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Research Article        Open Access 

Comparative analysis of functional components in Sakekasu (Sake 
lees) 

Yuto Nishidono1,2, Shingen Misaka3, Yuko Maejima3, Kenju Shimomura3, Ken Tanaka1* 

1College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Shiga 525-8577, Japan; 2Research 

Organization of Science and Technology, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Shiga 525-8577, Japan; 

3Department of Bioregulation and Pharmacological Medicine, School of Medicine, Fukushima Medical University, 1 

Hikarigaoka, Fukushima, Fukushima 960-1295, Japan 

*Corresponding author:  Ken Tanaka, Ph.D., College of Pharmaceutical Sciences, Ritsumeikan University1-1-1 Noji-Higashi, 
Kusatsu, Shiga 525-8577, Japan.   

Submission Date: December 18th, 2023; Acceptance Date: January12th, 2024; Publication Date: January 16th, 2024 

Please cite this article as: Nishidono Y., Misaka S., Maejima Y., Shimomura K., Tanaka K. Comparative analysis of functional 

components in Sakekasu (Sake lees). Functional Foods in Health and Disease 2024; 14(1):74-86. DOI: 

https://doi.org/10.31989/ffhd.v14i1.1272 

ABSTRACT 

Background: Sake lees (Sakekasu), a byproduct of sake production, has been recently attracting attention as a functional 

food. Sakekasu is rich in nutrients and contains glycerophosphocholine (GPC) and S-adenosylmethionine (SAM), which 

are well-known functional compounds. The content of these compounds in Sakekasu depends on a variety of factors, 

including fermentation conditions, especially the method and length of ripening. These differences are reflected 

prominently in the color of Sakekasu, which becomes darker due to the long ripening period and high drying 

temperature. 

Objective: This study aimed to clarify the contents of functional components in Sakekasu with different color tones (i.e., 

ripening period). 

Methods: Three types of Sakekasu with different color tones (white, ocher, and brawn) were collected from several 

breweries. The contents of multiple functional components in their extracts were determined by liquid chromatography 

coupled to high resolution ion-trap/time-of-flight mass spectrometry. 

Results: Sakekasu with white color had more abundant GPC, SAM, and fatty acids than those with darker color. However, 

ethyl glucoside and glyceryl glucosides did not differ significantly by color tone. Furthermore, the Maillard reaction 

products of sugar and dipeptide were mainly found in dark-colored Sakekasu, and their structures were annotated by 

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tandem mass spectrometry. 

Conclusions: This study has clarified many functional compounds in Sakekasu in relation to color tone (i.e., ripening 

period) and highlighted the potential of Sakekasu with white color tone as a functional food. 

Keywords: Sakekasu, functional components, glycerophosphocholine, S-adenosylmethionine, tandem mass 

spectrometer 

©FFC 2024.  This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License 

(http://creativecommons.org/licenses/by/4.0) ©FFC 2024. 

INTRODUCTION 

Consumers are growing increasingly aware of the 

significance of diet in human health leading to an 

increase in demand for functional foods [1, 2]. The 

debate over the definition of functional food has 

persisted for many years. In 2021, the Functional Food 

Center proposed the following definition of the term 

'functional food': “Natural or processed foods that 

contain biologically-active compounds, which, in defined, 

effective, non-toxic amounts, provide a clinically proven 

and documented health benefit utilizing specific 

biomarkers, to promote optimal health and reduce the 

risk of chronic/viral diseases and manage their 

symptoms” [3]. To place foods into the functional food 

category, various approaches have been proposed, such 

as a 16-step course [4] and Functional Food Development 

Cycle [1]. One important step in these processes is the 

identification of functional components, which are the 

element of classifying functional foods [4]. 

Sake is a traditional Japanese alcoholic beverage. 

During its production process, steamed rice is first 

fermented with Aspergillus oryzae to make “koji” mold. 

Then, koji mold, steamed rice, and Saccharomyces 

cerevisiae (Sake yeast) are mixed, and ethanol 

fermentation occurs at the oar. Subsequently, the 

fermented product is filtered and separated into liquid 

and solid components. The solid portion is called 

“Sakekasu” (sake lees). Sakekasu is rich in protein, 

peptides, amino acids, carbohydrates, dietary fiber, fat, 

ash, and vitamins and has traditionally been used as an 

ingredient in food processing and as a moisturizer in 

cosmetics in Japan [5]. 

Sakekasu has been recently attracting attention as 

a functional food, with numerous reported effects, such 

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as antidiabetic [6], osteoporosis-preventive [7], and anti–

colon cancer effects [8]. It also improves hepatic lipid 

accumulation [9, 10], inhibits acute alcohol-induced liver 

damage [11], alleviates pain sensitivity [12], and prevents 

allergic rhinitis–like symptoms [13]. Other than 

Sakekasu’s nutritional components, its functional 

ingredients have been widely studied. For example, 

peptides obtained from the hydrolyzed products of 

Sakekasu inhibit angiotensin-converting enzyme and 

suppress hypertension [14]; they also inhibit prolyl 

endopeptidase, which is involved in amnesia 

development [15]. Furthermore, ethyl glucoside (1) 

(Figure 1), which is relatively abundant in Sakekasu, has 

hepatoprotective [16] and diuretic effects [17] and 

improves the functions of the stratum corneum [18]. 

Sakekasu also contains S-adenosylmethionine (SAM) (3) 

(Figure 1), which is reportedly effective in treating 

diseases such as alcoholic liver dysfunction and 

depression [19]. In recent years, glycerophosphocholine 

(GPC) (6) found in Sakekasu has attracted attention for its 

anti-aging and brain function improvement effects 

(Figure 1) [20]. The GPC- and SAM-associated metabolic 

pathways (choline pathway and methionine pathway, 

respectively) are related via betaine, and the relationship 

between their content remains insufficiently understood. 

The composition of these functional components 

greatly varies depending on the fermentation conditions 

and especially length of aging of Sakekasu in different 

breweries. However, to date, only changes in Sakekasu 

composition caused by the drying method have been 

reported [21], and changes caused by aging remain 

uninvestigated. Differences in the degree of maturity of 

Sakekasu are reflected in the color. Therefore, in the 

present study, to clarify the relationship between the 

contents of the functional components in Sakakasu and 

color tones, we collected Sakekasu with white, ocher, and 

brawn color tones from several sake breweries and 

comprehensively analyzed their functional components. 

Figure 1. Structures of the compounds detected. 1: ethyl glucoside, 2: glyceryl glucosides, 3: S-adenosylmethionine (SAM), 4: 

glycerophosphocholine (GPC), 5: linolenoyl-glycero-phosphocholine, 6: linoleoyl-glycero-phosphocholine, 9: linoleic acid, 10: palmitic 

acid, 11: oleic acid, 12: 5'-deoxy-5'-methylthioadenosine, 13: fructose-leucine (isoleucine)-valine or fructose-valine-leucine (isoleucine), 

14: fructose-phenylalanine-valine or fructose-valine-phenylalanine, 15: fructose-leucine (isoleucine)-leucine (isoleucine), 16: fructose-

phenylalanine-leucine (isoleucine) or fructose-leucine (isoleucine)-phenylalanine. Glc, Glucose; L, leucine; I, isoleucine; V, valine, F, 

phenylalanine. 

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METHODS 

Specimens and reagents: We obtained eight samples of 

Sakekasu from Sasanokawa Shuzo Co. Ltd. (Fukushima, 

Japan) and two other brewery companies. (Aichi, Japan 

and Kyoto, Japan). Samples 1–4 were white (R: 255, G: 

242, B: 207), 5–6 were ocher (R: 223, G: 170, B: 105), and 

7–8 were brawn (R: 169, G: 98, B: 53). These color 

differences are caused by the ripening period and 

temperature of the Sakekasu. The color of samples 

becomes darker due to the long ripening period and high 

drying temperature [22]. All samples were lyophilized 

and stored at 4°C until analysis. We purchased 

analytically grade chemicals and chromatographic 

solvents (liquid chromatography–mass spectrometry 

[LC–MS] grade) from Fujifilm Wako Pure Chemical 

(Osaka, Japan), and SAM and GPC from Nacalai Tesque 

(Kyoto, Japan). 

Instrumentation and analysis: For LC–MS analyses, we 

used the mass spectrometer Shimadzu LC–IT–TOF 

(Shimadzu, Kyoto, Japan) equipped with an electrospray 

ionization (ESI) interface. The ESI parameters were as 

follows: source voltage, +4.5 kV in positive ion mode and 

−3.5 kV in negative ion mode; capillary temperature,

200°C; and nebulizer gas flow rate, 1.5 L/min. We used 

the mass spectrometer in positive and negative ion 

modes and recorded the scans from m/z 150 to 1500. 

GPC and its relating compounds were separated using the 

HILIC column Waters XBridge BEH amide (2.1 × 150 mm, 

5 μm) at 40°C consistently. The binary mobile phase 

consisted of (A) 5 mM CH3COONH4 in water and (B) 

CH3CN. These compounds were eluted using the 

following gradient conditions: 0–30 min, linear gradient 

from 95% to 45% B, and 30–40 min of isocratic solution 

at 45% B. Furthermore, SAM was separated using the 

column Thermo Fisher Scientific Hypercarb (2.1 × 100 

mm, 3 μm, 40°C), and the binary mobile phase consisted 

of (A) 0.1% HCOOH in water and (B) CH3CN. The 

compound was eluted using the following gradient 

conditions: 0–10 min, linear gradient from 0% to 60% B, 

10–12 min linear gradient from 60% to 80% B, and 12–15 

min of isocratic solution at 80% B. Other compounds 

were separated using the ODS column Waters Atlantis T3 

(2.1 × 150 mm, 5 μm, 40°C), and the binary mobile phase 

consisted of (A) 5 mM CH3COONH4 in water and (B) 

CH3CN. The compounds were eluted using the following 

gradient conditions: 0–30 min, linear gradient from 10% 

to 100% B, and 30–40 min of isocratic solution at 100% B. 

Extraction of constituents from Sakekasu: To extract 

GPC and SAM, we first pulverized freeze-dried Sakekasu 

specimens and mixed each 500 mg of the sample with 5 

mL of 50% methanol. These extraction mixtures were 

ultrasonicated for 10 min and then left overnight at room 

temperature. On the following day, we filtered the 

extracts through 0.45 μm Millipore filter units (Advantec, 

Tokyo, Japan) and injected 1 μL of the sample into the LC–

MS. Other compounds were extracted using other 

conditions. For example, 10 g of the fine powder of 

freeze-dried Sakekasu specimens was accurately 

weighted, and the constituents were extracted with 

methanol using the Extraction System B-811 LSV (BUCHI, 

Flawil, Switzerland) under reflux conditions for 100 min. 

The organic solvent was evaporated in vacuo to collect 

the methanol extract. Additionally, 2 mg of the extract 

was dissolved in 1 ml of methanol–water mixture (1:1 by 

vol.). All extracts were filtered through 0.45 μm Millipore 

filter units (Advantec), and 1 μL of the sample was 

injected into the LC–MS. 

RESULTS AND DISCUSSIONS 

Quantitation of GPC and SAM in Sakekasu: The total ion 

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chromatograms and the mass chromatograms of the 

extracts analyzed using the HILIC column are shown in 

Figure 2. Especially, Figure 2 (f) illustrates the mass 

chromatograms monitored by the M+ ion of GPC (4, m/z 

258.1102), while Table 1 lists the quantitation results of 

GPC in Sakekasu samples. The white Sakekasu in samples 

1–4, which are thought to have been aged for a shorter 

period, had higher GPC concentrations than aged 

samples 5–8 (Table 1). Notably, it was not detected in the 

brawn colored Sakekasu (samples 7 and 8), which 

appeared to have been aged for a long time (Table 1). It 

is well known that GPC is abundant in dried salmon and 

rainbow trout [23], and the GPC content of white 

Sakekasu was comparable to these products. In addition, 

samples with high GPC concentrations contained 

linolenoyl-glycero-phosphocholines (5) and linoleoyl-

glycero-phosphocholines (6) [Figure 2 (g) and (h)]. These 

compounds are active ingredients that inhibit 

inflammatory cytokine production [24] and contribute to 

Sakekasu functionality. In experiments using aged mice, 

Matsubara et al. reported that the deposition of 

transthyretin, an amyloidogenic protein, in the brain was 

reduced by additional 17% GPC intake [20]. In the present 

study, the GPC concentration in Sakekasu is very high and 

is considered to be a concentration at which efficacy can 

be expected with daily ingestion.

Table 1. Concentrations of glycerophosphocholine and S-adenosylmethionine in Sakekasu samples. 

Compounds Samples (mg/g) 

1 2 3 4 5 6 7 8 

Glycerophosphocholine 3.87 2.82 3.52 0.55 0.14 0.17 0.00 0.00 

S-Adenosylmethionine 2.84 3.62 3.77 2.95 0.00 0.00 0.00 0.00 

Figure 2 also shows a mass chromatogram 

monitored by the ion M+ of SAM (3) [Figure 2 (e)]. The 

peaks show a broad shape, making quantitation by this 

method difficult. Krijt et al. reported SAM quantitation 

using a Hypercarb column [25]. Hypercarb columns have 

unique properties that distinguish them from 

conventional columns, and they excel in retaining and 

separating highly polar compounds. Figure 3 shows the 

total ion chromatograms analyzed using a Hypercarb 

column and mass chromatograms monitored by the M+ 

ion of SAM. The amount of SAM in Sakekasu varied 

greatly from sample to sample, with a very high content 

in white Sakekasu (samples 1–4) [Figure 3 (c)]. 

Conversely, SAM was not detected at all in ocher and 

brawn-colored samples (samples 5–8) (Table 1). SAM is 

known for its mood-improving, anti-hepatotoxic, and 

anti-arthritic effects [26]. Sakekasu with a high SAM 

content is considered useful as a functional material. 

The metabolic pathways associated with GPC 

(choline pathway) and SAM (methionine pathway) are 

related via betaine. In our study, samples with high GPC 

also had high SAM; when Sakekasu containing high GPC 

and SAM contents is ingested, a synergistic effect of both 

functionalities can be expected. 

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Figure 2. Total ion chromatograms and mass chromatograms of the extracts analyzed using the HILIC column.  

Samples 1–4 were white, 5–6 were ocher, and 7–8 were brown. The following ions were monitored (numbers in brackets indicate 

magnification). a: positive ion, b: negative ion, c: m/z 231.0829 ([M+Na]+ ion of ethyl glucoside [1]), d: m/z 277.0897 ([M+Na]+ ion of 

glyceryl glucosides [2]), e: m/z 399.1460 (M+ ion of S‐adenosylmethionine [3]), f: m/z 258.1102 (M+ ion of glycerophosphocholine [4]), g: 

m/z 520.3428 (M+ ion of linolenoyl-glycero-phosphocholines [5]), h: m/z 522.3232 (M+ ion of linoleoyl-glycero-phosphocholines [6]), i: 

m/z 179.0575 ([M–H]– ion of monosaccharides [7]), j: m/z 341.1105 ([M–H]– ion of disaccharides [8]). 

(min)5 10 15 20 25 30 35

0

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Figure 3. Total ion chromatograms and mass chromatograms of the extracts analyzed using the Hypercarb column.  

Samples 1–4 were white, 5–6 were ocher, and 7–8 were brawn. The following ions were monitored (numbers in brackets 

indicate magnification). a: positive ion, b: negative ion, c: m/z 399.1460 (M+ ion of S‐adenosylmethionine [3]). 

0

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(x10,000,000)

Sample 4

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Figure 4. Total ion chromatograms and mass chromatograms of the extracts analyzed using the ODS column.  

Samples 1–4 were white, 5–6 were ocher, and 7–8 were brawn. The following ions were monitored (numbers in brackets 

indicate magnification). a: positive ion, b: negative ion, c: m/z 393.2227 ([M+H]+ ion of fructose-leucine (isoleucine)-valine 

and fructose-valine-leucine (isoleucine) [13]), d: m/z 427.2089 ([M+H]+ ion of fructose-phenylalanine-valine and fructose-

valine-phenylalanine [14]), e: m/z 407.2393 ([M+H]+ ion of fructose-leucine (isoleucine)-leucine (isoleucine) [15]), f: m/z 

441.2226 ([M+H]+ ion of fructose-phenylalanine-leucine (isoleucine) and fructose-leucine (isoleucine)-phenylalanine [16]), 

g: m/z 298.0950 ([M+H]+ ion of 5'-deoxy-5'-methylthioadenosine [12]). 

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Identification of other metabolites in Sakekasu: Figure 2 

(c), (d), (i), and (j) shows the mass chromatograms 

monitored by the [M+Na]+ ion of ethyl glucoside (1, m/z 

231.0829), [M+Na]+ ion of glyceryl glucosides (2, m/z 

277.0897), [M–H]– ion of monosaccharides (7, m/z 

179.0575), and [M–H]− ion of disaccharides (8, m/z 

341.1105). These sugar derivatives were detected in all 

samples, although their concentrations varied. Ethyl 

glucoside (1) is particularly abundant in sake; it originates 

from the unique fermentation method of sake wherein 

starch saccharification and alcohol fermentation occur 

simultaneously [27]. Ethyl glucoside not only affects the 

taste of sake but also possesses several functional 

properties, such as improving skin texture [27, 28]. Given 

that the amount of ethyl glucoside does not depend on 

the color-tone, it is expected to have a constant 

functionality. Furthermore, glyceryl glucoside (2), which 

has moisturizing properties, was also detected in sake 

[29]. Considering the differences in the position and 

conformation of sugar bonds in glycerol, several 

compounds have been detected, but the analytical 

conditions used in the present study did not allow them 

all to be separated. However, they were largely 

separated as two peaks [Figure 2 (d)], possibly owing to 

the difference in the position of sugar binding in glycerol 

(2). In addition, their content was not dependent on the 

color tone of Sakekasu. 

The methanol extracts of each sample were 

analyzed using an ODS column and the results are shown 

in Figure 4. Samples 1–4 contained higher amounts of 

fatty acids [linoleic acid (9), palmitic acid (10), and oleic 

acid (11)] than brown-colored samples 7 and 8. In Figure 

4, the mass chromatograms shown in c–f are Amadori or 

Heyns products derived from the Maillard reaction of 

sugar and dipeptide monitored by the [M+H]+ ion of the 

respective compounds, whereas that shown in Figure 4 

(g) is 5′-deoxy-5′-methylthioadenosine (MTA, 12)

monitored by the [M+H]+ ion. As shown clearly in Figure 

4, white Sakekasu samples 1–4 contained fewer 

compounds derived from the Maillard reaction of sugar 

and dipeptides (13–16) than the brown-colored samples 

7 and 8. In addition, MTA (12) was reduced in samples 7 

and 8 [Figure 4 (g)], which were ripened. Tadenuma et al. 

reported that MTA and its precursor SAM are unique to 

sake and not found in other brews, and that MTA is 

formed from SAM during sake storage [30, 31]. They also 

observed that the elution of SAM from yeast increased 

after the addition of brewing ethanol, indicating that the 

MTA and SAM amounts are proportional to the ethanol 

concentration [30, 31]. Considering that the detailed 

production methods for samples 1–4 analyzed in our 

study are unknown, we cannot confirm whether such 

result is a characteristic of the yeast used or of the 

production process; however, the high MTA and SAM 

contents in samples 1–4 may be a characteristic of these 

samples. Moreover, compounds derived from the 

Maillard reaction of sugar and dipeptide tended to be 

extremely low in samples 1–4 than in other Sakekasu 

samples. 

The compounds derived from the Maillard reaction 

were annotated by tandem mass spectrometry (MS/MS) 

analysis. Figure 5 shows the MS/MS analysis of the peak 

of compound 13 observed in the mass chromatogram 

depicted in Figure 4 (c). Compound 13 possessed a 

[M+H]+ ion at m/z 393.2227 with a composition of 

[C17H32N2O8+H]+. According to the composition of the 

ions, the Amadori or Heyns product of a dipeptide was 

assumed to be composed of hexose, leucine (isoleucine), 

and valine. Andruszkiewicz et al. reported that the 

reaction of fructose with peptides produces mainly the 

Heyns product and that the reaction of glucose with 

peptides produces mainly the Amadori product [32]. 

Furthermore, Yuan et al. investigated in detail the 

fragmentation attribution in the MS analysis of Heyns 

products [33, 34]. Based on these reports, the structure 

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of compound 13 was estimated by MS/MS analysis. The 

MS/MS analysis of compound 13 from the [M+H]+ ion at 

m/z 393.2227 detected a [M+H–3H2O–H2CO]+ ion at m/z 

309.1806 as the characteristic ion, and the MS3 analysis 

from the [M+H–3H2O–H2CO]+ ion provides fragment ions 

at m/z 150.0916 [Frucrose-L(I)-V+H–3H2O–H2CO–x2]+ 

and m/z 164.1070 [Frucrose-V-L(I)+H–3H2O–H2CO–x2]+, 

where x2 indicates a type of dipeptide cleavage (Figure 

5). Figure 5 also shows the results of the estimation based 

on Yuan et al.’s report [33, 34]. According to these 

results, compound 13 is a Heyns product composed of 

fructose (Fru) and leucine (isoleucine)-valine or valine-

leucine (isoleucine). Similarly, the MS analysis results 

considered Fru-F-V and Fru-V-F as compound 14, Fru-L(I)-

L(I) as compound 15, and Fru-F-L(I) and Fru-L(I)-F as 

compound 16.  

Figure 5. MSn spectra from m/z 393.2227 ([M+H]+ ion of fructose-leucine [isoleucine]-valine or fructose-valine-leucine 

[isoleucine] [13]) observed in Figure 4, and estimated structure of their ions. L, leucine; I, isoleucine; V, valine. 

CONCLUSION 

Several types of Sakekasu with different color tones were 

collected and analyzed for major functional ingredients 

such as GPC and SAM. Sakekasu with white color had 

large amounts of GPC and SAM. The concentration of 

GPC in Sakekasu was the highest ever found in a food 

product, and the daily consumption of this product may 

be sufficient for its efficacy. Moreover, compounds with 

suggested functionality, such as ethyl glucoside and 

glyceryl glucosides, were detected, and they were not 

309.1806 [M+H－3H2O－H2CO]+

100 150 200 250 300 350 400 450 (m/z)

MS2 (m/z 393.2227 →)

MS3 (m/z 309.1806 →)

150.0916 [Fructose-V-L(I)+H－3H2O－H2CO－x2]+

263.1761

210.1124

100 150 200 250 300 350 400 450 (m/z)

164.1070 [Fructose-L(I)-V+H－3H2O－H2CO－x2]+

196.0975

393.2227 [M+H]+

100 150 200 250 300 350 400 450 (m/z)

MS

393.2227

Fructose-L(I)-V

[C17H32N2O8+H]+

309.1806

[Fructose-L(I)-V+H

–3H2O–H2CO]+

164.1070

[Fructose-L(I)-V+H

–3H2O–H2CO–x2]+

393.2227

Fructose-V-L(I)

[C17H32N2O8+H]+

309.1806

[Fructose-V-L(I)+H

–3H2O–H2CO]+

150.0916

[Fructose-V-L(I)+H

–3H2O–H2CO–x2]+

http://www.ffhdj.com/


Functional Foods in Health and Disease 2023; 14(1):74-86       FFHD      Page 84 of 86            

greatly influenced by color tone (i.e., aging period). 

Furthermore, the dark colored Sakekasu samples 

contained more Maillard reaction products of sugars and 

dipeptides, whereas the white ones showed more fatty 

acids. Through MS/MS based analysis, the Maillard 

reaction products of sugars and dipeptides were 

assessed. Thus, this study has clarified many functional 

compounds in Sakekasu in relation to color tone (i.e., 

ripening period) and emphasized the potential of 

Sakekasu with white color tone, which are thought to 

have been aged for a shorter period, as a functional food. 

Further biological studies on Sakekasu containing 

functional components such as GPC and SAM are 

expected. 

Competing interest: There are no conflicts of interest to 

declare. 

Author contribution: YN analyzed data and editing and 

revised the manuscript. SM, YM, and KS designed the 

study. KT obtained fundings, designed the study, 

performed the experiments, analyzed data, wrote the 

manuscript, and provided overall supervision. All authors 

have read and approved the final manuscript. 

Acknowledgments and Funding: This work was 

supported by the AY2021 Program for Asia-Japan 

Research Development, Ritsumeikan University (Ibaraki, 

Osaka, Japan). 

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