar al gobierno de mi país (TOGO) y al gobierno cubano, los cuales me dieron la oportunidad de estudiar y de ser hoy un profesional más para esta sociedad. A mis tutores, la Dra C. Ana J. Rondón Castillo, la MsC. Marta E. Laurencio Silva, la Ing. Marvelis Socorro Ortega y el Dr C. Manuel Pérez Quintana, por brindarme su apoyo constante, su tiempo y paciencia para que este trabajo se realizara satisfactoriamente. A mi tío, mis padres por darme la vida, la atención y apoyo que siempre he necesitado. A mis hermanos y todos mis amigos de mi país. A todos los profesores por su apoyo en mi carrera estudiantil. A todos mis compañeros de estudio que de una forma u otra hicieron posible la culminación de este trabajo. Muchas Gracias. RESUMEN Las bacterias del género Bacillus producen sustancias que inhiben el desarrollo de microorganismos patógenos. El presente trabajo tuvo como objetivo evaluar la actividad antimicrobiana de la cepa Bacillus subtilis 21BMC, con potencial probiótico. Para ello se realizaron tres experimentos in vitro donde se enfrentó la cepa Bacillus subtilis (21BMC), procedente del tracto digestivo de cerdos a microorganismos patógenos y se evaluó la capacidad de coagregación de esta bacteria a células patógenas. Como resultado, Bacillus subtilis 21BMC produjo sustancias antimicrobianas o antibioticos que inhiben el crecimiento de Shigella spp. Se demostró además el efecto antimicrobiano de esta cepa frente a E. coli, Salmonella spp. y Shigella spp. a través del establecimiento de cocultivos en condiciones de simulación del TGI de cerdos. Se comprobó también, que las células de Bacillus inducen un alto grado de coagregación con las paredes celulares de los microorganismos patógenos, tanto en estado vegetativo como esporulado, lo cual significa que pudieran presentar un porciento elevado de adherencia de las fimbrias y arrastrar a los patógenos a través del tracto digestivo de los animales. Los resultados de este trabajo avalan el posible uso de este microorganismo como probiótico. INDICE INTRODUCCIÓN 1 CAPÍTULO 1. REVISIÓN BIBLIOGRÁFICA 1.1. Los probióticos 5 1.1.1. Reseña histórica 5 1.1.2. Evolución del concepto 5 1.2. Mecanismos de acción de los probióticos 7 1.3. Criterios de selección de los microorganismos probióticos 9 1.4. Actividad antimicrobiana 13 1.4.1. Producción de bacteriocinas y antibióticos 14 1.5. Género Bacillus. Características morfológicas, fisiológicas, 15 bioquímicas e inmunológicas. 1.5.1. Características morfológicas y fisiológicas del género Bacillus 15 1.5.2. Características bioquímicas del género Bacillus 18 1.5.3. Acción de las endosporas en el sistema inmunológico 18 1.6. Efecto de los probióticos elaborados con Bacillus spp en cerdos 19 CAPÍTULO 2. MATERIALES Y MÉTODOS GENERALES 2.1. Materiales y métodos 22 2.1.1. Cepas utilizadas 22 2.1.2. Medios de cultivos 22 2.2. Conservación de cepas 23 2.3. Procedimiento experimental para la evaluación in vitro de la actividad antimicrobiana de Bacillus subtilis 21BMC frente a cepas 23 potencialmente patógenas. 2.3.1. Experimento 1. Evaluación de la actividad antimicrobiana de la cepa de Bacillus subtilis (21BMC) frente a cepas patógenas, con la aplicación de la técnica de difusión de sustancias en agar. 24 2.3.2. Experimento 2. Determinación del efecto antimicrobiano de Bacillus subtilis a través del establecimiento de cocultivos con bacterias potencialmente patógenas. 25 2.3.3. Experimento 3. Comprobación del nivel de coagregación de Bacillus spp a células de bacterias patógenas. 26 2.4. Procesamiento estadístico 26 CAPÍTULO 3. RESULTADOS Y DISCUSIÓN 3.1. Evaluación de la actividad antimicrobiana de cepa de Bacillus subtilis frente a cepas patógenas, con la aplicación de la técnica de difusión de sustancias en agar. 27 3.2. Determinación del efecto antimicrobiano de Bacillus subtilis a través del establecimiento de cocultivos con bacterias potencialmente patógenas. 31 3.3. Comprobación del nivel de coagregación de Bacillus spp a células 35 de bacterias patógenas. CAPÍTULO 4. VALORACIÓN ECONÓMICA 4.1. Valoración Económica 39 CONCLUSIONES 40 RECOMENDACIONES 41 REFERENCIAS BIBLIOGRÁFICAS ANEXOS INTRODUCCIÓN La producción porcina moderna se caracteriza por una alta intensidad productiva que somete a los animales a situaciones estresantes, lo que trae como consecuencia, una mayor frecuencia en la aparición de enfermedades y una disminución de los niveles de producción. Antiguamente, para atenuar esta problemática, se incorporaron los antibióticos en las dietas como Aditivos Promotores de Crecimiento (APC), los cuales mostraron ser efectivos en la disminución de los trastornos diarreicos y en el aumento del comportamiento productivo de los animales (Jones et al., 2003; Coppola y Gil− Tuners, 2004). El uso indiscriminado de los antibióticos en el mundo, tanto en el hombre como en los animales, provocaron resistencia microbiana y crearon residuos en los animales, de ahí la necesidad de sustituir a los antibióticos como promotores del crecimiento en la producción animal por otros aditivos alternativos promotores de la respuesta productiva que sean compatibles con el medio ambiente y eviten efectos negativos en la salud humana, tales como los probióticos (Coppola y Gil−Tuners, 2004; Tim et al., 2006). Al finales del siglo pasado, en la década del 90, se desarrollaron los probióticos, conjunto de productos que no crean los problemas de resistencia microbiana o efecto residual que producen los antibióticos, los cuales se elaboran a partir de microorganismos, principalmente bacterias productoras de endosporas (Bacillus), bacterias lácticas, hongos y levaduras, que contribuyen a mantener el equilibrio ecológico favorable en el intestino y el buen funcionamiento del sistema inmune (Bengmark, 1998; Ferreira, 2003). Dentro de los productos que presentan actividad probiótica se encuentran los cultivos de Bacillus spp procedentes del tracto digestivo de los animales y el hombre, los cuales se destacan por su efectividad en mejoramiento de la microbiota intestinal (Rodríguez et al., 1996; Spring et al., 1996 y Bocourt et al., 2002). 1 En tal sentido, algunos cultivos del género Bacillus spp. y sus endosporas, reciben atención por el efecto probiótico que brindan en el balance de la microbiota intestinal, el mejoramiento de la digestión, la activación del sistema inmune, la absorción de nutrientes, la mejora de la fisiología digestiva, la mayor eficiencia en la conversión alimenticia y los mejores rendimientos productivos, en cerdos y en las aves (Lata et al., 2006; Mutus et al., 2006; Utiyama et al., 2006). Se conocen productos elaborados a partir de cepas de B. licheniformis, B. subtilis y sus endosporas como: el Biomin® P.E.P y el Protexin, BioPlus 2B®, BioGrow®, Biostart®, Toyocerin®, Liqualife®, Biosporin® y CenBiot®, con efectos probióticos en cerdos (Hong et al., 2004; Ugras et al., 2013). Existen instituciones científicas cubanas, tales como la Universidad de Matanzas “Camilo Cienfuegos” y el Instituto de Ciencia Animal (ICA), que ejecutan diferentes proyectos de investigación para el desarrollo de productos probióticos económicamente viables y que dentro de sus características tengan la capacidad de inhibir a microorganismos patógenos de los animales (Pérez, 2000; Milián, 2009; Rondón, 2009; Laurencio, 2010; Pérez,
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