Bioprospecting of wild yeast from Bio-Bio region for novel beer production.
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Date
2024
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Universidad de Concepción
Abstract
Con una producción global anual que ronda los 189 billones de litros, la cerveza se posiciona como la bebida fermentada más producida y consumida a nivel mundial. Además, ocupa el tercer lugar en consumo global de bebidas, después del agua y el té. Tradicionalmente, la cerveza se clasifica en dos estilos principales: cervezas de fermentación alta ("ale") y cervezas de fermentación baja ("lager").
Las cervezas lager representan aproximadamente el 90% de la producción total y su fermentación se realiza exclusivamente con la levadura Saccharomyces pastorianus. Esta levadura no corresponde una especie pura, sino que es el resultado de la hibridación entre la levadura cerveza y panificación, Saccharomyces cerevisiae, y una levadura salvaje tolerante al frío, Saccharomyces eubayanus. El sabor y aroma distintivamente limpio y fresco de las cervezas lager es uno de sus atributos más valorados. No obstante, la baja diversidad genética y aromática de estas levaduras se refleja en un restringido perfil aromático limitado en las cervezas lager.
En la actualidad, la demanda de cervezas con un perfil aromático y de sabor distintivos ha aumentado de manera importante durante los últimos años, generando un interés por la diversificación y diferenciación de las levaduras cerveceras utilizadas durante el proceso de elaboración. Además, las recientes iniciativas de salud pública que fomentan la reducción del consumo de alcohol para promover estilos de vida más saludables, junto con estrictas regulaciones sobre la conducción bajo los efectos del alcohol han impulsado la oferta y demanda de cervezas con un menor contenido de alcohol y un perfil de aroma y sabor deseables por el consumidor. En este escenario, el uso de levaduras no convencionales ha surgido como una alternativa prometedora para la producción de cervezas con un perfil aromático y de sabor más complejo y distintivo. Algunas de ellas, producen concentraciones de alcohol bajo el límite permitido y son consideradas cervezas de bajo contenido alcohólico.
En el capítulo uno, se presenta una revisión exhaustiva y detallada de la literatura sobre el papel de las levaduras convencionales y no convencionales en las fermentaciones industriales de cerveza y su efecto en las propiedades aromáticas y el sabor del producto final, así como la historia, las propiedades genéticas y fenotípicas y sus perspectivas futuras.
En el capítulo dos, se aislaron levaduras salvajes desde distintas fuentes naturales que se encuentran en la región del Bío-Bío y alrededores. Se identificaron cinco géneros de las levaduras salvajes aisladas Aureobasidium, Teunomyces, Yamadazyma, Metschnikowia y Saccharomyces. Debido a la importancia de Saccharomyces en la industria cervecera, se realizó el análisis de su genoma completo y fueron identificadas como Saccharomyces eubayanus. Se evaluaron 14 levaduras salvajes aisladas en relación con su capacidad de fermentación, su producción de compuestos aromáticos, glicerol y consumo de glucosa en pruebas de fermentación a escala de laboratorio. La producción de etanol de las levaduras salvajes no Saccharomyces fueron menores que 1.0 % ABV mientras que la producción de las levaduras salvajes Saccharomyces fue cercano a 4.0%. La producción de aroma de las levaduras no Saccharomyces también difirió significativamente de las levaduras salvajes Saccharomyces. De acuerdo a los resultados obtenidos, las levaduras salvajes pertenecientes al género Metschnikowia mostraron el perfil de compuestos más interesante dentro de las levaduras salvajes no Saccharomyces para la producción de cervezas con bajo contenido de alcohol. Paralelamente, las cepas de Saccharomyces eubayanus demuestran el potencial de estas levaduras salvajes para la producción de cervezas con un contenido de alcohol menor que las cervezas convencionales y de estilos en los que la presencia de 4-vinil guaiacol es considerada deseable.
En el capítulo tres, se utilizó la estrategia de cruzamiento espora-a-espora con el fin de generar nuevos híbridos interespecíficos entre dos cepas salvajes de S. eubayanus con una cepa de S. cerevisiae domesticada. Los 7 híbridos interespecíficos generados fueron expuestos a tres condiciones distintas durante las etapas tempranas de la estabilización de su genoma durante 20-25 generaciones. Se investigó cómo las distintas condiciones influyen en la trayectoria de estabilización del genoma y pueden conducir a variantes con diferentes fenotipos industriales. Los híbridos con la menor capacidad de transformar ácido ferúlico (precursor de 4-VG), fueron seleccionados para posteriormente analizar sus características de fermentación y la producción de metabolitos. Nuestros resultados revelan cómo la variación en el procedimiento de estabilización del genoma conduce a la variabilidad fenotípica y puede generar diversidad adicional después del proceso de hibridación inicial.
With a global annual production of around 189 billion liters, beer ranks as the most produced and consumed fermented beverage worldwide. Additionally, it occupies the third place in global beverage consumption, following water and tea. Traditionally, beer is classified into two main styles: top-fermented beers ("ale") and bottom-fermented beers ("lager"). Lager beers represent approximately 90% of total production, and their fermentation is carried out exclusively with the yeast Saccharomyces pastorianus. This yeast is not a pure specie but rather the result of hybridization between brewing and baking yeast, Saccharomyces cerevisiae, and a cold-tolerant wild yeast, Saccharomyces eubayanus. The distinctly clean and fresh flavor and aroma of lager beers is one of their most valued attributes. However, the low genetic and aromatic diversity of these yeasts is reflected in a limited aromatic profile in lager beers. Currently, the demand for beers with distinctive aromatic and flavor profiles has significantly increased in recent years, generating interest in the diversification and differentiation of brewing yeasts used during the production process. Furthermore, recent public health initiatives encouraging the reduction of alcohol consumption to promote healthier lifestyles, along with strict regulations on driving under the influence of alcohol, have driven the supply and demand for beers with lower alcohol content and desirable aroma and flavor profiles for consumers. In this context, the use of non-conventional yeasts has emerged as a promising alternative for producing beers with a more complex and distinctive aromatic and flavor profile. Some of these yeasts produce alcohol concentrations below the permitted limit and are considered low-alcohol beers. In chapter one, a thorough and detailed review of the literature on the role of conventional and non-conventional yeasts in industrial beer fermentations and their influence on the aromatic properties and flavor of the final product is presented, along with the history, genetic and phenotypic properties, and future prospects. In chapter two, wild yeasts were isolated from various natural sources found in the Bío-Bío region and its surroundings. Five genera of the isolated wild yeasts were identified: Aureobasidium, Teunomyces, Yamadazyma, Metschnikowia, and Saccharomyces. Due to the importance of Saccharomyces in the brewing industry, a complete genome analysis was performed, identifying them as Saccharomyces eubayanus. Fourteen isolated wild yeasts were evaluated for their fermentation capacity, production of aromatic compounds, glycerol, and glucose consumption in laboratory-scale fermentation tests. The ethanol production from non-Saccharomyces wild yeasts was less than 1.0% ABV, while the production from Saccharomyces wild yeasts was close to 4.0%. The aroma production from non Saccharomyces yeasts also differed significantly from that of Saccharomyces wild yeasts. According to the results obtained, wild yeasts belonging to the genus Metschnikowia showed the most interesting compound profile among non-Saccharomyces wild yeasts for producing low-alcohol beers. At the same time, strains of Saccharomyces eubayanus demonstrate the potential of these wild yeasts to produce beers with lower alcohol content than conventional beers, especially in styles where the presence of 4-vinyl guaiacol is considered desirable. In chapter three, a spore-to-spore crossing strategy was used to generate new interspecific hybrids between two wild strains of S. eubayanus and a domesticated strain of S. cerevisiae. The seven interspecific hybrids generated were exposed to three different conditions during the early stages of genome stabilization over 20-25 generations. The influence of different conditions on the genome stabilization trajectory was investigated to see how they could lead to variants with different industrial phenotypes. The hybrids with the lowest capacity to transform ferulic acid (a precursor of 4-VG) were selected for further analysis of their fermentation characteristics and metabolite production. Our results reveal how variations in the genome stabilization procedure lead to phenotypic variability and can generate additional diversity after the initial hybridization process.
With a global annual production of around 189 billion liters, beer ranks as the most produced and consumed fermented beverage worldwide. Additionally, it occupies the third place in global beverage consumption, following water and tea. Traditionally, beer is classified into two main styles: top-fermented beers ("ale") and bottom-fermented beers ("lager"). Lager beers represent approximately 90% of total production, and their fermentation is carried out exclusively with the yeast Saccharomyces pastorianus. This yeast is not a pure specie but rather the result of hybridization between brewing and baking yeast, Saccharomyces cerevisiae, and a cold-tolerant wild yeast, Saccharomyces eubayanus. The distinctly clean and fresh flavor and aroma of lager beers is one of their most valued attributes. However, the low genetic and aromatic diversity of these yeasts is reflected in a limited aromatic profile in lager beers. Currently, the demand for beers with distinctive aromatic and flavor profiles has significantly increased in recent years, generating interest in the diversification and differentiation of brewing yeasts used during the production process. Furthermore, recent public health initiatives encouraging the reduction of alcohol consumption to promote healthier lifestyles, along with strict regulations on driving under the influence of alcohol, have driven the supply and demand for beers with lower alcohol content and desirable aroma and flavor profiles for consumers. In this context, the use of non-conventional yeasts has emerged as a promising alternative for producing beers with a more complex and distinctive aromatic and flavor profile. Some of these yeasts produce alcohol concentrations below the permitted limit and are considered low-alcohol beers. In chapter one, a thorough and detailed review of the literature on the role of conventional and non-conventional yeasts in industrial beer fermentations and their influence on the aromatic properties and flavor of the final product is presented, along with the history, genetic and phenotypic properties, and future prospects. In chapter two, wild yeasts were isolated from various natural sources found in the Bío-Bío region and its surroundings. Five genera of the isolated wild yeasts were identified: Aureobasidium, Teunomyces, Yamadazyma, Metschnikowia, and Saccharomyces. Due to the importance of Saccharomyces in the brewing industry, a complete genome analysis was performed, identifying them as Saccharomyces eubayanus. Fourteen isolated wild yeasts were evaluated for their fermentation capacity, production of aromatic compounds, glycerol, and glucose consumption in laboratory-scale fermentation tests. The ethanol production from non-Saccharomyces wild yeasts was less than 1.0% ABV, while the production from Saccharomyces wild yeasts was close to 4.0%. The aroma production from non Saccharomyces yeasts also differed significantly from that of Saccharomyces wild yeasts. According to the results obtained, wild yeasts belonging to the genus Metschnikowia showed the most interesting compound profile among non-Saccharomyces wild yeasts for producing low-alcohol beers. At the same time, strains of Saccharomyces eubayanus demonstrate the potential of these wild yeasts to produce beers with lower alcohol content than conventional beers, especially in styles where the presence of 4-vinyl guaiacol is considered desirable. In chapter three, a spore-to-spore crossing strategy was used to generate new interspecific hybrids between two wild strains of S. eubayanus and a domesticated strain of S. cerevisiae. The seven interspecific hybrids generated were exposed to three different conditions during the early stages of genome stabilization over 20-25 generations. The influence of different conditions on the genome stabilization trajectory was investigated to see how they could lead to variants with different industrial phenotypes. The hybrids with the lowest capacity to transform ferulic acid (a precursor of 4-VG) were selected for further analysis of their fermentation characteristics and metabolite production. Our results reveal how variations in the genome stabilization procedure lead to phenotypic variability and can generate additional diversity after the initial hybridization process.
Description
Tesis presentada para optar al grado de Doctor en Ciencias y Tecnología Analítica
Keywords
Microorganismos, Innovación de proceso, Industria cervecera