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. 2018 Jan 3;13(1):e0190040.
doi: 10.1371/journal.pone.0190040. eCollection 2018.

Metabolite profiling of the fermentation process of "yamahai-ginjo-shikomi" Japanese sake

Yohei Tatsukami  1   2 , Hironobu Morisaka  1   3 , Shunsuke Aburaya  1   2 , Wataru Aoki  1   3 , Chihiro Kohsaka  3   4 , Masafumi Tani  3   5 , Kiyoo Hirooka  3   4 , Yoshihiro Yamamoto  3   4 , Atsushi Kitaoka  6 , Hisashi Fujiwara  6 , Yoshinori Wakai  6 , Mitsuyoshi Ueda  1   3
Affiliations

Metabolite profiling of the fermentation process of "yamahai-ginjo-shikomi" Japanese sake

Yohei Tatsukami et al. PLoS One. .

Abstract

Sake is a traditional Japanese alcoholic beverage prepared by multiple parallel fermentation of rice. The fermentation process of "yamahai-ginjo-shikomi" sake is mainly performed by three microbes, Aspergillus oryzae, Saccharomyces cerevisiae, and Lactobacilli; the levels of various metabolites fluctuate during the fermentation of sake. For evaluation of the fermentation process, we monitored the concentration of moderate-sized molecules (m/z: 200-1000) dynamically changed during the fermentation process of "yamahai-ginjo-shikomi" Japanese sake. This analysis revealed that six compounds were the main factors with characteristic differences in the fermentation process. Among the six compounds, four were leucine- or isoleucine-containing peptides and the remaining two were predicted to be small molecules. Quantification of these compounds revealed that their quantities changed during the month of fermentation process. Our metabolomic approach revealed the dynamic changes observed in moderate-sized molecules during the fermentation process of sake, and the factors found in this analysis will be candidate molecules that indicate the progress of "yamahai-ginjo-shikomi" sake fermentation.

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Conflict of interest statement

Competing Interests: The sake samples analyzed in this study were provided from Kizakura Co., Ltd. The sake samples were not commercial products, but prototypes. Atsushi Kitaoka, Hisashi Fujiwara and Yoshinori Wakai are employed by Kizakura Co., Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. PCA analysis of metabolic profiles.
(A) Score plot of the fermentation process. Metabolic profiles of samples at six time points were plotted using two analytical replicates from the same sample. (B) Loading scatter plot. Factors having values of PC1 from the top to the 20th at each plot are shown in Table 1.
Fig 2
Fig 2. Identification of peptides.
The factors that were predicted as peptides in Table 2 were analyzed using LC-MS/MS, and the amino acid sequences were identified using MS/MS spectra. [L/I] indicates leucine or isoleucine residue that could not be distinguished by MS analysis.
Fig 3
Fig 3. Validation of peptide leucine or isoleucine compositions.
(A) Chromatograms of sake samples with eight variations of synthetic [Leu/Ile]–[Leu/Ile]–[Leu/Ile] peptides. Extracted ion chromatograms (XIC) of m/z = 358.26–358.28 are presented. (B) Chromatograms of sake samples with two variations of synthetic Phe–Pro–[Leu/Ile] peptides. XIC of m/z = 376.22–376.23 are shown. Dashed lines with arrowheads indicate retention times of the main peaks of XIC for two samples. [Leu/Ile] indicates leucine or isoleucine residues that could not be determined by MS.
Fig 4
Fig 4. Quantification of peptides and associated compounds.
(A) Quantification of Leu–Leu–Leu (left) and Phe–Pro–Leu by LC-TQMS. (B) Relative quantification of four factors (ID 1, 2, 6, and 7) that were identified by metabolic profiling using nanoLC-MS.
See this image and copyright information in PMC

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