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La presente Tesis Doctoral estudia la viabilidad de nuevas elaboraciones de aceitunas de mesa con variedades autóctonas del Valle del Guadalhorce, como son el caso de la Aloreña de Málaga y la Campiñesa. Para ambas variedades se ha hecho un seguimiento fisicoquímico, microbiológico, y sensorial del proceso de elaboración como verdes al estilo español, principal tipo de elaboración a nivel mundial de aceitunas de mesa, además de la realización de estudios de genotipado al final de la etapa fermentativa. Esta Tesis Doctoral también incluye el estudio de la influencia de la metodología de envasado sobre la estabilidad fisicoquímica, microbiológica y organoléptica de aceitunas Aloreñas curadas ecológicas. En la actualidad las aceitunas Aloreñas de Málaga se procesan como verdes naturales, debido a su bajo contenido en oleuropeína, compuesto responsable del amargor de las aceitunas, no necesitando tratamiento alcalino. Existe un porcentaje de aceitunas de esta variedad que por su pequeño tamaño no cumplen los estándares de calidad que se establecen para ser procesadas con el sello de la DOP, se destinan a la producción de aceite, mismo destino del 100 % de la producción de aceitunas de la variedad Campiñesa, aceitunas con una producción muy localizada en este entorno. La gran demanda existente de aceitunas de mesa ha llevado a pensar que ambas variedades podrían ser elaboradas como aceitunas de mesa al estilo español innovando en el tipo de procesado que se emplea en la actualidad, aumentando así la oferta de productos en el mercado. Para evaluar la viabilidad de las dos variedades elaboradas al estilo español, durante la campaña 2018/2019 se estudiaron los parámetros influyentes en la calidad de las aceitunas de mesa, prestando especial atención a la etapa de cocido, ensayándose diferentes concentraciones de hidróxido de sodio. Asimismo, tras el cocido y el lavado, se hizo un seguimiento y estudio de los diferentes parámetros microbiológicos y fisicoquímicos durante la fermentación que fue dirigida por la adición del cultivo iniciador OleicaStarter® (compuesto por dos cepas de Lactiplantibacillus pentosus 13B4 y 119). Tras la fermentación se estudió el efecto de la conservación con altas concentraciones de cloruro de sodio, y la influencia del tipo de envasado, frasco de vidrio con salmuera (durante 8 meses) y bolsa de plástico con atmósfera modificada de nitrógeno (durante 6 meses), para conocer la posible viabilidad comercial de estas aceitunas. Durante la segunda campaña se desarrollaron modelos predictivos para la variedad Aloreña de Málaga calibre perdigón, que cuantificaron la evolución del comportamiento microbiano y los parámetros fisicoquímicos durante la fermentación para dos tratamientos de cocido, con el objetivo de conocer la influencia de los factores y formulaciones empleadas sobre la cinética en las salmueras de fermentación. Con respecto a las aceitunas Campiñesas, el ensayo de la segunda campaña se enfocó, en el uso de diferentes cultivos iniciadores de bacterias ácido lácticas (OleicaStarter® y Lp309) con el objetivo de favorecer el desarrollo de una fermentación láctica y conseguir una mayor estandarización del proceso. El estudio con aceitunas Aloreñas curadas ecológicas con sello DOP ha tenido como objetivo el desarrollo de nuevas técnicas de envasado, en las que se usaron diferentes líquidos de gobierno (agua de mar, agua de manantial o agua de red), diferentes aliños (oleorresinas o especias naturales), y solo una parte de los envasados fueron sometidos a tratamiento térmico de pasterización. A lo largo de la vida de mercado se evaluaron los parámetros fisicoquímicos y microbiológicos junto con estudios organolépticos de los nuevos productos elaborados. Se concluye, que las aceitunas de la variedad Aloreña de Málaga con calibre perdigón, pueden elaborarse como aceitunas verdes al estilo español, resultando un producto seguro que cumple con las exigencias de calidad y seguridad alimentaria. Además, la etapa de fermentación pudo ser descrita de forma precisa a través del desarrollo de modelos predictivos. Por el contrario, las aceitunas Campiñesas presentaron mayores dificultades en su elaboración, siendo necesario la utilización de cultivos iniciadores de bacterias ácido lácticas más resistentes a las condiciones de las salmueras que ayudaron al desarrollo correcto de la fermentación y a la rápida estabilización del producto. Asimismo, si por motivos logísticos estas variedades debieran ser conservadas en vez de envasadas, no existiría una merma sustancial en términos de calidad, pudiendo ser envasadas bajo demanda. El estudio de envasados de aceitunas curadas Aloreñas ecológicas, la no pasteurización de los distintos envasados produjo refermentaciones en algunas de las presentaciones estudiadas. Por tanto, se concluyó que el empleo de un tratamiento térmico mantiene el producto con mayor estabilidad durante el periodo de vida de mercado estudiado, obteniéndose mayor aceptación de consumo. This Doctoral Thesis studies the feasibility of new elaborations of table olives with autochthonous varieties of the Guadalhorce Valley, Aloreña de Málaga and Campiñesa. For both varieties, a physicochemical, microbiological, and sensory monitoring of the Spanish-style elaboration process has been carried out, being the main table olive processing worldwide. Besides, genotyping studies have been performed at the end of the fermentation stage. This Doctoral Thesis also includes the study of the influence of the packaging methods on the physicochemical, microbiological, and sensorial stability of organic cured Aloreña table olives. Table olives corresponding to the Aloreña de Málaga variety are currently processed as natural green olives, due to their low content of oleuropein, a compound responsible for the bitterness of the olives, not requiring alkaline treatment. There is a percentage of olives of this variety that, due to their small size, do not meet the quality standards established to be processed with the PDO certification. Thus, they are destined for production of olive oil, as 100% of the production of olives from the Campiñesa variety, having a very localized production in this environment. The increasing demand for table olives has led to think that both varieties could be elaborated as Spanish-style table olives. This would result in a processing type innovation, thus increasing the supply of products on the market. To assess the viability of the two varieties made in the Spanish-style, during the 2018/2019 season, the parameters influencing the quality of table olives were studied, deserving special attention to the lying treatment by using different concentrations of sodium hydroxide. Likewise, after lying and washing, the different microbiological and physicochemical parameters were monitored and studied during fermentation, which was directed by the addition of the OleicaStarter® starter culture (composed of two strains of Lactiplantibacillus pentosus 13B4 and 119). After fermentation, the effect of olives preservation was studied at different high concentrations of sodium chloride, as well as the influence of the type of packaging, i.e., glass jars with brine (for 8 months) and plastic bags with modified atmosphere of nitrogen (for 6 months), to know the commercial viability of these olives. During the second campaign, predictive models were developed for the small-sized Aloreña de Málaga variety. These models quantified the evolution of the microbial behavior and the physicochemical parameters during fermentation for two lying treatments, with the aim of understanding the influence of the factors and formulations used on the kinetics of fermentation brines. Regarding the Campiñesas olives, the trial of the second campaign focused on the use of different lactic acid bacteria starter cultures (OleicaStarter® and Lp309) with the aim of enhancing the development of lactic acid fermentation and process standardization. Regarding the study with organic cured Aloreñas olives with the PDO certification was the development of new packaging techniques using different brines (sea water, spring water or tap water), seasonings (oleoresins or natural spices), and the application or not of a pasteurization treatment. Throughout the shelf-life, the physicochemical and microbiological parameters were evaluated along with sensorial studies of the finished products. It is concluded that the small-sized Aloreña de Málaga can be produced as green Spanish-style table olives, resulting in a safe product meeting the quality and food safety standards. In addition, fermentation can be accurately described using different predictive models. On the contrary, the Campiñesas olives presented more difficulties for elaboration, being necessary the use of alternative lactic acid bacteria starter cultures more resistant to the conditions of the brines achieving a correct fermentation and a rapid stabilization of the product. Likewise, if for logistical reasons these varieties had to be preserved instead of packaged, there would be no substantial loss in terms of quality, and they could be packaged on demand. The study of packaging of organic Aloreña cured olives, the unpasteurized packages led to product refermentations in some of the studied presentations. Moreover, it was concluded that the use of a heat treatment maintains the product with greater stability during the studied shelf- life, achieving higher consumers’ acceptance.

La Aloreña de Málaga es una variedad de aceituna de mesa autóctona del Valle del Guadalhorce y la Sierra de las Nieves, con una demarcación muy concreta (19 municipios) siendo muy apreciada dentro y fuera de la provincia de Málaga. Fue la primera aceituna de mesa en España en obtener el sello de calidad de DOP (BOJA nº215, 2009). El volumen de producción de este tipo de especialidad de aceitunas de mesa es de aproximadamente 3.500- 4.000 ton/año según datos estimados por el Ministerio de Agricultura (http://www.aica.gob.es/) y contrastados con las declaraciones de los industriales, realizadas por el Consejo Regulador. Por lo tanto, la producción no es muy elevada comparada con otros tipos de elaboraciones como son las aceitunas verdes estilo español o sevillano. Sin embargo, su elaboración aún hoy en día se sigue realizando de manera muy artesanal (un aspecto este altamente valorado por los consumidores), y es llevado a cabo en industrias de pequeño tamaño (650). De este modo, se han estudiado los niveles poblacionales de aerobios mesófilos, levaduras, bacterias lácticas, enterobacterias, esporulados, presencia de Listeria, Salmonella, Clostridium y Staphylococcus coagulasa positivos. En base a los resultados obtenidos, se generó un modelo matemático que estimó el grado de cumplimiento higiénico-sanitario del proceso de elaboración de este tipo de aceituna de mesa mediante un sistema de ponderación (basado en la legislación presente y opiniones de expertos) y modelo probabilístico de gestión del riesgo. La contribución relativa de cada fase a la calidad y seguridad del producto final fue evaluada y ponderada por un panel de expertos (n=25) del ámbito científico y del sector de la Aloreña de Málaga. No se ha detectado en ninguna empresa la presencia de especies patógenas que puedan suponer un riesgo para los consumidores. La etapa de aliño ha resultado ser la más crítica dentro del proceso, ya que las hierbas aromáticas y diversos ingredientes que se utilizan en el mismo presentan una elevada carga microbiana que se añade directamente a los envasados sin ningún tipo de tratamiento previo. También es recomendable reducir el número de microorganismos en el ambiente y un mayor control de las aguas destinadas al consumo humano. Se observó asimismo que el grado de cumplimiento higiénico-sanitario determinado por el sistema de evaluación desarrollado aumenta conforme se acercan las etapas finales del proceso, las cuales resultan ser las más seguras. Con toda la información obtenida, se ha desarrollado un protocolo y procedimiento científico que puede ser utilizado por las empresas del sector para estandarizar sus procesos, valorar cuantitativamente el grado de cumplimiento higiénicosanitario de las diferentes etapas de producción y predecir los efectos de la introducción de medidas correctoras sobre la calidad y seguridad de los productos finales. The Aloreña de Málaga table olive is an autochthonous variety from Guadalhorce Valley and Sierra de las Nieves region, with a confined demarcation (19 municipalities) being well appreciated inside and outside Malaga province. It has been the first table olive with Protected Designation of Origin (PDO) in Spain (BOJA No. 215, 2009). The production volume of this table olive speciality is below 3,500-4,000 ton according to estimated records of the Ministry of Agriculture ((http://www.aica.gob.es/) and further verified with the interviews performed by the Regulatory Council to industrials. Thus, production is limited in comparison to other elaborations such as the Spanish table olive style. However, its elaboration is still very artisanal (which is highly appreciated by consumers), being carried out by SMEs with less than 20 operators. These industries have a great interest in optimizing and improving the transformation processes in order to obtain a valorised product with high quality and safety standards, which is currently requested by the large distribution chain. This Doctoral Thesis is focused on providing additional knowledge about the transformation processes of Aloreña de Málaga table olives as well as to evaluate the effect of new changes on quality and safety of final products. In this sense, the Doctoral Thesis is divided into two sections. In the first section, novel methodologies such as metagenomics and statistical modelling are applied with the aim to gain knowledge about how different modifications (reduction of sodium chloride, inclusion of resting periods to table olives, and application of heat treatments) can affect to the elaboration process. In the second section, the hazard analysis and critical control points from various industries were evaluated in different campaigns in order to determine the fulfilment of hygienic-sanitary conditions through the performance of physicochemical and microbiological analyses along the production chain. Information generated will allow the further development of a scientific procedure based on advanced mathematical models and different weighing values to improve food quality and safety management in industries. To study the effect of sodium chloride reduction and the addition of a resting period of fruits during fermentation and posterior table olive preservation, 1,500 kg of olive fruits were collected and placed in containers with different preservation brines and varying concentrations of acetic acid, citric acid and salt, either directly or with a resting period of table olives at room temperature (72 h, 25ºC). Afterwards, the main physicochemical and microbiological parameters were monitored during 1 year. The presence of a high amount of acetic acid together with the absence of sodium chloride did not negatively affect the evolution of the main physicochemical parameters, though differences existed according to the assayed treatments. The elaboration process was marked by a rapid inhibition of Enterobacteriaceae population, as well as by a growth of lactic acid bacteria (just at the end of the process) and specially, by the yeasts growth, which were continuously present reaching concentration levels up to 6 log10 CFU/mL. One of the first applications in food microbiology of a functional data analysis model programmed in R was presented. The model allowed a smart comparison of the different assayed treatments, providing additional information on the rate and acceleration of changes in the elaboration process through the calculation of means, maximal and minimal values. Model fitting showed that yeasts population was very similar among the evaluated treatments, though significant differences existed for some fermentation treatment times. Nevertheless, larger differences were found for pH evolution, specially during the first 3- month process, being then stabilized after all treatments until a pH of 4.0 units approximately. Next Generation Sequencing techniques were subsequently applied to study fermentation and preservation processes of Aloreña de Málaga table olives. To do this, four fermentation containers were monitored (approximately 500 kg of table olives) at the food industry during a 4-month period by performing physicochemical and microbiological (Enterobacteriaceae, lactic acid bacteria and yeasts) analyses. From a microbiological point of view, fermentation took place normally, being yeasts the only population group showing growth while lactic acid bacteria and Enterobacteriaceae were not present during fermentation. During the 4-month fermentation period metagenomic analysis of brines and fruit samples were carried out with the aim of studying the ecology of bacterial (16S) and fungal populations (ITS). This methodology allowed detecting with relative frequency bacteria genera not being previously described in this product such as Celerinatantimonas, Pseudomonas and Propionibacterium among the bacterial groups, and Penicillium, Pichia and Zygotorulaspora among yeasts and moulds. There was not any pathogenic microorganism in the fermented table olives studied by what microbial food safety was ensured. Therefore, the application of this culture independent technique has extended the microbial spectrum of bacterial species not being previously detected by conventional culture dependent techniques. The potential role of these microbial groups on the final product quality is still to be determined. One-hundred kg. of cracked olives (destined to the elaboration of traditional table olives) and whole olives (destined to the elaboration of cured table olives) were collected to study the effect of a previous heat treatments on the fermentation process of olive fruits together with their organoleptical characteristics. These fruits were submitted to a heat treatment which consisted on a water immersion at 60ºC for 5 min before placing them in brine. Afterwards, fermentations were monitored during a study period longer than 5 months. Performance of this heat treatment in table olives previously to their transferring to brines enhanced lactic acid fermentation, with a higher sugars consumption, free acidity production and pH drop, which guaranteed the stability of olive fruits. In the heat-treated fermented table olives, population levels of lactic acid bacteria reached 5 log10 CFU/mL, while in those brines without a heat treatment, this microbial group was not detected. Improvement of fruit coloration (luminosity, green colour etc.) was observed in those fruits undergoing a heat treatment during fermentation and packaging steps. The great advantage of the applied heat treatment was subsequently confirmed by the improvement in the stability and acceptability of table olives once packaged. Indeed, 41 days after packaging, the products having the highest proportion of olive tasters who would consume them and would purchase the packaging, corresponded to the traditional heat-treated table olives. The acceptability percentages sparsely varied between 4 and 41 after packaging, which did not occur with the rest of assayed treatments. Finally, the metagenomic analysis performed at the end of the fermentation period to all elaboration types, and between treated and non-treated olive fruits revealed that the bacteria genera Lactobacillus, Pediococcus and Celerinatantimonas were present in all elaborations in both fruits and brines, and that prevalence of Lactobacillus was significantly higher in heat-treated olive fruits, which confirmed the improvement of lactic acid fermentation by the heat treatment applied. Finally, in the present Doctoral Thesis three representative companies of the Aloreña de Málaga table olive sector were visited during three consecutive campaigns (13/14, 14/15, y 15/16). Physicochemical and microbiological analyses (>650) were performed corresponding to environmental control samples, equipment surfaces, food handlers, olive fruits, olive dressings, brines, and potable water. In this way, population levels of mesophilic bacteria, yeasts, lactic acid bacteria, sporulated bacteria, together with presence of Listeria, Salmonella, Clostridium and coagulase positive Staphylococci, were analysed. Based on the obtained results, a mathematical model was developed to estimate a Performance Hygiene and Safety Score (PHSS) of the elaboration process of this table olive type by means of a weighing system (based on current legislation applied and experts opinions) together with a probabilistic decision-making model. The relative contributions of each processing step to final product quality and safety were evaluated and weighted by a scientific expert panel (n=25) together with industrials from the Aloreña de Málaga table olive sector. There was not any food company having presence foodborne pathogens during the whole study period. Addition of olive dressings has resulted to be most critical processing step, since aromatic herbs and diverse ingredients used presented a high microbial load which is directly added to the packaged products without a posterior heat treatment. It is also recommended to reduce the environmental contamination as well as a more intensive control of the potable water destined to human consumption. Likewise, it was observed that the PHSS values determined by the developed mathematical model increased according to the final processing steps, being the safest ones. With the information obtained, a protocol and scientific procedure was performed, which can be used by industrials in order to standardize their elaboration processes, to quantitatively assess the degree of fulfilment of hygienic-sanitary conditions of the different production steps and to predict the effect of corrective measures on final products quality and safety.