Análisis de la diversidad microbiana asociada a la fermentación de aceitunas verdes de mesa por técnicas moleculares dependientes e independientes de cultivo

Programa de Doctorado en Biotecnología y Tecnología QuímicaLas aceitunas verdes de mesa al estilo Español son un componente básico de la dieta mediterránea y constituyen el alimento vegetal fermentado de mayor relevancia económica en España. La preparación de este producto se caracteriza por la recolección en verde de los frutos, que son endulzados mediante un tratamiento alcalino suave y, después de una etapa de lavado, se cubren con salmuera, donde se desarrollará una fermentación espontánea y principalmente láctica. A lo largo de la fermentación, la salmuera se comporta como un caldo de cultivo natural en la cual se produce una sucesión ecológica de las poblaciones microbianas presentes en el alimento o adquiridas en la manipulación y procesamiento del mismo. La acción de la diversa microbiota autóctona, que se ocupa de llevar a cabo esta fermentación natural, ejerce un papel fundamental en la calidad y propiedades organolépticas del producto final y en el rendimiento económico de la producción. Por ello el objetivo principal de este trabajo es rescatar, identificar molecularmente y conservar la biodiversidad microbiana inherente a la fermentación de aceitunas verdes de mesa. La idea central es no sólo contribuir a actualizar el conocimiento, dirigir y mejorar el proceso tradicional de fermentación, sino evaluar el potencial de este nicho ecológico para el hallazgo de nuevas especies o taxones microbianos. Para ello, se han analizado las salmueras de fermentación de dos grandes empresas de aderezo de aceitunas de la provincia de Sevilla. En ellas, se ha monitorizado el proceso fermentativo en 43 fermentadores mediante el control y seguimiento de las principales características físico-químicas (pH, acidez libre y concentración de sal), así como el estudio ecológico de la microbiota presente en las salmueras mediante técnicas moleculares dependientes e independitentes (PCR-DGGE) de cultivo. Fruto de estos trabajos, se ha obtenido una colección de más de 1250 aislados microbianos que han sido identificados molecularmente a nivel de especie y de cepa como pertenecientes, al menos, a 49 especies bacterianas y 12 especies de levaduras distintas. Más de la mitad de estas especies han sido asociadas por primera vez a este tipo de fermentación de aceitunas de mesa. También ha sido posible el aislamiento y descripción de cuatro nuevas especies bacterianas: Enterococcus olivae, Propionibacterium olivae, Propionibacterium damnosum y Vibrio olivae. Todo ello, pone de manifiesto la complejidad microbiana de este proceso fermentativo multifásico, pese a estar dominado en sus etapas esenciales por la especie Lactobacillus pentosus. Los resultados obtenidos mediante el análisis por PCR-DGGE de las muestras y posterior aislamiento en medios de cultivo específicos pusieron de manifiesto la diversidad, abundancia y ubicuidad de una serie de microorganismos halófilos y alcalófilos durante la primera fase de la fermentación. Entre estos, cabe subrayar la presencia de bacterias lácticas halófilas y alcalófilas (HALAB) de los tres géneros reconocidos que componen este grupo: Alkalibacterium, Marinilactibacillus y Halolactibacillus. Las HALAB son las únicas bacterias conocidas que pueden realizar una fermentación láctica en condiciones alcalinas, por lo que podrían contribuir decisivamente a la creación de las condiciones adecuadas para el posterior desarrollo de bacterias lácticas comunes, como L. pentosus, en fases más avanzadas de esta fermentación.Universidad Pablo de Olavide. Departamento de Biología Molecular e Ingeniería Bioquímic

Diversity and enumeration of halophilic and alkaliphilic bacteria in Spanish-style green table-olive fermentations

© 2015 Elsevier Ltd. The presence and enumeration of halophilic and alkaliphilic bacteria in Spanish-style table-olive fermentations was studied. Twenty 10-tonne fermenters at two large manufacturing companies in Spain, previously studied through both culture dependent and independent (PCR-DGGE) methodologies, were selected. Virtually all this microbiota was isolated during the initial fermentation stage. A total of 203 isolates were obtained and identified based on 16S rRNA gene sequences. They belonged to 13 bacterial species, included in 11 genera. It was noticeable the abundance of halophilic and alkaliphilic lactic acid bacteria (HALAB). These HALAB belonged to the three genera of this group: Alkalibacterium, Marinilactibacillus and Halolactibacillus. Ten bacterial species were isolated for the first time from table olive fermentations, including the genera Amphibacillus, Natronobacillus, Catenococcus and Streptohalobacillus. The isolates were genotyped through RAPD and clustered in a dendrogram where 65 distinct strains were identified. Biodiversity indexes found statistically significant differences between both patios regarding genotype richness, diversity and dominance. However, Jaccard similarity index suggested that the halophilic/alkaliphilic microbiota in both patios was more similar than the overall microbiota at the initial fermentation stage. Thus, up to 7 genotypes of 6 different species were shared, suggesting adaptation of some strains to this fermentation stage. Morisita-Horn similarity index indicated a high level of codominance of the same species in both patios. Halophilic and alkaliphilic bacteria, especially HALAB, appeared to be part of the characteristic microbiota at the initial stage of this table-olive fermentation, and they could contribute to the conditioning of the fermenting brines in readiness for growth of common lactic acid bacteria.This research was funded by the Spanish Ministry of Science and Innovation (MICINN), through Projects AGL2009-07861 and AGL2012-33400, and by the Junta de Andalucía Excellence Projects AGR-04621 and AGR-07345. All these projects included FEDER funds. AMB was the recipient of a post-doctoral grant awarded by the Junta de Andalucía as part of the Project AGR-07345. HLP was the recipient of a contract funded by the Spanish Ministry of Economy and Competitiveness as part of the Project AGL2012-33400. We want to express our most sincere gratitude to Juan Carlos Roldán, from JOLCA S.A., and Antonio Martín and Marta Sánchez, from GOYA en España S.A.U., for their invaluable collaboration in this study.Peer Reviewe

Aislamiento, diversidad y evolución de bacterias lácticas halófilas y alcalófilas (HALAB) asociadas a fermentaciones de aceitunas verdes de mesa al estilo >Español>

Este estudio es continuación de trabajos anteriores sobre la biodiversidad microbiana inherente a la fermentación de aceitunas verdes de mesa al estilo >Español> mediante técnicas moleculares dependientes e independientes (PCR-DGGE) de cultivo. El estudio se centró en un total de 20 termentadores de 10.000 Kg de aceitunas localizados en dos industrias de gran tamaño. Nuestro objetivo final es rescatar esta biodiversidad para potenciales usos biotecnológicos. Como resultado de PCR-DGGE, observamos que una parte importante, por su ubicuidad al menos, de la microbiota podría haberse pasado por alto al no emplear medios de cultivo específicos. Concretamente, bacterias del ácido láctico halófilas y alcalófilas (HALAB) aparecieron en los perfiles de PCR-DGGE de todos los fermentadores analizados. Así, identificamos bandas específicas de los tres géneros reconocidos que componen este grupo: Alkalibacterium, Marinilactibacillus y Halolactibacillus. Para corroborar la presencia viable de especies de estos géneros y conocer su número y evolución en la fermentación usamos medios selectivos específicos para el aislamiento de bacterias halófilas y alcalófilas. Así obtuvimos un total de 203 aislados que fueron genotipados por RAPD y que pudieron agruparse en 66 cepas distintas que fueron identificadas molecularmente. Estas cepas pertenecieron a 15 especies bacterianas, siendo notable la abundancia de HALABs entre ellos, incluyendo especies de los tres géneros adscritos a este grupo y pronosticados por PCR-DGGE. Entre ellas destaca una especie identificada preliminarmente como Marinilactibacillus sp. que podría en realidad constituir un género nuevo en base a la homología de la secuencia del ADNr 16S completo (<95%). Estos aislados HALAB resultaron circunscritos a la denominada primera fase de la fermentación, donde las condiciones iniciales de alta salinidad (10-12 % NaCl) y pH alcalino (pH 10-11) son las idóneas para favorecer su crecimiento. Las HALAB son las únicas bacterias conocidas que pueden realizar una fermentación láctica en condiciones alcalinas, por lo que podrían contribuir decisivamente a la creación de las condiciones adecuadas para el posterior desarrollo de bacterias lácticas comunes, como Lactobacillus pentosus, en la segunda fase de esta fermentación. Este estudio nos permitió además aislar por primera vez especies de los géneros Amphibacillus, Natronobacillus, Catenococcus y Streptohalobacillus de fermentaciones de aceitunas de mesa. PALABRAS CLAVE: Fermentación de aceitunas, HALAB, biodiversidad, genotipadoPeer Reviewe

Microbial biogeography of Spanish-style green olive fermentations in the province of Seville, Spain

58 Páginas.-- 7 Tablas.-- 1 Figura.-- 1 Tabla suplementaria.-- 4 Figuras suplementariasThe aim of this study is to examine the biogeography of the microbial communities associated to the Spanish-style green olive fermentations in the province of Seville (Andalucía, south-western Spain). Also, to understand how microorganisms colonize and persist in non-sterile food fermentations across a specific table olive producing area, i.e. a specific “agroecosystem”. The microbial diversity, bacteria and yeast, in 30 ten-ton fermenters of three different fermentations yards (patios) along the olive fermentation was studied. A total of 951 microbial isolates were obtained which were clustered according to their RAPD profile. A total of 376 distinct genotypes were identified, belonging to 57 different microbial species, 41 bacterial and 16 yeast species. Up to 16 bacterial species had not been described before in table olives. Only the species Lactobacillus pentosus showed a ubiquitous presence in all 30 fermenters. Pediococcus parvulus, Lactobacillus collinoides/paracollinoides, Lactobacillus coryniformis, Lactobacillus plantarum, Pichia manshurica and Candida thaimueangensis were found in every patio. Cosmopolitan strains, up to 15, were shared by the three patios and belonged to the species L. pentosus (12 strains), P. parvulus (1), L. collinoides/paracollinoides (1) and P. manshurica (1). To expand our biodiversity analyses to the “regional” level, we have compared our results with those obtained from two previously studied patios of similar characteristics and in the same geographical area. PERMANOVA analysis of the microbial community composition revealed significant differences among different patios in their structure at every fermentation stage. In contrast, SIMPER analyses showed that, as fermentation progressed, the overall dissimilarities among patios were reduced. Discriminant species were identified for each fermentation stage. Among these, L. pentosus and P. parvulus were “eu-constant” species, while L. collinides/paracollinoides and Marinilactibacillus psychrotolerans group were “constant” species that could be considered microbial key taxa based on the occurrence stability index. The characteristic and, presumably, well adapted microbiota associated to the Spanish-style olive fermentations at the specific geographic area described here is a valuable natural resource which should be preserved conveniently. To our knowledge, this is the first study on the microbial biogeography of table olive fermentations, both at the species and strain levels.This research was funded by the Spanish Ministry of Science and Innovation through the Project AGL2009-07861, by the Spanish Ministry of Economy and Competitiveness by Project AGL2012-33400, by the Junta de Andalucía Excellence Projects AGR-04621 and AGR-07345, as well as by CSIC Project 201670E028. These projects included FEDER funds. HLP was the recipient of a contract funded by the Spanish Ministry of Economy and Competitiveness as part of the Project AGL2012-33400.Peer reviewe

Genetic diversity and dynamics of bacterial and yeast strains associated to Spanish-style green table-olive fermentations in large manufacturing companies

© 2014 Elsevier B.V. We have genotyped a total of 1045 microbial isolates obtained along the fermentation time of Spanish-style green table olives from the fermentation yards (. patios) of two large manufacturing companies in the Province of Sevilla, south of Spain. Genotyping was carried out using RAPD-PCR fingerprinting. In general, isolates clustered well into the relevant phylogenetic dendrograms, forming separate groups in accordance to their species adscription. We could identify which bacterial and yeast genotypes (strains) persisted throughout the fermentation at each patio. Also, which of them were more adapted to any of the three stages, i.e. initial, middle and final, described for this food fermentation. A number of genotypes were found to be shared by both patios. Fifty seven of these belonged to five different bacterial species, i.e. Lactobacillus pentosus, Lactobacillus paracollinoides/. collinoides, Lactobacillus rapi, Pediococcus ethanolidurans and Staphylococcus sp., although most of them (51) belonged to L. pentosus. Four yeast genotypes were also shared, belonging to the species Candida thaimueangensis, Saccharomyces cerevisiae and Hanseniaspora sp. Two genotypes of L. pentosus were found to be grouped with those of two strains used in commercially available starter cultures, one of them bacteriocinogenic, which were used up to three years before this study in these patios, demonstrating the persistence of selected strains in this environment. Biodiversity was assessed though different indexes, including richness, diversity and dominance. A statistically significant decrease in biodiversity between the initial and final stages of the fermentation was found in both patios. However, values of biodiversity indexes in the fermenters were very similar, and no significant differences were found in the total biodiversity between both patios. This study allowed us to identify a range of well adapted strains (genotypes), especially those belonging to the lactic acid bacteria, which could be useful to improve safety and quality of table olive fermentations.This research was funded by the Spanish Ministry of Science and Innovation (MICINN), which included FEDER funds, through Projects AGL2009-07861 and AGL2012-33400, and by the Junta de Andalucía Excellence Projects AGR-04621 and AGR-07345. All these projects included FEDER funds. AMB and HLP were the recipients of grants awarded by the Spanish National Research Council (CSIC) through the JAE Doc (ref. JAEDoc 07-00163) and JAE Pre (ref. JAEPre 09-01649) Programmes, respectively. We want to express our most sincere gratitude to Juan Carlos Roldán, from JOLCA S.A., and Antonio Martín and Marta Sánchez, from GOYA en España S.A.U., for their invaluable collaboration in this study.Peer Reviewe

Microbial diversity and dynamics of Spanish-style green table-olive fermentations in large manufacturing companies through culture-dependent techniques

We have studied the microbiota associated to Spanish-style green olive fermentations, attending to its dynamics along the time. Twenty 10-tonne fermenters were selected from two large table-olive manufacturing companies in southern Spain. While culture-dependent methodology was used to isolate the microorganisms, molecular methods were used to identify the isolates. A total of 1070 isolates were obtained, resulting in 929 bacterial and 141 yeast isolates. Thirty seven different bacterial species were isolated, belonging to 18 different genera, while 12 yeast species were isolated, belonging to 7 distinct genera. This fermentation was dominated by the species Lactobacillus pentosus, while its accessory microbiota was variable and depended on the fermentation stage and the actual fermentation yard (>. patio>). It was noticeable the abundance of lactic acid bacteria isolates, belonging to 16 different species. Twenty bacterial species were isolated for the first time from Spanish-style green olive fermentations, while 17 had not been described before in any table olive preparation. The genera Brachybacterium, Paenibacillus, Sporolactobacillus, Paracoccus and Yersinia had not been cited before from any table olive preparation. Saccharomyces cerevisiae and Candida thaimueangensis appeared to dominate the yeast microbiota. Candida butyri/. asseri and Rhodotorula mucilaginosa had not been described before from Spanish-style preparations, while Saturnispora mendoncae was isolated for the first time from any table olive preparation. Biodiversity was analysed through different alpha and beta indexes which showed the evolution of the microbiota over time. © 2014 Elsevier Ltd.This research was funded by the Spanish Ministry of Science and Innovation (MICINN) through Projects AGL2009-07861 and AGL2012-33400, and by the Junta de Andalucía Excellence ProjectsAGR-04621 and AGR-07345. AMB and HLP were the recipients of grants awarded by the Spanish National Research Council (CSIC) through the JAE DocJAEDoc 07-00163 and JAE predoctoral ProgrammesJAEPre 09-01649, respectively. We want to express our most sincere gratitude to Juan Carlos Roldán, from JOLCA S.A., and Antonio Martín and Marta Sánchez, from GOYA en España S.A.U., for their invaluable collaboration in this study. We thank Dr. Antonio Higinio Sánchez and Mrs. Elena Cabello, from Instituto de la Grasa-CSIC at Sevilla, for their assistance in the analyses of the physico-chemical parameters.Peer Reviewe

Genome Sequence of Lactobacillus pentosus IG1, a Strain Isolated from Spanish-Style Green Olive Fermentations

Lactobacillus pentosus is the most prevalent lactic acid bacterium in Spanish-style green olive fermentations. Here we present the draft genome sequence of L. pentosus IG1, a bacteriocin-producing strain with biotechnological and probiotic properties isolated from this food fermentations.This research was funded by the Spanish Ministry of Science and Innovation (MICINN) through Project AGL2009-07861 and by the Junta de Andalucía Excellence Project AGR-04621. A.M.-B. and H.L.-P. were the recipients of grants awarded by the Spanish National Research Council (CSIC) through the JAE Doc and JAE predoctoral Programmes, respectivelyPeer reviewe

PCR-DGGE assessment of the bacterial diversity in Spanish-style green table-olive fermentations

© 2015 Elsevier B.V. The bacterial ecology associated to Spanish-style green olive fermentations has been studied, attending to its dynamics along the time and its distribution, by a culture-independent approach based on PCR-DGGE. Forty-three 10-tonne fermenters were selected from the fermentation yards (patios) of two large table-olive manufacturing companies in southern Spain. The fermenting brines of 20 of these fermenters were previously analyzed through culture-dependent methods, allowing comparisons of both methodologies. A statistical analysis of DGGE banding profiles obtained using bacteria universal primers demonstrated significant evidences of discrimination of bacterial communities by location (patio) and fermentation stage. Specific microbial >fingerprints> could be established for these variables. At least 17 bacterial species were detected, most of them previously isolated from the same fermenters. Most of these species belonged to the lactic acid bacteria (LAB) group. Dominance of species within the Lactobacillus plantarum group was confirmed. Marinilactibacillus sp. and Propionibacterium olivae, which were not isolated in the previous culture-dependent study, were detected. Alkalibacterium sp. and Halolactibacillus sp. were detected for the first time in table olive fermentations. Using Lactobacillus-group specific primers, significant clustering within the DGGE banding profiles was observed, allowing discrimination regarding the actual fermentation stage. These results corroborated the previous culture-dependent study, and added the detection of Alkalibacterium sp. and Pediococcus acidilactici. The species Alkalibacterium sp., Marinilactibacillus sp. and Halolactibacillus sp. are characterized by their ability to carry out lactic acid fermentation under alkaline conditions and thus ascribed within the halophilic and alkaliphilic lactic acid bacteria (HALAB). Their ubiquitous presence suggests that they could play an important role in Spanish-style olive fermentations, especially at the initial fermentation stage. Thus, they could contribute to brine conditioning before L. plantarum group-driven lactic acid fermentation takes place.This research was funded by the Spanish Ministry of Science and Innovation (MICINN), through projects AGL2009-07861 and AGL2012-33400, and by the Junta de Andalucía Excellence projects AGR-04621 and AGR-07345. All these projects included FEDER funds. AMB was the recipient of a post-doctoral grant awarded by the Junta de Andalucía as part of the project AGR-07345. HLP was the recipient of a contract funded by the Spanish Ministry of Economy and Competitiveness as part of the project AGL2012-33400. We want to express our most sincere gratitude to Juan Carlos Roldán, from JOLCA S.A., and Antonio Martín and Marta Sánchez, from GOYA en España S.A.U., for their invaluable collaboration in this study.Peer Reviewe