Residual mitochondrial transmembrane potential decreases unsaturated fatty acid level in sake yeast during alcoholic fermentation

Abstract

Oxygen, a key nutrient in alcoholic fermentation, is rapidly depleted during this process. Several pathways of oxygen utilization have been reported in the yeast Saccharomyces cerevisiae during alcoholic fermentation, namely synthesis of unsaturated fatty acid, sterols and heme, and the mitochondrial electron transport chain. However, the interaction between these pathways has not been investigated. In this study, we showed that the major proportion of unsaturated fatty acids of ester-linked lipids in sake fermentation mash is derived from the sake yeast rather than from rice or koji (rice fermented with Aspergillus). Additionally, during alcoholic fermentation, inhibition of the residual mitochondrial activity of sake yeast increases the levels of unsaturated fatty acids of ester-linked lipids. These findings indicate that the residual activity of the mitochondrial electron transport chain reduces molecular oxygen levels and decreases the synthesis of unsaturated fatty acids, thereby increasing the synthesis of estery flavors by sake yeast. This is the first report of a novel link between residual mitochondrial transmembrane potential and the synthesis of unsaturated fatty acids by the brewery yeast during alcoholic fermentation.

Keywords: Alcoholic fermentation; Anaerobiosis; Mitochondria; Oxygen; Sake yeast; Unsaturated fatty acid.

Figure 1. Residual mitochondrial transmembrane potential decreases unsaturated fatty acid level in sake yeast during alcoholic fermentation oxidoreductive status and mitochondrial morphology of sake yeast during alcoholic fermentation.

Sake yeasts with visualized mitochondria (RAK1536 K7 his3/his3 +pRS413-GPDmitoGFP) were incubated by shaking in selective synthetic media, inoculated into media with a layer of liquid paraffin on top, and incubated statically. (A-D) Rezarulin (0.004 % w/v) was added to the culture, and the oxidoreductive state of the culture was monitored by its color. (A) 2 h without yeast (B) 2 h with sake yeast (C) 2 h with laboratory yeast (D) 4 h without yeast (E) 4 h with sake yeast (F) 4 h with laboratory yeast (G) 8 h without yeast (H) 8 h with sake yeast (I) 8 h with laboratory yeast (J) 16 h without yeast (K) 16 h with sake yeast (L) 16 h with laboratory yeast. Sake yeast RAK1536 K7 his3/his3 +pRS413-GPDmitoGFP and laboratory yeast CEN.PK2 + pRS413-GPDmit were used. Sake yeasts cultured under alcoholic fermentation for 4 h (M; DIC, N; GFP), 12 h (O; DIC, P; GFP), and 24 h (Q; DIC, R; GFP) were fixed with formaldehyde and observed under a fluorescent microscope. Detailed methods are described under Materials and Methods. The results shown are representative of at least two independent fermentation experiments.

Figure 2. Fatty acid composition of sake yeast during alcoholic fermentation challenged with or without mitochondrial inhibitor.

Sake yeast (RAK1536 K7 his3/his3 + pRS413-GPDmitoGFP) were incubated by shaking in selective synthetic media. Yeast cells (1.0 × 10⁶ cells/ml) were inoculated into 10 ml media, with or without 20 μM carbonyl cyanide m-chlorophenylhydrazone. The culture was covered by adding a layer of liquid paraffin on top of the media, and incubated statically. Yeast cells were collected by centrifugation and lipids were extracted with chloroform/methanol. Extracted lipids were subjected to derivatization and analyzed by GC analysis. (A) Palmitic acid (B) Palmitoleic acid (C) Stearic acid (D) Oleic acid. Closed squares represent the results without 20 μM carbonyl cyanide m-chlorophenylhydrazone and open squares represent the results with 20 μM carbonyl cyanide m-chlorophenylhydrazone. Experiments were performed in triplicate from the respective starter cultures. The results are expressed as mean values ± standard errors of means. Significant differences were calculated by unpaired two-tailed Student’s t-test (^**, p < 0.01, ^*, p < 0.05). Detailed methods are described under Materials and methods.