Identificación taxonómica y clonal de bacterias acéticas, y estudio del efecto de la nisina frente a biofilms de bacterias enológicas
The community of microorganisms involved in wine and vinegar elaborations is very diverse, complex and scarcely characterized, and precisely microorganisms are responsible for the fermentations that take place, i.e. the transformations of grape juice into wine and wine into vinegar. During fermentat...
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Universidad de La Rioja (España)
2015
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The community of microorganisms involved in wine and vinegar elaborations is very diverse, complex and scarcely characterized, and precisely microorganisms are responsible for the fermentations that take place, i.e. the transformations of grape juice into wine and wine into vinegar. During fermentations microorganisms produce numerous changes in the composition, colour, and organoleptic attributes of the fermented final product that will determine its quality. Lactic acid bacteria (LAB) and acetic acid bacteria (AAB) are the two bacterial families involved in these transformations within the oenological context. The overall objectives of this thesis are: a) to perform clonal and taxonomic characterization of AAB responsible for the acetic fermentation of a variety of industrial vinegars elaborated by the submerged method, and b) to study the possibility of using the bacteriocin nisin in winemaking to achieve wine microbiological control and against the formation of undesired bacterial biofilms.
In this thesis methodologies based on DNA analysis have been developed for clonal and taxonomic identification of AAB isolates from samples of wine- cider- and ethanol vinegars in full acetic fermentation. Moreover, the methodology of fluorescence microscopy has been developed for quantification of AAB in vinegar samples and LAB in wine samples. Regarding clonal identification, results showed as well that restriction analysis with SpeI of genomic DNA and pulsed field gel electrophoresis is an appropriate method for studying AAB strains, whereas the rapid method ERIC-PCR is appropriate for monitoring a previously characterized AAB strain and to determine its implantation in acetic fermentations. 43 new AAB clones from vinegar samples were identified, which became part of the bacteria collection of the University of La Rioja of indigenous strains responsible for fermentations. Two G. europaeus strains prevailed and could be excellent candidates for selection of starters for vinegar elaboration.
An active extract was obtained from the LAB strain with the QPS status (qualified presumption of safety) by the EFSA Lactococcus lactis LM29. The extract inhibited the growth of 83.5% of the oenological LAB collection and contained 60 % white grape must and 200 ?g/ml of nisin equivalent. Nisin in this concentration prevented biofilm formation by LAB that presented this wine spoilage and microbial contamination feature. The amino acid metabolism of L. lactis in presence of ethanol was also studied and it was unveiled that this LAB utilizes the ADI (arginine deiminase) pathway of arginine degradation as mechanism of response to the presence of ethanol. It generated ornithine and ammonium, but it did not generate any biogenic amine (agmatine, spermidine, phenylethylamine, histamine, or putrescine) either in presence or in absence of ethanol in the culture broth. These results indicate that nisin preparations with the appropriate concentrations could inhibit undesired LAB and prevent biofilm formation, therefore they could constitute useful tools for the microbiological control of wines and could help to decrease the levels of sulphites that are currently used in winemaking. |
| author2 |
Ruiz Larrea, Fernanda (Universidad de La Rioja) |
| author_facet |
Ruiz Larrea, Fernanda (Universidad de La Rioja) Fernández Pérez, Rocío |
| format |
text (thesis) |
| author |
Fernández Pérez, Rocío |
| spellingShingle |
Fernández Pérez, Rocío Identificación taxonómica y clonal de bacterias acéticas, y estudio del efecto de la nisina frente a biofilms de bacterias enológicas |
| author_sort |
Fernández Pérez, Rocío |
| title |
Identificación taxonómica y clonal de bacterias acéticas, y estudio del efecto de la nisina frente a biofilms de bacterias enológicas |
| title_short |
Identificación taxonómica y clonal de bacterias acéticas, y estudio del efecto de la nisina frente a biofilms de bacterias enológicas |
| title_full |
Identificación taxonómica y clonal de bacterias acéticas, y estudio del efecto de la nisina frente a biofilms de bacterias enológicas |
| title_fullStr |
Identificación taxonómica y clonal de bacterias acéticas, y estudio del efecto de la nisina frente a biofilms de bacterias enológicas |
| title_full_unstemmed |
Identificación taxonómica y clonal de bacterias acéticas, y estudio del efecto de la nisina frente a biofilms de bacterias enológicas |
| title_sort |
identificación taxonómica y clonal de bacterias acéticas, y estudio del efecto de la nisina frente a biofilms de bacterias enológicas |
| publisher |
Universidad de La Rioja (España) |
| publishDate |
2015 |
| url |
https://dialnet.unirioja.es/servlet/oaites?codigo=45994 |
| work_keys_str_mv |
AT fernandezperezrocio identificaciontaxonomicayclonaldebacteriasaceticasyestudiodelefectodelanisinafrenteabiofilmsdebacteriasenologicas |
| _version_ |
1718346629518458880 |
| spelling |
oai-TES00000085602019-07-14Identificación taxonómica y clonal de bacterias acéticas, y estudio del efecto de la nisina frente a biofilms de bacterias enológicasFernández Pérez, RocíoThe community of microorganisms involved in wine and vinegar elaborations is very diverse, complex and scarcely characterized, and precisely microorganisms are responsible for the fermentations that take place, i.e. the transformations of grape juice into wine and wine into vinegar. During fermentations microorganisms produce numerous changes in the composition, colour, and organoleptic attributes of the fermented final product that will determine its quality. Lactic acid bacteria (LAB) and acetic acid bacteria (AAB) are the two bacterial families involved in these transformations within the oenological context. The overall objectives of this thesis are: a) to perform clonal and taxonomic characterization of AAB responsible for the acetic fermentation of a variety of industrial vinegars elaborated by the submerged method, and b) to study the possibility of using the bacteriocin nisin in winemaking to achieve wine microbiological control and against the formation of undesired bacterial biofilms. In this thesis methodologies based on DNA analysis have been developed for clonal and taxonomic identification of AAB isolates from samples of wine- cider- and ethanol vinegars in full acetic fermentation. Moreover, the methodology of fluorescence microscopy has been developed for quantification of AAB in vinegar samples and LAB in wine samples. Regarding clonal identification, results showed as well that restriction analysis with SpeI of genomic DNA and pulsed field gel electrophoresis is an appropriate method for studying AAB strains, whereas the rapid method ERIC-PCR is appropriate for monitoring a previously characterized AAB strain and to determine its implantation in acetic fermentations. 43 new AAB clones from vinegar samples were identified, which became part of the bacteria collection of the University of La Rioja of indigenous strains responsible for fermentations. Two G. europaeus strains prevailed and could be excellent candidates for selection of starters for vinegar elaboration. An active extract was obtained from the LAB strain with the QPS status (qualified presumption of safety) by the EFSA Lactococcus lactis LM29. The extract inhibited the growth of 83.5% of the oenological LAB collection and contained 60 % white grape must and 200 ?g/ml of nisin equivalent. Nisin in this concentration prevented biofilm formation by LAB that presented this wine spoilage and microbial contamination feature. The amino acid metabolism of L. lactis in presence of ethanol was also studied and it was unveiled that this LAB utilizes the ADI (arginine deiminase) pathway of arginine degradation as mechanism of response to the presence of ethanol. It generated ornithine and ammonium, but it did not generate any biogenic amine (agmatine, spermidine, phenylethylamine, histamine, or putrescine) either in presence or in absence of ethanol in the culture broth. These results indicate that nisin preparations with the appropriate concentrations could inhibit undesired LAB and prevent biofilm formation, therefore they could constitute useful tools for the microbiological control of wines and could help to decrease the levels of sulphites that are currently used in winemaking.La comunidad de microorganismos implicados en los procesos de elaboración del vino y del vinagre es muy diversa, compleja y poco caracterizada, siendo precisamente esos microorganismos los responsables de las fermentaciones, es decir, de las transformaciones del zumo de uva en vino y de éste en vinagre. Durante estas fermentaciones, los microorganismos producen numerosos cambios en la composición, color y en los atributos organolépticos que finalmente marcarán la calidad del producto. Las bacterias lácticas (BL) y las bacterias acéticas (BA) son las dos familias bacterianas implicadas en estas transformaciones del contexto enológico. Los objetivos generales de esta tesis son: a) la caracterización a nivel taxonómico y clonal de BA responsables de la fermentación acética de distintos vinagres industriales elaborados por el método sumergido, y b) estudiar la posibilidad del empleo de la bacteriocina nisina en la elaboración y control microbiológico del vino y como agente contra la formación de biofilms bacterianos no deseados en el ámbito de la bodega. En esta tesis se han desarrollado y puesto a punto metodologías basadas en el análisis del DNA para la identificación taxonómica y clonal de BA aisladas de muestras en plena fermentación acética de vinagres de vinos, sidras y alcohol etílico. Asimismo, se ha puesto a punto el método de microscopía de fluorescencia para la cuantificación de BA en muestras de vinagres y de BL en muestras de vinos, resultando ser un método rápido y eficaz. Respecto a la identificación clonal, los resultados obtenidos mostraron que el método de análisis de restricción del DNA genómico con la enzima SpeI y la electroforesis de campos pulsados es el método adecuado para estudiar cepas de BA, mientras que el método rápido ERIC-PCR es un método apropiado para el seguimiento de una cepa específica de BA previamente caracterizada y que se desee seguir su implantación en un proceso de acetificación. Se identificaron de las muestras de vinagres un total de 43 nuevos clones de BA que pasaron a formar parte de la colección de la Universidad de La Rioja de cepas bacterianas autóctonas responsables de fermentaciones, destacando dos clones G. europaeus que podrían constituir excelentes candidatos para la selección de cultivos iniciadores para la elaboración de vinagre. A partir de la BL con el estatus QPS (qualified presumption of safety) de la EFSA, Lactococcus lactis LM29, se consiguió un extracto que inhibía el crecimiento del 83,5 % de las BL enológicas y que contenía 60 % mosto de uva blanca y 200 ?g/ml equivalentes de nisina. En esta concentración la nisina impidió la formación de biofilms de BL enológicas que presentaban esa capacidad alterante de los vinos y contaminante. Asimismo se estudió el metabolismo aminoacídico de L. lactis en presencia de etanol y se comprobó que esta BL utiliza la vía ADI (arginina deiminasa) de degradación de la arginina como mecanismo de respuesta a la presencia del etanol, generando ornitina y el ion amonio, si bien no generó en presencia ni en ausencia de etanol en el medio de cultivo ninguna de las aminas biógenas: agmatina, espermidina, feniletilamina, histamina, ni putrescina. Estos resultados indican que la nisina puede inhibir posibles BL contaminantes del vino y en las concentraciones adecuadas puede impedir la formación de biofilms indeseados, constituyendo por tanto una herramienta útil para el control microbiológico de los vinos y podría ayudar a la disminución de los niveles de sulfitos que actualmente se utilizan para la elaboración del vino.Universidad de La Rioja (España)Ruiz Larrea, Fernanda (Universidad de La Rioja)2015text (thesis)application/pdfhttps://dialnet.unirioja.es/servlet/oaites?codigo=45994(Tesis) ISBN 978-84-617-5277-5 spaLICENCIA DE USO: Los documentos a texto completo incluidos en Dialnet son de acceso libre y propiedad de sus autores y/o editores. Por tanto, cualquier acto de reproducción, distribución, comunicación pública y/o transformación total o parcial requiere el consentimiento expreso y escrito de aquéllos. Cualquier enlace al texto completo de estos documentos deberá hacerse a través de la URL oficial de éstos en Dialnet. 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