Two-carbon metabolites, polyphenols and vitamins influence yeast chronological life span in winemaking conditions

Abstract

Background: Viability in a non dividing state is referred to as chronological life span (CLS). Most grape juice fermentation happens when Saccharomyces cerevisiae yeast cells have stopped dividing; therefore, CLS is an important factor toward winemaking success.

Results: We have studied both the physical and chemical determinants influencing yeast CLS. Low pH and heat shorten the maximum wine yeast life span, while hyperosmotic shock extends it. Ethanol plays an important negative role in aging under winemaking conditions, but additional metabolites produced by fermentative metabolism, such as acetaldehyde and acetate, have also a strong impact on longevity. Grape polyphenols quercetin and resveratrol have negative impacts on CLS under winemaking conditions, an unexpected behavior for these potential anti-oxidants. We observed that quercetin inhibits alcohol and aldehyde dehydrogenase activities, and that resveratrol performs a pro-oxidant role during grape juice fermentation. Vitamins nicotinic acid and nicotinamide are precursors of NAD+, and their addition reduces mean longevity during fermentation, suggesting a metabolic unbalance negative for CLS. Moreover, vitamin mix supplementation at the end of fermentation shortens CLS and enhances cell lysis, while amino acids increase life span.

Conclusions: Wine S. cerevisiae strains are able to sense changes in the environmental conditions and adapt their longevity to them. Yeast death is influenced by the conditions present at the end of wine fermentation, particularly by the concentration of two-carbon metabolites produced by the fermentative metabolism, such as ethanol, acetic acid and acetaldehyde, and also by the grape juice composition, particularly its vitamin content.

Figure 1

Physical determinants of the wine yeast life span. The CLS analyses of strain EC1118 in SC medium were done. The assays were performed by making serial dilutions, plating an aliquot and counting colonies. CFU (colony-forming units)/mL were expressed in relation to the values obtained from the day-3 post-inoculation, thatwere considered to be 100% survival. A) Effect of temperature on CLS. B) Effect of hyperosmorality on CLS. C) Effect of pH on CLS. Experiments were done in triplicate, and the mean and standard deviation are provided.

Figure 2

Role of two-carbon metabolites in the wine yeast life span.A) The CLS analyses were performed with strain EC1118 from a completed fermentation in synthetic grape juice placed in another completed fermentation (wine), from which ethanol was removed by evaporation (evaporated wine), and with evaporated wine containing 2% and 10% ethanol. The time when cells were placed in the new medium is considered 0. B) CLS analysis of the T73 strain in water containing ethanol (8 g/L), acetate (0.4 g/L) and acetaldehyde (0.12 g/L). The assays were performed as described in Figure 1. Experiments were done in triplicate, and the mean and standard deviation are provided.

Figure 3

Analysis of the effect of resveratrol and quercetin on wine fermentation.A) Cell viability and sugar consumption of a fermentation on synthetic grape juice by strain EC1118 with the addition of resveratrol (2 mg/L) and quercetin (9 mg/L). B) CLS profile by taking day 4 of the fermentation described in panel A) as 100% survival. Experiments were done in triplicate, and the mean and standard deviation are provided.

Figure 4

Polyphenols influence enzymatic activity.A) The alcohol dehydrogenase activity of wine yeast extracts in the presence of resveratrol (2 mg/L) and quercetin (9 mg/L). B) Cytosolyc aldehyde dehydrogenase Ald6p activity under the same conditions. C) Mitochondrial aldehyde dehydrogenase Ald4p activity under the same conditions. D) Lipid peroxidation levels on day 6 of a fermentation on synthetic grape juice with and without resveratrol. Experiments were done in triplicate, and the mean and standard deviation are provided. *p < 0.05, **p < 0.01, unpaired t-test, two-tailed.

Figure 5

Nicotinamide and nicotinic acid exert a negative effect during wine fermentation. The cell viability of a fermentation on synthetic grape juice by strain EC1118 with the addition of nicotinic acid and nicotinamide at 3.75 mg/L. Experiments were done in triplicate, and the mean and standard deviation are provided.

Figure 6

Effect of micronutrients on life span during wine fermentation.A) The CSM strain’s CLS profile by taking the day when sugars were consumed as 100% survival. Cell viability after adding amino acids, vitamins and anaerobic factors was followed. B) Cell integrity measured as propidium iodide staining was followed under the same conditions. Experiments were done in triplicate, and the mean and standard deviation are provided.