Improving freshness and acidity, in warm zone wines through fermentation biotechnologies, and emerging non-thermal technologies

The world of oenology is booming and so is the creation of unique wines. But there is a big problem worldwide, which is the climate change that increasingly affects the vineyards and the quality of their grapes. In Spain, we are suffering this problem in the central and southern parts of the country. This influences the pH of the wines, which are increasing significantly, affecting the stability and ageing of the wines. The aim of this thesis was to further investigate non-Saccharomyces yeasts and in particular two of them, Lachancea thermotolerans (naturally acidifying) and Hanseniaspora vineae (with very particular aromatic characteristics), in monocultures and in co-inoculations (also known as mixed inoculation), to observe their fermentative power, their interactions, their nutrient demand, their level of implantation with the use of emerging non-thermal technologies, and their organoleptic characteristics, among other parameters. This thesis is a compendium of article. In it, a first trial was carried out to observe, the lactic acid production and other relevant characteristics of various L. thermotolerans at two different temperatures (17 and 27 °C), and with two different concentrations of sulphites (25 and 75 mg/L). It was observed that at higher temperatures, and with a lower amount of sulphites, more lactic acid and more fermentative esters were generated, while at lower temperatures more alcoholic strength was produced (~11% v/v). In the second trial, the objective was to know the interactions that occurred at the microbiological level, and how they affected the metabolism of each other, for which different co-inoculations with non-Saccharomyces yeasts were performed; L. thermotolerans was co-inoculated with Hanseniaspora vineae, Torulaspora delbrueckii or Metschnikowia pulcherrima, and the fermentation process was later completed with the sequential inoculation of Saccharomyces cerevisiae (ternary fermentation). These fermentations were also carried out in monoculture but using sequentially a S. cerevisiae to finish the fermentation (binary fermentation). Regarding acidification by L. thermotolerans in co-inoculations some metabolic interactions were observed, such as the inhibition of acidification when L. thermotolerans/H. vineae was used, generating slightly more than 0.13 g/L of lactic acid and, conversely, a synergistic effect when L. thermotolerans/M. pulcherrima was used, achieving 3.2 g/L of lactic acid and a pH value reduction of up to 0.33. We have observed a decrease in alcoholic strength of more than 0.6 % v/v in the co-inoculation with L. thermotolerans/M. pulcherrima yeasts, with total sugar consumption and very slow completion of fermentation in the inoculations with H. vineae and T. delbrueckii. We analysed the aromatic composition of the wines, and we found that both L. thermotolerans strains and the co-inoculations retained more aromatic esters over time, and had less evolution towards the yellow tones typical of oxidation. Both L. thermotolerans and co-inoculations produced wines with low levels of volatile acidity (< 0.4 g/L). In the third trial, we studied the potential of emerging non-thermal technologies, such as pulsed electric fields (PEF), to reduce the wild microbial load in the must, primarily to improve the implantation of selected yeasts (L. thermotolerans, M. pulcherrima, H. vineae, T. delbrueckii, S. pombe, and S. cerevisiae as a control), and secondarily to increase colour extraction more rapidly. For this purpose, we applied two PEF treatments; one with an intensity of 5 kV/cm was applied continuously to the must for higher colour extraction, and a second treatment was applied only to the must (without skins) after a 24-hour maceration with an intensity of 17.5 kV/cm. With the PEF treatment, wild yeasts were reduced by up to 2 log CFU/mL and, therefore all selected yeasts were better implanted giving higher amounts of their own metabolites. A higher number of total esters were retained. Also, L. thermotolerans produced four times more lactic acid (3.62 ± 0.84 g/L) than the same yeast without treatment, and H. vineae with PEF treatment produced almost three times more 2-phenylethyl acetate than the rest. We conducted the fourth trial on a pilot scale with the objective of comparing, on one hand, a conventional fermentation with a Saccharomyces and acidification with tartaric acid (1.5 g/L), and on the other hand, with two other fermentations with two strains of L. thermotolerans that naturally acidified (1 g/L) with lactic acid. To achieve tartaric acid-like acidification, S. cerevisiae was inoculated sequentially on the third day. The use of non-Saccharomyces yeasts generated wines with a higher amount of volatiles including carbonyl compounds, organic acids, lactones, fumaric compounds, and phenols, increasing their aromatic profile. Finally, in the fifth trial, the objective was to study the biocompatibility between L. thermotolerans species and two Hanseniaspora spp. These yeasts were fermented in co-inoculation with the addition of two different nutrients (Nutrient Vit BlancTM and Nutrient Vit NatureTM) at high doses (2.11 g/L for the first nutrient and 3.76 g/L for the second) to observe whether lactic acid formation was determined by the species or by the strain. Significant results were obtained in the population count (CFU/mL), with higher populations of Hanseniaspora spp. compared to L. thermotolerans. Fermentations with L. thermotolerans/H. vineae showed an inhibition of acidification, generating up to 0.41 g/L of lactic acid. In contrast, a synergistic effect when using L. thermotolerans/H. opuntiae, achieved 2.44 g/L lactic acid and a pH reduction of up to 0.16 and always more significant with Nutrient Vit BlancTM, perhaps due to the fact that this nutrient contains diammonium phosphate. At the same time, the ethanol concentration decreased by 0.34%. It was found that all fermentations retained more aromatic esters compared to the control with both nutrients and that, on day 7, the amount of 2-phenylethyl acetate was at least 3 times higher in all fermentations compared to the control (Sc + Nutrient Vit BlancTM) which was 5.96 mg/L. In view of the different results obtained in the articles, it would be interesting to continue investigating L. thermotolerans to know exactly what influences its higher or lower lactic acid production, and also to investigate the best way to form a consortium of two or more non-Saccharomyces yeasts to get the best organoleptic benefit from them and to continue promoting and evaluating non-thermal technologies for sterilisation and shelf-life extension of foodstuffs. RESUMEN El mundo de la enología está en auge y la creación de vinos únicos también. Pero hay un gran problema a nivel mundial, que es el cambio climático que afecta cada vez más a los viñedos y a la calidad de sus uvas, en España estamos sufriendo este problema en la zona centro y sur del país. Esto influye en el pH de los vinos que están aumentando de forma significativa, afectando a la estabilidad y envejecimiento de los vinos. El objetivo de esta tesis fue profundizar en la investigación de las levaduras no-Saccharomyces y en particular de dos de ellas, Lachancea thermotolerans (naturalmente acidificante) y Hanseniaspora vineae (con características aromáticas muy particulares), en monocultivos y en co-inoculaciones (también conocidas como inoculación mixta), para observar su poder fermentativo, sus interacciones, su demanda de nutrientes, su nivel de implantación con el uso de tecnologías emergentes no térmicas, sus características organolépticas, entre otros parámetros. En esta tesis por compendio de artículo, se realizó el primer ensayo para observar, la producción de ácido láctico y otras características relevantes de varias L. thermotolerans a dos temperaturas diferentes (17 y 27 °C), y con dos concentraciones diferentes de sulfitos (25 y 75 mg/L). Se observó que a mayor temperatura, y menor cantidad de sulfitos, se generaba más ácido láctico y más ésteres fermentativos, mientras que a menor temperatura más grado alcohólico (~11% v/v). En el segundo ensayo, el objetivo era conocer las interacciones que se producían a nivel microbiológico y cómo afectaban al metabolismo de unos y otros, para lo cual se realizaron diferentes co-inoculaciones con levaduras no-Saccharomyces; L. thermotolerans se co-inoculó con Hanseniaspora vineae, Torulaspora delbrueckii o Metschnikowia pulcherrima, y el proceso de fermentación se completó posteriormente con la inoculación secuencial de Saccharomyces cerevisiae (fermentación ternaria). Estas fermentaciones también se realizaron en monocultivo pero utilizando secuencialmente un S. cerevisiae para finalizar la fermentación (fermentación binaria). En cuanto a la acidificación por parte de L. thermotolerans en co-inoculaciones, se mostraron algunas interacciones metabólicas, como la inhibición de la acidificación cuando se utilizó L. thermotolerans/H. vineae, generando algo más de 0,13 g/L de ácido láctico y, por el contrario, un efecto sinérgico cuando se utilizó L. thermotolerans/M. pulcherrima, consiguiendo 3,2 g/L de ácido láctico y una reducción del valor del pH de hasta 0,33. Se observó una disminución del grado alcohólico de más de 0,6 % v/v en la co-inoculación con levaduras L. thermotolerans/M. pulcherrima, con un consumo total de azúcar y una finalización muy lenta de la fermentación en las inoculaciones con H. vineae y T. delbrueckii. Se analizó la composición aromática de los vinos y se comprobó que tanto las cepas de L. thermotolerans como las co-inoculaciones retuvieron más ésteres aromáticos a lo largo del tiempo, y tuvieron una menor evolución hacia los tonos amarillos típicos de la oxidación. Tanto L. thermotolerans como las co-inoculaciones produjeron vinos con bajos niveles de acidez volátil (< 0,4 g/L). En el tercer ensayo, se estudió el potencial de las tecnologías emergentes no térmicas, como los campos eléctricos pulsados (PEF), para reducir la carga microbiana silvestre en el mosto, con el fin de mejorar, en primer lugar, la implantación de levaduras seleccionadas (L. thermotolerans, M. pulcherrima, H. vineae, T. delbrueckii, S. pombe y S. cerevisiae como control) y, en segundo lugar, para aumentar la extracción de color más rápidamente. Para ello, se aplicaron dos tratamientos PEF; uno con una intensidad de 5 kV/cm se aplicó de forma continua al mosto para una mayor extracción de color, y un segundo tratamiento se aplicó sólo al mosto (sin hollejos) después de una maceración de 24 horas con una intensidad de 17,5 kV/cm. Con el tratamiento PEF, las levaduras silvestres se redujeron hasta en 2 log UFC/mL y, por tanto, todas las levaduras seleccionadas se implantaron mejor dando mayores cantidades de sus propios metabolitos viéndose un mayor número de ésteres totales. También L. thermotolerans produjo cuatro veces más ácido láctico (3,62 ± 0,84 g/L) que la misma levadura sin tratamiento, y H. vineae con tratamiento PEF produjo casi tres veces más acetato de 2-feniletilo que el resto. En el cuarto ensayo se realizó a escala piloto con el objetivo de comparar una fermentación convencional con un Saccharomyces y acidificación con ácido tartárico (1,5 g/L) y por otro lado, otras dos fermentaciones con dos cepas de L. thermotolerans que acidificaban de forma natural (1 g/L) con ácido láctico. Para conseguir una acidificación similar a la del ácido tartárico, se inoculó S. cerevisiae de forma secuencial el tercer día. El uso de levaduras no-Saccharomyces generó vinos con una mayor cantidad de volátiles, incluyendo compuestos carbonílicos, ácidos orgánicos, lactonas, compuestos fumáricos y fenoles, aumentando su perfil aromático. Por último, en el quinto ensayo, el objetivo fue estudiar la biocompatibilidad entre la especie L. thermotolerans y dos Hanseniaspora spp. Estas levaduras se fermentaron en co-inoculación con la adición de dos nutrientes diferentes (Nutrient Vit BlancTM y Nutrient Vit NatureTM) a dosis elevadas (2,11 g/L para el primer nutriente y 3,76 g/L para el segundo), todo ello para observar si la formación de ácido láctico se determinaba por la especie o por la cepa. Se obtuvieron resultados significativos en el recuento de la población (UFC/mL), con mayores poblaciones de Hanseniaspora spp. en comparación con L. thermotolerans. Las fermentaciones con L. thermotolerans/H. vineae mostraron una inhibición de la acidificación, generando hasta 0,41 g/L de ácido láctico. Por el contrario, se observó un efecto sinérgico al utilizar L. thermotolerans/H. opuntiae, que logró 2,44 g/L de ácido láctico y una reducción del pH de hasta 0,16 y siempre más significativa con Nutrient Vit BlancTM, quizás debido a que este nutriente contiene fosfato diamónico. Al mismo tiempo, la concentración de etanol disminuyó en un 0,34%. Se comprobó que todas las fermentaciones retenían más ésteres aromáticos en comparación con el control con ambos nutrientes y que en el día 7 la cantidad de acetato de 2-feniletilo era al menos 3 veces mayor en todas las fermentaciones en comparación con el control (Sc + Nutrient Vit BlancTM), que era de 5,96 mg/L. A la vista de los diferentes resultados obtenidos en los artículos, sería interesante seguir investigando L. thermotolerans para saber exactamente qué influye en su mayor o menor producción de ácido láctico, también, investigar la mejor manera de formar un consorcio de dos o más levaduras no-Saccharomyces para obtener el mejor beneficio organoléptico de las mismas, además de seguir promoviendo y evaluando las tecnologías no térmicas para la esterilización y extensión de la vida útil de los alimentos

Use of natural anthocyanins of grape, lees and wood extracts as dyes and flavourings in the elaboration of wine vermouth

Vermouth is a wine derivative fortified and flavored with botanicals (Fig. 1) [1]. Traditionally, red vermouth is pigmented with caramel. We used natural grape anthocyanins to improve the color and, at the same time, increase flavonoid contents, molecules with positive repercussion on health. The anthocyanins were extracted from grape skins using acidified water as solvent and physically assisted by ultrasounds. Commercial anthocyanins extracts from grape by-products were also used to improve vermouth color. Vermouths were aged using yeast lees biomass and also using chips from different types of woods: ?palo rojo?, cherry, cedar, oak and ebony. During 3 months

Use of natural anthocyanins of grape, lees and wood extracts as dyes and flavourings in the elaboration of wine vermouth

Vermouth is a wine derivative fortified and flavored with botanicals (Fig. 1) [1]. Traditionally, red vermouth is pigmented with caramel. We used natural grape anthocyanins to improve the color and, at the same time, increase flavonoid contents, molecules with positive repercussion on health. The anthocyanins were extracted from grape skins using acidified water as solvent and physically assisted by ultrasounds. Commercial anthocyanins extracts from grape by-products were also used to improve vermouth color. Vermouths were aged using yeast lees biomass and also using chips from different types of woods: ?palo rojo?, cherry, cedar, oak and ebony. During 3 months

XIV Congreso de estudiantes de Ciencia, Tecnología e Ingeniería agronómica

Compendio de las comunicaciones orales y pósters presentados por los estudiantes de Ciencias, Agronomía, Ingeniería y Biotecnología en el XIV Congreso de estudiantes de la Escuela Técnica de Ingeniería Agronómica, Alimentaria y de Biosistemas

High PEEP with recruitment maneuvers versus Low PEEP During General Anesthesia for Surgery -a Bayesian individual patient data meta-analysis of three randomized clinical trials

Background: The influence of high positive end-expiratory pressure (PEEP) with recruitment maneuvers on the occurrence of postoperative pulmonary complications after surgery is still not definitively established. Bayesian analysis can help to gain further insights from the available data and provide a probabilistic framework that is easier to interpret. Our objective was to estimate the posterior probability that the use of high PEEP with recruitment maneuvers is associated with reduced postoperative pulmonary complications in patients with intermediate-to-high risk under neutral, pessimistic, and optimistic expectations regarding the treatment effect. Methods: Multilevel Bayesian logistic regression analysis on individual patient data from three randomized clinical trials carried out on surgical patients at Intermediate-to-High Risk for postoperative pulmonary complications. The main outcome was the occurrence of postoperative pulmonary complications in the early postoperative period. We studied the effect of high PEEP with recruitment maneuvers versus Low PEEP Ventilation. Priors were chosen to reflect neutral, pessimistic, and optimistic expectations of the treatment effect. Results: Using a neutral, pessimistic, or optimistic prior, the posterior mean odds ratio (OR) for High PEEP with recruitment maneuvers compared to Low PEEP was 0.85 (95% Credible Interval [CrI] 0.71 to 1.02), 0.87 (0.72 to 1.04), and 0.86 (0.71 to 1.02), respectively. Regardless of prior beliefs, the posterior probability of experiencing a beneficial effect exceeded 90%. Subgroup analysis indicated a more pronounced effect in patients who underwent laparoscopy (OR: 0.67 [0.50 to 0.87]) and those at high risk for PPCs (OR: 0.80 [0.53 to 1.13]). Sensitivity analysis, considering severe postoperative pulmonary complications only or applying a different heterogeneity prior, yielded consistent results. Conclusion: High PEEP with recruitment maneuvers demonstrated a moderate reduction in the probability of PPC occurrence, with a high posterior probability of benefit observed consistently across various prior beliefs, particularly among patients who underwent laparoscopy