Reduction of ethanol content in wine with an improved combination of yeast strains and process conditions

One interesting strategy to address the increasing alcohol content of wines, associated with climate change, is to reduce the ethanol yield during fermentation. Within this strategy, the approach that would allow the clearest reduction in alcohol content is the respiration of part of the grape sugars by yeasts. Non-Saccharomyces species can be used for this purpose but suffer from a limited ability to dominate the process and complete fermentation. In turn, Saccharomyces cerevisiae shows a high production of acetic acid under the growth conditions required for respiration. Previously proposed procedures used combinations of non-Saccharomyces and S. cerevisiae starters, or a strain of S. cerevisiae (PR1018), with unique metabolic properties. In both cases, precise management of oxygen availability was required to overcome the acetic acid problem. In this work, we have developed a laboratory scale process to take advantage of the properties of PR1018 and a strain of Metschnikowia pulcherrima. This process is more robust than the previous ones and does not rely on strict control of oxygenation or even the use of this particular strain of S. cerevisiae. Aeration can be interrupted instantly without impairing the volatile acidity. Under the selected conditions, an ethanol reduction of around 3% (v/v) was obtained compared to the standard fermentation control.The authors would like to thank Cristina Juez, Laura López, and Rufino Aguirrezábal for technical assistance. This work was funded by the Spanish Government through grant, PCI2018-092949 (ERA-CoBioTech) funded by MCIN/AEI/10.13039/501100011033 and co-funded by the European Union; and a research contract with AZ3Oeno (Spain). AMG predoctoral contract was funded by Consejería de Desarrollo Económico e Innovación de la Comunidad Autónoma de La Rioja.Peer reviewe

Biotechnological approaches to lowering the ethanol yield during wine fermentation

One of the most prominent consequences of global climate warming for the wine industry is a clear increase of the sugar content in grapes, and thus the alcohol level in wines. Among the several approaches to address this important issue, this review focuses on biotechnological solutions, mostly relying on the selection and improvement of wine yeast strains for reduced ethanol yields. Other possibilities are also presented. Researchers are resorting to both S. cerevisiae and alternative wine yeast species for the lowering of alcohol yields. In addition to the use of selected strains under more or less standard fermentation conditions, aerobic fermentation is increasingly being explored for this purpose. Genetic improvement is also playing a role in the development of biotechnological tools to counter the increase in the wine alcohol levels. The use of recombinant wine yeasts is restricted to research, but its contribution to the advancement of the field is still relevant. Furthermore, genetic improvement by non-GMO approaches is providing some interesting results, and will probably result in the development of commercial yeast strains with a lower alcohol yield in the near future. The optimization of fermentation processes using natural isolates is, anyway, the most probable source of advancement in the short term for the production of wines with lower alcohol contents

Cribado de cepas de S. cerevisiae para su aplicación en vinificación aeróbica

Resumen del trabajo presentado en el VIII Congreso Nacional de Microbiología Industrial y Biotecnología Microbiana, celebrado en Valencia (España), del 1 al 3 de junio de 2022La utilización del metabolismo respiratorio de S. cerevisiae podría ser una solución al problema del incremento del grado alcohólico del vino. La mayor dificultad en su aplicación no es la baja tasa de respiración de S. cerevisiae (debida al efecto Crabtree) sino la sobreproducción de ácido acético bajo condiciones aeróbicas. En este trabajo hemos realizado un cribado de 25 cepas enológicas de S. cerevisiae con el objetivo de identificar cepas aplicables en vinificación aeróbica. Las cepas presentaron homogeneidad en la producción de etanol, pero una gran diversidad para el rendimiento de ácido acético. Las tres cepas más prometedoras fueron seleccionadas para una caracterización en biorreactor. Bajo condiciones aeróbicas la producción de ácido acético fue significativamente baja para dos de ellas. En cuanto al perfil volátil, se hallaron compuestos tanto cepa-dependientes como dependientes de las condiciones de fermentación. También se ha optimizado el proceso para reducir la producción de etanol manteniendo una acidez volátil aceptable. El cambio de ambiente aeróbico-anaeróbico actúa como desencadenante para la producción de ácido acético, incluso en las cepas con menor producción. Con una disminución paulatina de la aireación se consiguió una reducción significativa del contenido alcohólico con una acidez volátil dentro de los estándares enológicos.Este trabajo se ha financiado con los proyectos AGL 2015-63629-R (cofinanciado con fondos FEDER), y PCI 2018-092949 (financiado por MCIN/AEI /10.13039/501100011033 y cofinanciado por la Unión Europea). JT es contratado del programa COMFUTURO de FGCSIC. AMG tiene un contrato predoctoral de la Consejería de Desarrollo Económico e Innovación de la C.A de La Rioja. EC tiene una beca JAE-Intro del CSIC

Is Saccharomyces cerevisiae suitable for winemaking under aerobic conditions?

Trabajo presentado en el 3rd Iberoamerican Congress on Biotechnology, celebrado en Braga (Portugal), del 7 al 9 de abril de 2022The increasing ethanol content in wine, due both climate change and market trends, has been a matter of concern for winemaking industry for the last two decades. The use of yeast respiratory metabolism has been proposed as a promising approach to solve this problem. Contrary to initial expectation, the use of S. cerevisiae for this purpose is not hampered because Crabtree effect -causing low respiration rates- but by acetic acid overproduction generated under the necessary aerobic conditions. In this work, we performed a screening of 25 S. cerevisiae oenological strains, with the aim to identify suitable strains for aerobic fermentation, and the optimization of their fermentation conditions to obtain a reduced ethanol yield with an acceptable volatile acidity (1). The data showed a low variability in ethanol yield but a great diversity in acetic acid yield. Some strains with very low volatile acidity under aerobic conditions were identified. The three more promising strains were selected for further characterization in bioreactor under both aerobic and anaerobic conditions, compared with a regular oenological strain. As expected, the ethanol yield of all the strains was lower under aerobic than anaerobic conditions. Two of the strains showed low acetic acid production under aerobic conditions. The volatile profile analysis showed differences strain-dependent and aeration conditions-dependent. The aeration process was optimized in laboratory bioreactors for the most promising strain. Even for this low acetate producer strain, the transition from aerobic to anaerobic fermentation triggers acetic acid production. A gradual decrease of aeration allowed a significant ethanol reduction with an acceptable volatile acidity (1).This work was funded by the Spanish Government through grants AGL 2015-63629-R (co-financed by FEDER funds), PCI 2018-092949 (funded by MCIN/AEI /10.13039/501100011033 and the European Union). JT is funded by FGCSIC by the COMFUTURO program. AMG predoctoral contract is funded by Consejería de Desarrollo Económico e Innovación de la C.A de La Rioja, EC was recipient of JAE-Intro fellowship from the Spanish National Research Council (CSIC)

Nuevas vías de disminución del grado alcohólico del vino mediante evolución dirigida

Resumen del trabajo presentado en el VIII Congreso Nacional de Microbiología Industrial y Biotecnología Microbiana, celebrado en Valencia (España), del 1 al 3 de junio de 2022En los últimos años, los vinos han aumentado su porcentaje en etanol debido principalmente al desequilibrio entre la madurez tecnológica (concentración azúcares) de la uva, adelantada por el cambio climático, y la madurez fenólica óptima, exigida por los consumidores, que prefieren vinos con cuerpo y estructura. Este escenario es especialmente problemático en los países cálidos, más afectados por el cambio climático y los productores tradicionales de vino. Para paliar esta tendencia, las fermentaciones aeróbicas se han propuesto como método para reducir el grado alcohólico. Teóricamente, la adición de oxígeno en el proceso de fermentación permitiría a la levadura S. cerevisiae respirar los azúcares en lugar de fermentarlos, disminuyendo así el contenido final de etanol. Esta propuesta tiene una serie de problemas, siendo uno de ellos, la alta producción de ácido acético que S. cerevisiae alcanza en estas condiciones. Para solucionar esta desventaja, utilizando cepas de S. cerevisiae comerciales, hemos puesto a punto una metodología, basada en la evolución dirigida en laboratorio que nos ha permitido obtener levaduras capaces de fermentar el mosto en condiciones aeróbicas con baja producción de ácido acético y con una disminución del rendimiento en la producción de etanol.Este trabajo se ha financiado con los proyectos AGL 2015-63629-R (cofinanciado con fondos FEDER), y PCI 2018-092949 (financiado por MCIN/AEI /10.13039/501100011033 y cofinanciado por la Unión Europea). JT ha sido contratado del programa COMFUTURO de FGCSIC. AMG tiene un contrato predoctoral de la Consejería de Desarrollo Económico e Innovación de la C.A de La Rioja

Directed evolution of Saccharomyces cerevisiae for low volatile acidity during winemaking under aerobic conditions

The use of yeast respiratory metabolism has been proposed as a promising approach to solve the problem of increasing ethanol content in wine, which is largely due to climate change. The use of S. cerevisiae for this purpose is mostly hampered by acetic acid overproduction generated under the necessary aerobic conditions. However, it was previously shown that a reg1 mutant, alleviated for carbon catabolite repression (CCR), showed low acetic acid production under aerobic conditions. In this work directed evolution of three wine yeast strains was performed to recover CCR-alleviated strains, expecting they will also be improved concerning volatile acidity. This was done by subculturing strains on galactose, in the presence of 2-deoxyglucose for around 140 generations. As expected, all evolved yeast populations released less acetic acid than their parental strains in grape juice, under aerobic conditions. Single clones were isolated from the evolved populations, either directly or after one cycle of aerobic fermentation. Only some clones from one of three original strains showed lower acetic acid production than their parental strain. Most clones isolated from EC1118 showed slower growth. However, even the most promising clones failed to reduce acetic acid production under aerobic conditions in bioreactors. Therefore, despite the concept of selecting low acetic acid producers by using 2-deoxyglucose as selective agent was found to be correct, especially at the population level, the recovery of strains with potential industrial utility by this experimental approach remains a challenge.The authors would like to thank Cristina Juez and Laura López for technical assistance. This work was funded by the Spanish Government through grants AGL2015-63629-R (co-funded by MCIN/AEI/10.13039/501100011033 and the ERDF A way to make Europe), PCI 2018-092949 (co-funded by ERA-CoBioTech MCIN/AEI/10.13039/501100011033 and the European Union). JT is funded by FGCSIC by the COMFUTURO program. AMG predoctoral contract is funded by Consejería de Desarrollo Económico e Innovación de la C.A de La Rioja

Exploring the suitability of Saccharomyces cerevisiae strains for winemaking under aerobic conditions

Aerobic fermentation was previously proposed to reduce the ethanol content of wine. The main constraint found for Saccharomyces cerevisiae to be used under these conditions was the high levels of acetic acid produced by all S. cerevisiae strains previously tested. This work addressed the identification of S. cerevisiae wine yeast strains suitable for aerobic fermentation and the optimization of fermentation conditions to obtain a reduced ethanol yield with acceptable volatile acidity. This approach unveiled a great diversity in acetic acid yield for different S. cerevisiae strains under aerobic conditions, with some strains showing very low volatile acidity. Three strains were selected for further characterization in bioreactors, with natural grape must, under aerobic and anaerobic conditions. Ethanol yields were lower under aerobic than under anaerobic conditions for all strains, and acetic acid levels were low for two of them. Strain-dependent changes in volatile compounds were also observed between aerobic and anaerobic conditions. Finally, the process was optimized at laboratory scale for one strain. This is the first report of S. cerevisiae wine strains showing low acetic acid production under aerobic conditions and paves the way for simplified aerobic fermentation protocols aimed to reducing the alcohol content of wines.This work was funded by the Spanish Government through grants AGL 2015-63629-R (co-financed by FEDER funds), PCI 2018-092949 (co-funded by ERA-CoBioTech). JT is funded by FGCSIC by the COMFUTURO program. AMG predoctoral contract is funded by Consejería de Desarrollo Económico e Innovación de la Comunidad Autónoma de La Rioja, EC was recipient of JAE-Intro fellowship from the Spanish National Research Council (CSIC)