Physiological characterization of two native yeasts in pure and mixed culture using fermentations of agave juice

Cien. Inv. Agr. 46(1):1-11. 2019 www.rcia.uc.cl food technology DOI 10.7764/rcia.v46i1.1880 research paper Physiological characterization of two native yeasts in pure and mixed culture using fermentations of agave juice Martha E. Nuñez-Guerrero1,2, Elizabeth Salazar-Vázquez2, Jesús B. PáezLerma2, Raúl Rodríguez-Herrera1, and Nicolás O. Soto-Cruz2 Universidad Autónoma de Coahuila, Faculty of Chemical Sciences, Laboratory of Molecular Biology. Blvd. V. Carranza e Ing. José Cardenas s/n, Saltillo Coahuila, México. 2 Tecnológico Nacional de México/Instituto Tecnológico de Durango. Felipe Pescador 1830 Ote. 34080, Durango, Dgo., México. 1 Abstract M.E. Nuñez-Guerrero, E. Salazar-Vázquez, J.B. Páez-Lerma, R. Rodríguez-Herrera, and N.O. Soto-Cruz. 2019. Physiological characterization of two native yeasts in pure and mixed culture using fermentations of agave juice. Cien. Inv. Agr. 46(1): 1-11. Yeast cells are subjected to diverse environmental conditions during the alcoholic fermentation of agave juice, causing different kinetic behaviors. Agave juice was used as culture medium to evaluate the kinetic behavior of Saccharomyces cerevisiae ITD-00185 and Torulaspora delbrueckii ITD00014a, as pure and mixed cultures, under different inoculum sizes (1×105, 1×106, 1×107 and 1×108 cells mL-1), and combined pH levels (3.5, 4.0 and 4.5) and temperatures (18 °C, 28 °C and 38 °C). Saccharomyces cerevisiae displayed high fermentation capacities at all inoculum concentrations assayed. However, T. delbrueckii required a high inoculum concentration (≥1×107 cells mL-1) to perform at fermentation levels similar to S. cerevisiae. Low temperatures (18 °C) slowed fermentation, while high temperatures (38 °C) adversely affected the development of the yeast strains, especially T. delbrueckii. The best temperature was 28 °C in all fermentations. The pH level had a strong effect on the performance of the coculture, since the fermentation kinetics suggested a synergistic effect at pH 4.5, while an antagonistic effect was postulated at pH 3.5. In all of the mixed culture cases, a positive effect at 28 °C, especially at pH 4.0 and 4.5, was demonstrated by greater levels of sugar consumption and ethanol production (~20%, p<0.05) compared to fermentations of the S. cerevisiae monoculture. The coculture results allow us to postulate that a complex interaction exists between the two yeasts, which could be synergistic or antagonistic, as the environmental conditions change. Key-words: Coculture, inoculum concentration, pH, Temperature. Introduction Several different alcoholic beverages are derived from agave plants such as pulque, tequila, bacanora and mezcal. However, only tequila, and more recently mezcal, have achieved internaReceived Jul 26, 2018. Accepted Mar 04, 2019. Corresponding author: tional recognition. Mezcal is a Mexican regional beverage with an origin denomination (Norma Oficial Mexicana: NOM-070.SCFI-1994). Tequila and mezcal are both protected under the North American Free Trade Agreement (NAFTA) and an “Agreement between the European Union and the United Mexican States on the mutual recognition and protection of designations for spirit drinks”. 2 ciencia e investigación agraria Tequila is obtained by distilling fermented sugar from Agave tequilana, whereas mezcal is obtained from Agave duranguensis juice as well as other Agave species (Soto-García et al., 2009). Fermentation is a critical stage for the quality and productivity of the mezcal production process. This stage is performed spontaneously during artisan processes, which can cause fermentation to occur over different lengths of time, the presence of undesirable products (e.g., methanol and furfural) and leave high amount of residual, nontransformed sugar. Saccharomyces and non-Saccharomyces yeasts are responsible for the alcoholic fermentation of agave juice (Escalante-Minakata et al., 2008; LappeOliveras et al., 2008; Narváez-Zapata et al., 2010; Páez-Lerma et al., 2013). Yeast population changes that occur during the alcoholic fermentation of A. duranguensis juice for mezcal production were studied by Páez-Lerma et al. (2013). They found that the early stage of the fermentation process was conducted by highly diverse yeast populations, which contained species such as Kluyveromyces marxianus, Torulaspora delbrueckii, Candida diversa, Pichia fermentans and Hanseniaspora uvarum; however, S. cerevisiae was the only yeast found at the end of the fermentation process. This finding is similar to the yeast behavior reported during grape fermentation for wine production (Esteve-Zarzoso et al., 1998; Pretorius et al. 2003; Caridi, 2003). Standardizing the fermentation process could be achieved by using a starter culture (inoculant), formulated with selected yeast strains, which could possibly achieve better utilization of the fermentable sugars (Nuñez-Guerrero et al., 2016) while meeting the requirements of the Mexican Official Norm. Nuñez-Guerrero et al. (2016) demonstrated that a yeast mixture composed of 75% S. cerevisiae and 25% T. delbrueckii produced mezcal with a high yield and richness of volatile compounds, which resulted in better acceptability in sensory tests. Therefore, the aim of this work was to evaluate the kinetic behavior of the abovementioned inoculant (75% S. cerevisiae and 25% T. delbrueckii), as well as the individual yeasts, with reference to pH, temperature and inoculum size, during the alcoholic fermentation of agave juice. Materials and Methods Microorganisms Saccharomyces cerevisiae ITD-00185 and Torulaspora delbrueckii ITD-00014a strains were obtained from the yeasts collection of the Microbial Biotechnology Laboratory at the Instituto Tecnológico de Durango. These yeast strains were selected in a previous work (Nuñez-Guerrero et al., 2016) and were activated on Yeast-Peptone-Dextrose (YPD) agar (1% yeast extract, 2% dextrose, 2% casein peptone, 2% agar), incubated at 28 °C for 12 h and recultured in YPD broth (1% yeast extract, 2% dextrose, 2% casein peptone) at 28 °C for 12 h. The cells were stained with methylene blue and counted using a Neubauer chamber. Agave juice Agave duranguensis juice with an initial sugar concentration of 120 ± 5 g L-1 was kindly donated by Productora Mexicana de Mezcal S. DE R.L.M.I. Its total nitrogen was measured using the MicroKjeldahl method as described previously in De los Ríos-Deras et al., (2015). The agave broth had a nitrogen concentration of 0.49 g N L-1, and ammonium sulfate was added to reach 1.64 g N L-1, which corresponds to a C/N ratio of 73 g C/g N. This C/N ratio has been demonstrated as the best C/N ratio for agave must fermentation during mezcal production (De los Ríos-Deras et al., 2015). Effect of inoculum concentrations Fermentation assays were performed using pure cultures of each yeast strain. A mixture culture VOLUME 46 Nº1 JANUARY – APRIL 2019 (75% S. cerevisiae and 25% T. delbrueckii) was also used as inoculum as previously assayed in (...truncated)