Identificación y selección de Saccharomyces cerevisiae nativas para mejorar el proceso productivo del Pisco a partir de uva Quebranta

El Pisco es el destilado del Perú, elaborado a partir de mostos recientemente fermentados con uvas criollas denominadas "pisqueras". Las levaduras son los microorganismos clave en la fermentación y el uso de cepas nativas seleccionadas presenta ventajas competitivas para la tipicidad del producto, así como también para la estandarización del producto y el control microbiológico del proceso. El objetivo fue identificar y seleccionar las cepas de levaduras nativas para la producción del Pisco de uva Quebranta aisladas de procesos productivos de Pisco en el valle de Ica. Para ello, se emplearon técnicas microbiológicas y moleculares mediante el análisis de ITS1-5.8S-ITS2 PCR-RFLP para la identidad taxonómica. La evaluación de las cepas para producir Pisco consistió en el análisis fisicoquímico y organoléptico del destilado obtenido con las cepas seleccionadas. Se evaluaron 3 aislados para la producción de Pisco identificados como Saccharomyces cerevisiae: UNA SC - 25, UNA SC - 49 y UNA SC - 54, de los cuales la cepa UNA SC-49 destacó por mostrar aptitudes enológicas diferentes a las otras cepas. Este trabajo constituye el primer registro de levaduras nativas del Perú para mostos procedentes de uva Quebranta.

El Pisco stands as Peru's distilled spirit, crafted from recently fermented musts derived from native grape varieties known as "pisqueras". Yeasts serve as decisive microorganisms in the fermentation process, and the utilization of selected native strains confers competitive advantages for product typicity, standardization, and microbiological control within the production process. The aim was to identify and select native yeast strains for Quebranta grape Pisco production, isolated from Pisco production processes in the Ica Valley. Microbiological and molecular techniques were employed, utilizing ITS1-5.8S-ITS2 PCR-RFLP analysis for taxonomic identification. The assessment of yeast strains for Pisco production involved the physicochemical and organoleptic analysis of the distilled product obtained using the selected strains. Three isolates were evaluated for Pisco production, identified as Saccharomyces cerevisiae: UNA SC - 25, UNA SC - 49, and UNA SC - 54. Among these, the UNA SC-49 strain stood out due to displaying oenological characteristics distinct from the other strains. This work is the first documentation of native yeasts in Peru for musts derived from Quebranta grapes.

Prospection of indigenous yeasts from Uruguayan Tannat vineyards for oenological applications.

Prospection of yeasts from oenological environments can provide knowledge of new native strains that are capable of fermenting must and positively influence the composition and sensory characteristics of the wine. This work addressed the biotechnological characterization of indigenous yeasts of Tannat must, an emblematic and widespread vineyard of Uruguay. Fifty-three yeast isolates were morphologically characterized and further identified by amplification and sequencing of ITS and D1-D2 regions, grouping into a total of fifteen species. One isolate of each species was randomly chosen and evaluated for its technological traits. In presence of ethanol (6 to 16% v/v) and sulfur dioxide (40 mg/L), native Saccharomyces cerevisiae 3FS presented the best growth rates and minor lag phase. Regarding non-Saccharomyces strains, Starmerella bacillaris 3MS stood out for its behavior in vinification conditions, more closely related to S. cerevisiae strains. Saccharomyces cerevisiae 3FS, Starmerella bacillaris 3MS, and Saturnispora diversa 1FS conducted a successful fermentation process reaching a final ethanol concentration ≥ 10% v/v and presenting a killer and resistant phenotype, suggesting that they could be used as pure starter cultures, as well as in mixed culture fermentations. This preliminary screening and oenological characterization of indigenous Saccharomyces cerevisiae and non-Saccharomyces yeasts might be a useful tool to identify some strains as potential candidates for wine vinification.

Native Yeasts and Lactic Acid Bacteria Isolated from Spontaneous Fermentation of Seven Grape Cultivars from the Maule Region (Chile).

Grapes are a source of native yeasts and lactic acid bacteria (LAB); however, the microbial make up is dependent on the grape cultivar and the regional growth conditions. Therefore, the aim of this study was to characterize the yeast and LAB in seven grape cultivars cultivated in Chile. Grape juices were fermented at 25 °C for 7 days. Samples were collected to analyze sugar, organic acids, and ethanol. Microbial evolution was measured with culture-dependent and molecular approaches. Then, a native isolated Candida oleophila was selected for further sequential fermentations with Saccharomyces cerevisiae. The grape cultivars in the Maule showed a diversity of non-Saccharomyces yeasts, with a greater diversity observed at the beginning of the fermentation. However, species from the Hansenasporia, Metschnikowia, Torulaspora, Lachancea, and Candida genera were detected after 7 days, suggesting tolerance to environments rich in ethanol, capability may be associated to the terroir studied, which is characterized by torrid weather and antique and traditional vineyards. The alcoholic fermentation negatively impacted the LAB population, and after 7 days only Leuconostoc mesenteroides was isolated. In the sequential fermentations, C. oleophila was able to produce fermented grape juices with <1.5 g/L glucose, 12.5% (v/v) alcohol, and low concentrations of malic (<1.00 g/L) and succinic (2.05 g/L) acids, while acetic acid reached values >0.3 (g/L). To our knowledge this is the first time C. oleophila has been reported as a potential starter culture for wine production. However, more studies are necessary to fully characterize the potential of C. oleophila on wine attributes.

Optimization of fermentation-relevant factors: A strategy to reduce ethanol in red wine by sequential culture of native yeasts.

Current consumer preferences are determined by well-structured, full-bodied wines with a rich flavor and with reduced alcohol levels. One of the strategies for obtaining wines with reduced ethanol content is sequential inoculation of non-Saccharomyces and Saccharomyces cerevisiae yeasts. However, different factors affect the production of metabolites like ethanol, glycerol and acetic acid by inoculated yeasts. In order to obtain low alcohol wines without quality loss, the aims of our study were: i) to determine optimum conditions (fermentation temperature and time of permanence and initial inoculum size of the non-Saccharomyces population at the beginning of the process, prior to inoculation with S. cerevisiae); ii) to validate the optimized factors; and iii) to assess sensory quality of the wines obtained after validation. Two combinations of yeasts were used in this study: Hanseniaspora uvarum BHu9/S. cerevisiae BSc114 and Candida membranaefaciens BCm71/S. cerevisiae BSc114. Optimization of three fermentation factors that affect to non-Saccharomyces yeasts prior to S. cerevisiae inoculation was carried out using a Box-Behnken experimental design. Applying the models constructed by Response Surface Methodology, the lowest ethanol production by H. uvarum BHu9/S. cerevisiae BSc114 co-culture was obtained when H. uvarum BHu9 was inoculated 48 h 37 min prior to S. cerevisiae inoculation, at a fermentation temperature of 25 °C and at an initial inoculum size of 5 × 106 cells/mL. Lowest alcohol production with C. membranaefaciens BCm71/S. cerevisiae BSc114 was observed when C. membranaefaciens BCm71 was inoculated 24 h 15 min prior to S. cerevisiae at a fermentation temperature of 24.94 °C and at an initial inoculum size of 2.72 × 106 cells/mL. The optimized conditions of the two co-cultures were subsequently submitted to lab-scale validation. Both proposed strategies yielded ethanol levels that were significantly lower than control cultures (S. cerevisiae). Wines fermented with non-Saccharomyces/Saccharomyces co-cultures under optimized conditions were also associated with higher aromatic complexity characterized by the presence of red fruit aromas, whereas wines obtained with S. cerevisiae BSc114 were described by parameters linked with high ethanol levels.

Population and oenological characteristics of non-Saccharomyces yeasts associated with grapes of Northwestern Argentina.

Yeasts population associated with grapes from Northwest Argentina, a region with a significant vine-growing increase over the past years, was evaluated. Ten species of non-Saccharomyces yeasts were identified from four grape varieties (Malbec, Merlot, Syrah and Torrontes) being Hanseniaspora uvarum the dominant species. Typing of isolates revealed genetic variability within Hanseniaspora genus and also high variability was observed according to their oenological characteristics. Based on the oenological properties, the most adequate strains as starter cultures were H. uvarum HuT7, HuMe15, HuS16, H. vineae HvT-mc1 and Metschnikowia pulcherrima MpT2/MpT3. These selected yeasts exhibited moderate resistance to SO2, reduced values of volatile acidity, null or low production of H2S, high levels of enzymes related to aroma and did not produce killer toxins. Further studies using mixed cultures of these non-Saccharomyces strains and S. cerevisiae are needed to validate the contribution of selected indigenous yeasts on wine organoleptic characteristics.

Evaluación de levaduras nativas productoras de etanol presentes en el bagazo de caña de azúcar / Evaluation of producing ethanol native yeasts present in sugar cane bagasse

RESUMEN La hidrólisis química o enzimática del bagazo de caña de azúcar permite la obtención de azúcares fermentables, utilizados en la producción biotecnológica de etanol, mediante el empleo de levaduras comerciales o autóctonas obtenidas de diferentes materiales lignocelulósicos. El objetivo de este trabajo fue valorar la capacidad de producción de e tanol de cepas de levaduras nativas, aisladas en medio YPD e hidrolizado de bagazo de caña de azúcar, concentrado hasta un 75 %. Utilizando como variables de estudio el tipo de cepa y el tiempo de proceso, se realizó un análisis multifactorial (ANOVA) para su evaluación. Los resultados obtenidos con la cepa seleccionada UAT-3, fueron para Yp/s de 0.441 7 g/g y QP de 0.076 7 g/L-h a las 120 h. Las condiciones de proceso utilizadas en el presente estudio permitieron aislar y seleccionar cepas nativas de Sacharomyces cereviseae, con características adecuadas para ser utilizadas en procesos biotecnológicos industriales de producción de etanol, utilizando como sustrato residuos o subproductos derivados de la in dustria azucarera como el bagazo de caña de azúcar.

ABSTRACT The chemical or enzymatic hydrolysis of sugar cane bagasse, allows the obtaining of fermentable sugars used in the biotechnological production of ethanol by using commercial or native yeasts obtained from different lignocellulosic materials. The purpose of this study was to assess the production capacity of ethanol from a native yeast strain isolated in YPD and hydrolyzed sugar cane bagasse concentrated up to 75 %. Using as study variables the type of strain and processing time, a multivariate analysis (ANOVA) was performed for its evaluation. The results achieved with the selected strain UAT-3, were 0.441 7 g/g for Yp/s and 0.076 7 g/L-h to 120 h for QP. The process conditions used in the present study allowed to isolate and select native strains of Sacharomyces cereviseae, with characteristics suitable to be used in industrial biotechnological proceses of ethanol production, using as substrate residues or by-products derived from the sugar industry such as bagasse of sugar.

How native yeasts may influence the chemical profile of the Brazilian spirit, cachaça?

Cachaça is the typical Brazilian spirit, obtained by distillation of fermented sugarcane must, whose production is closely related to regional heritage and geographical traits. In this work, we investigate the influence of different autochthonous yeast species, in comparison to other commercial strains, on the chemical profile of cachaça. Fermentations were performed with four different wild species, a mixed starter culture with native yeasts, and two commercial strains. Procedures were carried out simulating traditional practices, the distillates analyzed by gas chromatography, and data analyzed by multivariate statistics. Results show that the overgrowth of some non-Saccharomyces during fermentation can lead to higher concentration of contaminant compounds in the distillate. However, those microorganisms do not impress negative traits when working in cluster association, and may indeed contribute to the distinctive chemical quality and flavor of cachaça. Spontaneous fermentation are still the traditional method used for the production of cachaça, and our results indeed suggest that higher diversity, and evened fermentative environments, enable yeasts to act as a complex cluster and imprint a distinctive, quality chemical profile to the distillate. Finally, this study may help to understand the role of the native microbiota and its influence on cachaça's chemical and sensory profiles, besides demonstrating the importance of adopting insightful handling practices and strict control of technical guidelines.