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16 Páginas.-- 9 Figuras.-- 3 Tablas, Vida de mercado de aceitunas tradicionales Aloreña de Málaga aliñadas basado en la apariencia del envase y características del fruto. Pocos estudios han sido llevados a cabo para determinar la vida de mercado de aceitunas de mesa Aloreña de Málaga envasadas desde un punto de vista físico-químico, microbiológico y sensorial. Este trabajo muestra, cómo, bajo las actuales condiciones de envasado, los productos comerciales están libres de enterobacterias, la población inicial de levaduras se inhibe progresivamente, mientras que las bacterias ácido lácticas son las únicas que se desarrollan durante el envasado. Entre las características físico-químicas, el pH descendió, se formó ácido láctico mientras que el manitol y el ácido cítrico fueron consumidos. Estos cambios dieron lugar a una progresiva degradación de la textura y a una depreciación del color verde de los frutos. El análisis multivariante realizado mostró que los envasados entre los 6 y 42 días de vida de mercado fueron los mejores valorados por los panelistas, mientras que después de los 74 días, el producto envasado fue perdiendo aceptabilidad, lo cual fue especialmente evidente tras 131 días de envasado (la predisposición de compra fue solo del 50%). Una completa estabilización microbiológica del producto requeriría del uso de conservantes alternativos, ya que el tratamiento térmico no es muy conveniente para este tipo de elaboración., Few studies have been carried out to determine the shelf-life of the Aloreña de Málaga table olive packaging from a physicochemical, microbiological and sensorial point of view. This study showed that under the current packaging conditions, commercial products were free from Enterobacteriaceae, the initial yeast population was progressively inhibited, and only lactic acid bacteria grew during shelf-life. Among the physicochemical characteristics, pH decreased, lactic acid was formed while citric acid and mannitol were consumed. These changes resulted in gradual olive texture degradation and green color fading during packaging. A multivariate analysis showed that the packaged olives with storage time between 6 and 42 days enjoyed the highest acceptance; while after the 74th day, they were progressively losing acceptability, which was mainly evident at the 131st day of packaging (willingness-to-buy attribute was reduced to 50%). A complete microbiological stabilization would require the use of alternative preservatives since thermal treatment is not convenient for this type of olive speciality., The research leading to these results has received funding from Junta de Andalucía Government through the PrediAlo project (AGR-7755: www.predialo.science.com.es) and FEDER European funds. VRG would like to thank ceiA3, Spanish Government, Bank of Santander, IG-CSIC and ‘Aloreña de Málaga’ Olive Manufacturing Association for her pre-doctoral fellowship.

La Aloreña de Málaga fue la primera aceituna de mesa en obtener el sello de calidad de Denominación de Origen Protegida (DOP) en España (BOJA nº215, 2009). Se trata de una aceituna de mesa autóctona del Valle del Guadalhorce, muy apreciada en la costa del sol (provincia de Málaga) donde tienen lugar la mayor parte de sus ventas. Esto es debido, en gran parte, a la gran inestabilidad que presentan los envasados de ciertos tipos de elaboraciones como las verdes frescas y tradicionales, lo que dificulta que puedan ser comercializados en lugares alejados de su zona de producción. Por lo tanto, aumentar la vida de mercado de sus envasados es, actualmente, una prioridad para las empresas. Además, el sector también desea conocer el efecto que el uso de diferentes condiciones de envasado (pH, sal y conservantes) podría tener sobre la calidad y seguridad microbiológica del producto final. Por tanto, la presente Tesis Doctoral se orienta al estudio de los envasados de aceitunas de mesa con DOP Aloreña de Málaga con el objetivo de mejorar la calidad y seguridad microbiológica de los mismos. Está dividida en tres secciones. En la primera de ellas se realiza un estudio fisicoquímico, microbiológico y sensorial de los envasados para determinar su vida de mercado y actuales causas de alteración. En la segunda, se evalúa in silico, mediante la aplicación de modelos matemáticos (dosis respuesta y logístico/probabilístico), el empleo de diferentes conservantes (sales de zinc, metabisulfito de sodio, cinamaldehido, ácido pirúvico, ácido fumárico y natamicina) como alternativas a los habituales sorbatos y benzoatos, con la finalidad de mejorar la estabilidad y calidad de los productos mediante la inhibición del desarrollo microbiano. En la tercera sección, se evalúa la seguridad microbiológica de los envasados mediante la inoculación artificial con patógenos alimentarios (Listeria monocytogenes, Escherichia coli, Staphylococcus aureus y Salmonella enterica), determinando su supervivencia y su relación con la presencia de ciertos compuestos inhibidores presentes en el fruto. The Aloreña de Málaga was the first table olive speciality to obtain the Protected Designation of Origin (PDO) in Spain (BOJA nº215, 2009). Its production is located in Guadalhorce Valley and is highly appreciated in the Costa del Sol (Malaga, Spain), where it is mainly commercialised. The restriction of its delivery is due, at least in part, to the commercial packaging instability, which prevents its distribution to markets far from the production area. Thus, increasing the shelf life of packaging is nowadays a priority for companies. Moreover, the sector also wishes information on the effect that the different packaging conditions (pH, salt, and preservatives) may have on the quality and microbiological safety of the final products. This Doctoral Thesis is focused on the study of the packaging conditions of Aloreña de Málaga table olives to improve the quality and microbiological safety of their commercial products. It is divided into three sections. In the first, a physicochemical, microbiological and sensory study of commercial packaging is carried out to determine its shelf life and possible causes of spoilage. In the second, the use of different preservatives (zinc salts, sodium metabisulfite, cinnamaldehyde, pyruvic acid, fumaric acid, and natamycin) as alternatives for the substitution of sorbates and benzoates is evaluated, using mathematical models. In the last section, the microbiological safety of the final products is evaluated using challenge tests. They consisted of inoculating the commercial packaging with foodborne pathogens (Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Salmonella enterica) for determining the influence of specific inhibitory compounds from the fruits on their survival.

This work focuses on the persistence of the putative probiotic bacteria Lactobacillus pentosus TOMC‐LAB2 on green Spanish‐style Manzanilla olives according to different packaging conditions and storage temperatures. The lactic acid bacteria population decreased with time but the highest survival counts (and lowest yeasts) at the end of storage (8 months) were observed in plastic pouches under nitrogen atmosphere and glass jars with brine stored at 20°C. Molecular techniques showed a 100% presence of the putative probiotic bacteria in biofilms adhered to olive epidermis, while it was absent in PPB (plastic pouches with brine) and in olives stored at 7°C. No changes in NaCl, pH or combined acidity were observed during the storage except for a slight increase in titratable acidity at 20°C. The color of the fruits was stable but degraded at 20°C for olives in plastic pouches with brine.

Trabajo presentado en la 4th International Food Safety, Quality & Policy Conference, celebrada en Dubai los días 5 y 6 de diciembre de 2016, This work has received funding from the Spanish Government through the Olifilm project (AGL2013-48300-R)

Trabajo presentado en la 3rd International Conference on Microbial Diversity (MD2015, "The Challenge of Complexity"), celebrada en Perugia del 27 al 29 de octubre de 2015., The biochemistry and microbial ecology of PDO Aloreña de Málaga table olive fermentations were investigated in two different industries (1 and 2) of province of Málaga (Spain). Monitoring of the physicochemical parameters such as pH, acidity, salt, color and texture of fruits was carried out during the entire process. No differences were detected in physicochemical arameters, which were characteristics for this type of elaboration. The microbial ecology was investigated using both culture-dependent and culture-independent methods in fruit and brine. The initial yeast population was different between both industries. While industry (1) showed counts around 4 - 5 log10 cfu/mL during the first month of fermentation, industry 2 presented lower oncentrations (1 – 3 log 10 cfu/mL), although, both locations reached the same yeast populations at the end of storage period (>5 log 10 cfu/mL). Bar - coded pyrosequencing analysis of the ITS rDNA region identified 109 and 118 operational taxonomic units (OTUs) in fruits and brines, espectively, and revealed the presence of yeast genera previously identified in table olives such as Pichia, Candida, Debaryomyces and Sacharomyces sp., but also, this is the first time that other predominant genera such as Zygostorulaspora, Penicillium and Aspergillus have been identified in this type of table olives. Small differences were found between most abundant yeasts in fruits and brines, during the fermentation process and between both industries.

El presente estudio ha utilizado un modelo de dosis/respuesta para evaluar el efecto inhibidor del metabisulfito sódico, cinamaldehido y ácido pirúvico sobre el crecimiento de los principales grupos de microorganismos relacionados con el procesado de aceitunas de mesa

Trabajo presentado en la 3rd International Conference on Microbial Diversity (MD2015, "The Challenge of Complexity"), celebrada en Perugia del 27 al 29 de octubre de 2015., The objective of this research was to characterize the biochemistry and microbiology of spontaneous PDO Aloreña de Málaga table olive fermentations and define the bacterial biodiversity during the process. The study was carried out in two different locations in Malaga province (Spain) following the fermentation during 4 months. Biochemical analysis of olives showed a progressive increase in free acidity as consequent of a spontaneous fermentation, with pH and salt values characteristic of this type of product. Both locations showed a similar trend in microbiology enumeration by plating in selective media. Yeasts were the most abundant microorganisms during the time, while lactic acid bacteria (LAB) cell count revealed low concentration or even were not detected. The novel of this research is the study of the microbial ecology of olives fermentation using bar-coded pyrosequencing of 16S rDNA to characterize bacterial diversity. The bacterial α- and β- diversity differed between fruits and brines being higher for fruits especially at the beginning of the fermentation process and the opposite for the brines. Bacterial diversity changed during fermentation time, and a total of 323 and 150 operational taxonomic units (OTUs) were identified in fruits and brines, respectively. The predominant genera were Celerinantantimonas, Pseudomonas, Propionibacterium, and diverse Acetobacteraceae. Some differences in the bacterial diversity were found between industries, special at the beginning of the process, whereas no differences were found for the rest of physicochemical parameters such as color and texture in the final product.

This work examines the microbial communities adhered to the epidermis of natural green olives, Gordal variety, in order to study the microbial biodiversity present in olive biofilms. The fermentation process was microbiologically monitored for 90 d. At this time, formation of true biofilms in the surface of fermented fruits was confirmed by scanning electron microscopy. Then, samples of olive fruits were withdrawn and treated by means of stomacher and sonication for biofilm disaggregation and subsequent recovery of the lactic acid bacteria and yeast populations. The use of the stomacher for 1 min was the most effective treatment to release the lactic acid bacteria from biofilms (6.6 log10 cfu·g-1), whereas sonication for 5 min was the most efficient method for recovery of yeasts (up to 3.5 log10 cfu·g-1). Molecular identification and characterization of isolates revealed that among lactic acid bacteria, Lactobacillus pentosus was the only species found, showing two different genotypes. In the other hand, Pichia membranifaciens was the predominant yeast species. A greater predisposition of the bacteria (due to their higher counts obtained) than the yeast for integration into these biofilms was noticed.