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

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.


Introduction
Sake is a traditional Japanese rice wine prepared by multistep fermentation of rice, also called multiple parallel fermentation, in which conversions of rice starch to sugar by Aspergillus oryzae and sugar to alcohol by sake yeast (Saccharomyces cerevisiae) spontaneously occur [1]. The process of brewing sake begins with polishing of rice grains to remove bran-containing proteins, lipids, and minerals that could impair the aroma and flavor of sake. The sake using rice polished to at least 60% of the original grain was called as "ginjo" sake. Generally, a fermentation starter is prepared using steamed rice, malted rice, and sake yeast. Several types were used PLOS ONE | https://doi.org/10.1371/journal.pone.0190040 January 3, 2018 1 / 11 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 as 'starter' of the fermentation process, mainly classified to sokujo-type starter and yamahaitype (or more original type; kimoto-type) starter. For sokujo-type starter, yeast and lactic acid are simultaneously added to avoid the growth of contaminant bacteria. This process typically takes 1-2 weeks. In contrast, for yamahai-type starter, air-bone or certain Lactobacilli and yeast are added, and the inhibition of contaminant bacterial growth depends on lactic acid naturally produced by Lactobacilli. This process requires careful management of the fermentation process by a master brewer, and usually takes approximately 1 month, which is 2-4 times greater than the time used for sokujo-type. After the preparation of fermentation starter, a process called "tome-shikomi" (commencement of fermentation) are performed, in which fermentation starter and steamed rice are combined to yield the sake mash, and further fermented for a month. Finally, refined sake is obtained by filtration of sake mash and then bottled with or without pasteurization. "Yamahai-shikomi" sake using yamahai-type starter needs a more complex and long-term fermentation process than "sokujo-shikomi" sake, and management of the fermentation process requires fully-experienced master brewer. Japanese sake is a target of metabolomic investigation as it usually contains many metabolites derived from both the primary rice substrate and microbial activity which are produced during parallel multiple fermentation to establish an easier quality control system instead of master brewers. There are many reports that investigated the compounds found in sake and their relationship to its sensory properties [2][3][4][5]. These studies targeted the small molecules and volatile metabolites such as amino acids, sugars, organic acids, and so on, using the capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) or gas chromatography coupled with mass spectrometry (GC-MS). There are also a few reports that found characteristic peptides in sake [6,7] and that targeted time-course variations of some unique peptides [8,9]. However, there are no comprehensive reports that have focused on moderate mass compounds such as peptides and secondary metabolites during the progress of sake fermentation.
In the present study, we performed time-course metabolic profiling of "yamahai-ginjo-shikomi" sake to identify novel metabolites which characterize the progress of fermentation process. We used the nanoLC-MS system equipped with a meter-long monolithic silica capillary column and an orbitrap mass spectrometer, which is capable of accurate mass determination. We targeted molecules with mass-to-charge ratios of 200-1000 to identify the moderate-sized ones. Our research identified certain leucine-or isoleucine-rich peptides, which are the candidate molecules which characterize the fermentation process. Demonstration of brewing "yamahai-ginjo-shikomi" sake with metabolic analysis will contribute to comprehensive understanding of Japanese sake.

Preparation of sake samples
In this study, we prepared two sake samples, sake_1 and sake_2. For the preparation of sake_1 sample, 250 kg of "Yamada Nishiki" rice harvested at Hyogo Prefecture was used with a polishing ratio of 35%. Starter of "yamahai-ginjo-shikomi" was prepared using 21.2% aliquot of rice. For preparation of starter, the malted rice was kept under around 6˚C for 5 days. The temperature of starter was then gradually increased to the room temperature for 10 days. Then yeast was added and grew for 27 th day from starter preparation started. In total, 339 L of sake containing 15.9% of ethanol was produced, and samples were collected at 6, 11, 16, 21, 26, and 34 days after "tome-shikomi".
For the preparation of sake_2 sample, 200 kg of "Yamada Nishiki" rice harvested at Tottori Prefecture was used with a polishing ratio of 35%. Starter was prepared using 20.5% aliquot of rice by the same way described above. For efficient saccharification of rice starch, 10 g of glucoamylase "Amano" SD (Amamo Enzyme Inc., Nagoya, Japan) was added at an early stage of fermentation. In total, 270 L of sake containing 16.3% of ethanol was produced, and samples were taken at 1,5,11,19,26, and 34 days after "tome-shikomi". All fermentation processes of sake mash were performed under conditions of 5.8-10.4˚C for both kinds of sake.
Microbial strains used for fermentation were Leuconostoc mesenteroides and Lactobacillus plantarum for starter Lactobacillales, Saccharomyces cerevisiae for sake yeast, and Aspergillus oryzae for seed malted rice. Parameters of fermentation process are shown in S1 Table. nanoLC-MS analysis Non-targeted metabolomic analysis was performed using a nanoLC (Ultimate 3000, DIONEX, Sunnyvale, CA, USA)-MS system (LTQ Orbitrap Velos, Thermo Fisher Scientific). Five μl volumes of samples were injected and separated by reversed-phase chromatography using a monolithic column (2,000 mm, 0.1 mm ID, Kyoto Monotech Co., Ltd., Kyoto) [10]. The gradient was obtained by changing the mixing ratio of two eluents: A, 0.1% (v/v) formic acid and B, 80% (v/v) acetonitrile containing 0.1% (v/v) formic acid. The 120-min gradient separation at a flow rate of 500 nL/min and 40˚C was as follows: (i) 5% B for 10 min, (ii) a 60-min gradient to 95% B, (iii) a 10-min hold at 95% B, (iv) a 1-min gradient return to 5% B, and (v) a 40-min hold at 5% B. The separated metabolites were detected by mass spectrometry using a full scan range of 200-1000 m/z at a resolution of 100,000. MS was performed with an ESI voltage of 2.3 kV and a transfer tube temperature of 280˚C. MS data acquisition was set at 30-115 min.
For data-dependent MS/MS spectrum acquisition, which was used for peptide identification, the method was set to automatically analyze the top 10 most intense ions observed during the MS scan. Target peptides were synthesized using Genscript (Piscataway, NJ, USA).

Data analysis
Data were acquired using the following procedures: 1) raw data obtained by LTQ Orbitrap Velos (which comprised 3,000 spectrum counts and 800 Th of mass-to-charge ratio) were delimited by 50 spectrum counts and a mass-to-charge ratio of 1, 2) the intensities in each compartment were totaled, and 3) data were exported in text file format by an in-house developed C-script named OrbitrapDataRead (Raw data and OrbitrapDataRead are available at http://www.ebi.ac.uk/metabolights/MTBLS451). The data obtained from sake samples were analyzed and integrated into the same CSV file. In this step, all ion signals are included in numeric information of CSV file without discrimination of noise signals from metaboliterelated signals. The detailed workflow of OrbitrapDataRead was described in S1 Fig. Global median normalization was conducted to standardize the total intensities of compounds injected into the LC-MS. Finally, principal component analysis (PCA) was performed using R (https://www.r-project.org/).

Quantification of compounds
Quantification of the two peptides of interest (see in Results) was performed by LC-triple quadrupole mass spectrometry (LC-TQMS; LC-MS-8060, Nexela system, Shimadzu, Kyoto, Japan). Samples were injected and separated by reversed-phase chromatography using Inert-Sustain AQ-C18 (150 mm, 2.1 mm ID, 2.0 μm particle size, GL Science, Osaka, Japan) at a flow rate of 400 μL/min. The gradient was obtained by changing the mixing ratio of two eluents: A, 0.1% (v/v) formic acid and B, acetonitrile containing 0.1% (v/v) formic acid. The 23 min gradient separation at a flow rate of 400 μL/min and 40˚C was as follows: (i) 5% B for 2 min, (ii) a 15-min gradient to 45% B, (iii) an immediate increase to 95% B, (iv) a 3 min hold at 95% B, (v) an immediate decrease back to 5% B, and (vi) a 3-min hold at 5% B.

Results
Metabolomics of "yamahai-ginjo-shikomi" sake during fermentation process We used examples of sake mash from two samples identified as sake_1 and sake_2. To profile the metabolite changes of the fermentation processes, we prepared the sake mash samples taken during sake fermentation process at 6, 11, 16, 21, 26, and 34 days after "tome-shikomi" for sake_1 sake mash sample, and at 1, 5, 11, 19, 26, and 34 days after "tome-shikomi" for sake_2 sake mash samples. Supernatants of the prepared samples were then analyzed using LC-MS. PCA of the acquired data showed that the spectral profiles of the sake mash changed in a phased manner during fermentation, as well as exhibited high levels of similarity between the two technical replicates (Fig 1A). The PCA loading scatter plot exhibited certain features that profile the progress of fermentation process (Fig 1B). Factors having values of principal component 1 (PC1) from the top to the 20th were extracted (Table 1). Then the common factors in both sake_1 and sake_2 were focused (described in underlined bold numbers in Table 1). For detailed analysis, the main exact mass occupying each factor was extracted from  Table 1.

Identification of characteristic peptides
To identify candidate compounds which can characterize the progress of fermentation process, we performed nanoLC-MS/MS analysis. We particularly focused on the leucine-or isoleucine-rich peptides, and the sequences of these peptides were analyzed.  (Fig 2). All peptides contained leucine or isoleucine (represented as [Leu/Ile]). However, leucine and isoleucine could not be distinguished by MS analysis because these two amino acid residues share the same molecular weight.
Next, we tried to distinguish leucine and isoleucine residues by their retention time on chromatographic columns using (as an example) the tripeptides [Leu/Ile]-[Leu/Ile]-[Leu/Ile], which display 2 3 patterns of leucine and/or isoleucine sequence. The long-duration gradient program employed for LC using the long monolithic column was able to distinguish these tripeptides (Fig 3A). From the chromatogram, the sample contained more than one species of Factors shared between sake_1 and sake_2 (represented in underlined bold characters in Table 1 peptide, and the predominant one was found to be Leu-Leu-Leu, indicating that peptides possessing a poly-leucine motif may be important. Moreover, putative Phe-Pro-[Leu/Ile] was validated as Phe-Pro-Leu by the retention time analysis using LC (Fig 3B), suggesting that leucine-containing peptides are the compounds that showed highest PC1 scores in our samples.

Quantification of fermentation-characteristic peptides
To confirm quantitative changes involving the peptides found by metabolic profiling using our LC-MS analytical system, polypeptides were quantified by LC-triple quadrupole (TQ) MS.
Because eight variants of [Leu/Ile]-[Leu/Ile]-[Leu/Ile] cannot be separated by the LC-TQMS system, the quantity of peptides was determined using a standard calibration curve of Leu-Leu-Leu, which was the main component of the peptides (Fig 3A). From the results of quantification (Fig 4A), it was observed that the levels of Leu-Leu-Leu and Phe-Pro-Leu dramatically increased to the level of micrograms per milliliter during the early stages of fermentation process. Relative abundances of C 19 (Fig 4B).

Discussion
In this study, we performed the time-course metabolic profiling of "yamahai-ginjo-shikomi" Japanese sake and first identified factors that indicate the progress of the fermentation process. By targeting moderate-sized molecules with mass-to-charge ratios of 200-1000, signature biomolecules produced during the fermentation process were revealed by PCA (Fig 1A). Analysis indicates that the profiles of these moderate-sized molecules changed during fermentation. Moreover, the metabolite profile of moderate-sized molecules changed more dramatically during the early fermentation stages (<11 days) than in later stages (!11 days), indicating that early fermentation stages determine the metabolic profile of sake. Four peptides which contain leucine or isoleucine were identified by metabolic profiling (Fig 2). From the separation of [Leu/Ile]-[Leu/Ile]-[Leu/Ile] (Fig 3A), the dominant peptide was found to be Leu-Leu-Leu, suggesting that the peptides identified in this study share leucine-rich motifs. Therefore, the other two peptides were suggested to be Leu-Leu-Leu-Pro and Leu-Leu-Leu-Leu-Pro. These three peptides may be derived from the same protein precursor. Leucine is a hydrophobic amino acid, and leucine-rich motifs are typically associated with transmembrane α-helical structures [11] and membrane-anchor domains of proteins [12]. The leucine-rich motif, including Leu-Leu-Leu-Leu-Pro is more abundant in proteins encoded in the genome of Oryza sativa (rice) than those of S. cerevisiae (sake yeast), A. oryzae, or Lactobacilli as revealed by BLAST homology identification, suggesting that the peptides found in this study could be generated during digestion of rice proteins by proteases of inoculated yeast, Lactobacilli, A. oryzae, or externally added glucoamylase which is crude enzyme mixture and has peptidase and protease activity.
The peptides revealed in this study possibly affect the taste of sake. Branched chain amino acids such as valine, leucine, and isoleucine are generally considered to confer bitter taste sensations [13]. Leucine-containing peptides are also known to confer bitterness [14][15][16], but the bitterness thresholds of these peptides are the milligram per milliliter level, and the concentration of the peptides in our sample was microgram per litter level. There is another report that indicates Leu-Leu-Leu has a taste-masking effect of sourness [17], indicating that the peptides identified in this study may affect the taste of "yamahai-ginjo-shikomi" sake. Our results indicated that the amount of Leu-Leu-Leu and Phe-Pro-Leu were elevated up to the order of μg/ mL, indicating that these peptides likely characterize the taste of sake.
In this study, two factors other than peptides were also identified by metabolic profiling ( Table 2). ID 1 (m/z: 273-274, # spectra: 1350-1400), identified as C 19 H 28 O, was categorized as a likely steroidal agent, including androstenone. Androstenone, a well-known mammalian sex hormone, can also be detected in plants [18], and is reported to be resemble a sweaty, urine or woody smell, or even a pleasant floral odor, this perceptual variation most likely depends on the olfactory receptor variant genotypes of individuals subjected to exposure [19]. The factor ID 2 (m/z: 307-308, # spectra: 1400-1450) identified as C 17 H 26 N 2 O 3 was predicted to be angeloyloxylupanine or acetoxymatrine. Angeloyloxylupanine and acetoxymatrine are plant alkaloids [20,21], which generally exert bitter taste sensations, and they are predicted to appear during rice seed decomposition by yeast or A. oryzae. These factors require further researches with authentic standards.
Here, we first demonstrated time-course metabolic profiling targeting moderate-sized molecules during fermentation of "yamahai-ginjo-shikomi" sake. Metabolic profiling revealed that the abundance profiles of these moderate-sized molecules were dependent on the specific fermentation stage employed. Analysis of characteristic and distinguishing ions by LC-MS/MS revealed that four leucine-(or isoleucine-) containing short peptides became elevated during the fermentation process. These peptides would be likely candidate biological molecules that reveal and indicate the progress of sake fermentation, and require further investigation and validation.