The quality score and the number of counts of <i>ginjo</i> sake by 15 sensory evaluation panelists.

<p>The quality score and the number of counts of <i>ginjo</i> sake by 15 sensory evaluation panelists.</p

Protein profiles of 13 sake samples showing that yeast cellular protein leakage is linked to low sake quality.

<p>A) Overall protein profiling by Coomassie brilliant blue (CBB) staining after SDS-PAGE. For each sample, 10 μL of denatured protein was applied to a lane, and 5 μL of marker was utilized. Protein bands indicated by double black triangles are <i>Saccharomyces cerevisiae</i> triosephosphate isomerase (TPI). B) Immunoblot analysis for <i>S</i>. <i>cerevisiae</i> TPI1 in sake. Pyruvic acid concentrations in sake (n = 2) determined by colorimetric assay is shown in the upper rectangle. C) <i>Upper panel</i>; Immunoblot analysis for glucoamylase (GlaB) derived from <i>Aspergillus oryzae</i> in sake. The <i>N</i>-glycosylated forms of GlaB were detected at 47 kDa and as a blurred band beyond 65 kDa. <i>Middle panel</i>; Immunoblot analysis for α-amylase. <i>Lower panel</i>; Immunoblot analysis for the acid protease, PepA. D) Protein profiling following PNGaseF treatment by CBB staining after SDS-PAGE. For each sample, 10 μL of denatured protein was applied to a lane, and 5 μL of marker was applied. The asterisk at 34 kDa shows PNGaseF. E) Immunoblot analysis for glucoamylase (GlaB) following PNGaseF treatment of sake. The blurred band and multiple bands at 55–70 kDa and 37–50 kDa correspond to GlaB.</p

The patterns of representative metabolites associated with sake quality.

<p>A) Compounds involved in the methionine cycle. 5′-deoxi-5′-methylthioadenosine is shown as relative value. <i>S</i>-Adenosylmethionine and methionine are shown as μmol/L. B) A simple summarization of the methionine cycle pathway. C) Compounds involved in glutathione or ophthalmic acid biosynthesis pathways. Cysteine and glutathione (GSSG; oxiglutathione) are shown as μmol/L. Gamma-glutamyl-2-aminobutyric acid, ophthalmic acid, and cysteine glutathione disulfide are shown as relative values. D) The metabolites that participate in part of the glutathione (GSH) and ophthalmic acid biosynthesis pathways. E) Other amino acid-related chemical compounds associated with lower sake quality. Saccharopine and cystine are shown as relative values. Homoserine is shown as μmol/L. Data are shown as means ± SEM (n = 2).</p

Principal component analysis (PCA) of Japanese sake.

<p>After selecting 202 compounds using Fisher ratio criteria, PCA was performed for 13 sake samples in duplicate. Same symbols indicate the same sake samples. Highly ranked sake (n = 7) are shown in red, sake with fatty acid odor (n = 3) are shown in green, and inharmonious bitter tasting sake (n = 3) are shown in blue.</p

Chemical compounds whose levels were positively or negatively correlated with either taste or flavor quality.

<p>Chemical compounds whose levels were positively or negatively correlated with either taste or flavor quality.</p

Patterns of identified metabolites from the central metabolic pathway of the sake yeast, <i>Saccharomyces cerevisiae</i>.

<p>A) A typical schematic for the central metabolic pathway. In the sake brewing process, lactic acid can be also added as a food additive at an early stage of the yeast starter making process. Alpha-lipoic acid can facilitate acetyl-CoA production with vitamin B₁. B) The concentrations of the indicated chemical compounds involved in the glycolytic pathway in sake. Highly ranked sake, inharmonious bitter tasting sake, and sake with fatty acid odor are shown in red, blue, and green, respectively. Glucose is shown as a percentage (n = 4). Glucose 6-phosphate, glycerol 3-phosphate, pyruvic acid, and lactic acid are shown as μmol/L (n = 2). C) The concentrations of the indicated chemical compounds involved in the tricarboxylic acid (TCA) cycle in sake. All metabolites are shown as μmol/L (n = 2). D) The relative values of pyridoxamine and pyridoxine (n = 2). Data are shown as means ± SEM.</p

Chemical compounds contributing to fatty acid odor and inharmonious bitter taste.

<p>A) Representative chemical compounds involved in fatty acid odor generation are medium-chain fatty acids and medium-chain fatty acid analogues. B) Two representative polyamines (cadaverine and agmatine) involved in inharmonious bitter taste generation. All chemical compounds are shown as relative values. Data are shown as means ± SEM (n = 2).</p

Chemical compounds whose levels were positively or negatively correlated with the off-flavors of either fatty acid odor or inharmonious bitter taste.

<p>Chemical compounds whose levels were positively or negatively correlated with the off-flavors of either fatty acid odor or inharmonious bitter taste.</p

Relative heatmap for the metabolites of 13 sake samples.

<p>Heatmap showing the metabolic profiles of 13 sake samples (7 of highly ranked sake, 3 of inharmonious bitter tasting sake, and 3 of sake with fatty acid odors) analyzed in duplicate. Maximum to minimum level is represented by a red-gray-green color scheme. The values of <i>m/z</i> of the unknown compounds XA0004, XA0012, XA0017 are 144.0292, 166.0154, and 186.1124, respectively, by the anion detection mode. The values of <i>m/z</i> of the unknown compounds XC0016, XC0017, XC0040, and XC0120 are 129.0646, 130.0977, 174.0861, and 298.0518, respectively, by the cation detection mode.</p

Different Polar Metabolites and Protein Profiles between High- and Low-Quality Japanese <i>Ginjo</i> Sake

<div><p>Japanese <i>ginjo</i> sake is a premium refined sake characterized by a pleasant fruity apple-like flavor and a sophisticated taste. Because of technical difficulties inherent in brewing <i>ginjo</i> sake, off-flavors sometimes occur. However, the metabolites responsible for off-flavors as well as those present or absent in higher quality <i>ginjo</i> sake remain uncertain. Here, the relationship between 202 polar chemical compounds in sake identified using capillary electrophoresis coupled with time-of-flight mass spectrometry and its organoleptic properties, such as quality and off-flavor, was examined. First, we found that some off-flavored sakes contained higher total amounts of metabolites than other sake samples. The results also identified that levels of 2-oxoglutaric acid and fumaric acid, metabolites in the tricarboxylic acid cycle, were highly but oppositely correlated with <i>ginjo</i> sake quality. Similarly, pyridoxine and pyridoxamine, co-enzymes for amino transferase, were also highly but oppositely correlated with <i>ginjo</i> sake quality. Additionally, pyruvic acid levels were associated with good quality as well. Compounds involved in the methionine salvage cycle, oxidative glutathione derivatives, and amino acid catabolites were correlated with low quality. Among off-flavors, an inharmonious bitter taste appeared attributable to polyamines. Furthermore, protein analysis displayed that a diversity of protein components and yeast protein (triosephosphate isomerase, TPI) leakage was linked to the overall metabolite intensity in <i>ginjo</i> sake. This research provides insight into the relationship between sake components and organoleptic properties.</p></div