Rational Selection of Mixed Yeasts Starters for Agave Must Fermentation

Abstract

Tequila and mezcal are both traditional Mexican liquors that are produced from cooked Agave spp. must fermentation and usually rely on spontaneous or pure Saccharomyces cerevisiae strain inoculation. In order to contribute to the rational selection of yeast starters for tequila and mezcal productions, we tested a collection of 25 yeasts originally isolated from mezcal musts, spanning 10 different yeast species. These strains were first characterized in a semi synthetic medium (labeled as M2, having 90 g/L fructose and 10 g/L glucose of initial hexoses) at 48 h of culture, observing a differential pattern in the consumption of sugars and productivity. Selected Saccharomyces strains left around 10 g/L of fructose and showed higher fermentation performance. However, some non-Saccharomyces strains, specifically from Torulospora (Td), Kluyveromyces (Km), and Zygosaccharomyces (Zb) genera, consumed almost all the sugar (i.e., Km1Y9 with <5 g/L) and had a high productivity of ethanol. In general, all Saccharomyces strains presented a high production of ethyl-butyrate, ethyl-decanoate, and ethyl-hexanoate with peaks of 10, 38, and 3 μg/L, respectively. In addition, some Kluyveromyces and Torulospora strains showed a high production of phenyl ethyl acetate (i.e., Km1D5 with up to 1400 μg/L); isoamyl acetate (i.e., Km1D5 and Td1AN2 with more than 300 μg/L), and hexyl acetate (i.e., Td1AN2 with 0.3 μg/L). Representative strains of the most productive genera (Saccharomyces, Torulospora, and Kluyveromyces) were selected to evaluate their fermentative performance and survival in a mixed culture on a medium based on Agave tequilana must, and their population kinetics was characterized using specific fluorescent in situ hybridization (FISH) probes in a qualitative and semi-quantitative analysis during fermentation. We observed that the mixture ratios of 0.1:1:1 or 1:1:1 (Saccharomyces:Kluyveromyces:Torulospora), maintained good fermentation productivities, with alcohol yields above 0.45 g/g, and allowed a high survival rate of the non-Saccharomyces strains during the fermentation process. Finally, mixed inoculum fermentations on A. tequilana must medium, including different Saccharomyces strains and the finally selected Torulospora and Kluyveromyces strains, showed the best production parameters in terms of ethanol, carbon dioxide, glycerol, and acetic acid values, as well as improved volatile metabolite profiles as compared to the pure cultures. All these data were used to propose a methodology of selection of strains to be used as a pure or mixed starter for tequila and mezcal fermentations, with high primary metabolite productivity and desired aromatic profile.

Effects of different lactic acid bacteria on the characteristic flavor profiles of Chinese rice wine

“…Some of esters come from raw materials, but most are produced by the esterase‐catalyzed reaction of alcohols and acids produced from glucose and amino acids during microbial metabolism 32 . Generally, the latter is the primary pathway in the formation of esters 33‐35 . Compared to the control CRW, adding L. lactis enriched the content of esters in CRW, and adding P. pentosaceus increased the relative content of alcohols by 9.87%, while adding W. confusa increased the relative content of alcohols and esters by 3.23% and 3.55%, respectively.…”

“…32 Generally, the latter is the primary pathway in the formation of esters. [33][34][35] Compared to the control CRW, adding L. lactis enriched the content of esters in CRW, and adding P. pentosaceus increased the relative content of alcohols by 9.87%, while adding W. confusa increased the relative content of alcohols and esters by 3.23% and 3.55%, respectively. In addition, LAB have facilitated the formation of three new esters from CRW: ethyl 4-hydroxybutanoate, 2-phenethyl acetate and ethyl pentadecanoate.…”

Yeast competition during alcoholic fermentation of agave. Its comprehension as a way to reach sustainable mezcal production

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Enhanced protein glutaminase production from Chryseobacterium proteolyticum combining physico‐chemical mutagenesis and resistance screening and its application to soybean protein isolates

“…24,25 In the process of mutation breeding, the reasoning selection technology that can directionally screen the target strain in the large-capacity mutation library is also a key step for improving the efficiency. 26 Its aim is to make mutant strains retain more strains with application value under the action of screening pressure. The general process of reasoning selection is to set appropriate screening pressures according to the metabolic pathways and growth characteristics of the production strains, so as to eliminate the inferior strains.…”

“…In the process of mutation breeding, the reasoning selection technology that can directionally screen the target strain in the large‐capacity mutation library is also a key step for improving the efficiency 26 . Its aim is to make mutant strains retain more strains with application value under the action of screening pressure.…”