༤ኈㄽᩥ
Studies on the sake brewing
properties of the sake rice

Koshitanrei㓇㐀ዲ㐺⡿࣭㉺ῐ㯇ࡢ㔊㐀≉ᛶ࡟㛵ࡍࡿ◊✲ᕷ ᕝ ⤮ ᲍ᗈᓥ኱Ꮫ኱Ꮫ㝔ඛ➃≀㉁⛉Ꮫ◊✲⛉2020 ᖺ 9 ᭶

┠ḟ
㸯㸬୺ㄽᩥ
    Studies on the sake brewing properties of the sake rice Koshitanrei     㸦㓇㐀ዲ㐺⡿࣭㉺ῐ㯇ࡢ㔊㐀≉ᛶ࡟㛵ࡍࡿ◊✲㸧    ᕷᕝ ⤮᲍㸰㸬බ⾲ㄽᩥ
(1) Analysis of metabolites in Japanese alcoholic beverage sake made from the sake rice
Koshitanrei.
Eri Ichikawa, Shougo Hirata, Yuko Hata, Hisashi Yazawa, Hiroyashu Tamura,
Mitsuoki Kaneoke, Kazuhiro Iwashita, and Dai Hirata
Bioscience, Biotechnology, and Biochemistry, 83(8), 1570-1582 (2019).

(2) Effect of koji starter on metabolites in Japanese alcoholic beverage sake made fromthe sake rice Koshitanrei.
Eri Ichikawa, Shougo Hirata, Yuko Hata, Hisashi Yazawa, Hiroyashu Tamura,
Mitsuoki Kaneoke, Kazuhiro Iwashita, and Dai Hirata
Bioscience, Biotechnology, and Biochemistry, 84(8), 1714-1723 (2020).

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୺ㄽᩥ

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CONTENTS
Page
Introduction

Chapter I.

4Analysis of metabolites in Japanese alcoholic beverage sake made fromthe sake rice Koshitanrei.

7

1.1.

Abstract

8

1.2.

Introduction

8

1.3.

Materials and Methods

10

1.4.

Results and Discussion

13

Chapter II.

Effect of koji starter on metabolites in Japanese alcoholic beverage sakemade from the sake rice Koshitanrei.

33

2.1.

Abstract

34

2.2.

Introduction

34

2.3.

Materials and Methods

36

2.4.

Results and Discussion

38

Concluding remarks

55

References

58

Acknowledgments

61

Related publications

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Introduction
Alcoholic beverages have been made from the major agricultural products of each country.
Sake is the traditional Japanese alcoholic beverage brewed from raw materials, rice, andwater (Yoshizawa, 1999). In this process, 2 microorganisms, the koji-fungus Asperugillus
oryzae and the sake yeast Saccharomyces cerevisiae, are utilized. The starch of rice isdecomposed to glucose by enzymes produced by A. oryzae. Then, the glucose is converted
to alcohol by the metabolism of S. cerevisiae. In the sake mash (main mash, moromi), 2
bioconversions, saccharification from rice starch to glucose and fermentation from glucose
to alcohol, take place simultaneously. This unique process is known as parallel fermentation
and a key mechanism to achieve high alcohol production. This sake brewing process, which
achieves high alcohol production, is a unique process in the sake brewing.
In Japan, the production quantity of sake has decreased since its peak in 1973. Oneof the reasons is that the consumer began to select other alcoholic beverages (beer, wine,liqueurs, etc.) as a result of diversification of lifestyles. In order to improve the quality ofsake and develop new products, many sake companies have been studying the raw materials,
A. oryzae, S. cerevisiae, as well as new brewing technologies, etc. As a result, various typesof sake have been developed.
The main raw material for sake brewing is the rice, and the characteristic features ofrice are one of most important factors determining the quality of the final product, sake. In
the sake brewing process, brown rice is polished to white rice, steamed, and then used in 2
ways: for rice-koji making and directly added to sake mash (as kake-mai). Various ricecultivars have been used for sake brewing, and it is empirically known that thecharacteristics of rice cultivars affect the aroma/ taste of the sake. Therefore, the selectionof the rice cultivar is one of the most important factors to produce high-quality sake.
On the other hand, in sake-brewing process, rice-koji making is one of the important
steps, because it is a determinant of the aroma/ taste components of sake, being the supplierof rice-degrading enzymes and essential vitamins for yeast fermentation. In the koji-making
process, A. oryzae grows on steamed rice and produces a number of enzymes, such as
saccharification enzymes and proteolytic enzymes, as well as others. The activity of eachenzyme is affected by rice cultivar and koji starters (Takahashi et al., 2008; Yanagiuchi etal., 1993). The difference in the levels of enzyme activities and other components such asvitamins directly or indirectly affects the aroma/ taste component of sake. Thus, theselection of koji starter for rice cultivar is important.
4

In general, 2 kinds of rice (Oryza sativa L), sake rice and cooking rice, have been used
for sake brewing. Officially, sake rice has been defined as shuzo-koteki-mai in the
Agricultural Products Inspection Act. The grain size of sake rice is larger than that of
cooking rice, has a white core (called shinpaku), and a low protein content. Breeding ofsake rice cultivars has been carried out in many areas of Japan (Maeshige and Kobayashi,
2000), and approximately 100 types of sake rice have been developed for use in sakebrewing. Among them, Yamadanishiki (YAM, developed in Hyogo Prefecture),
Gohyakumangoku (GOM, in Niigata Prefecture), and Miyamanishiki (MYN, in Nagano
Prefecture) have been the major sake rice varieties widely used for high-quality sakebrewing.
In Niigata Prefecture, the originally developed sake rice GOM has been used widely.
GOM has contributed to high-quality sake having a clear and dry taste (“Tanrei”, one ofthe perceivable attributes in the refined sake), although the hardness of its grains is
insufficient for high polishing (especially to less than the 50% polishing ratio for Daiginjoshu), due to the shape and size of its white core (Anzawa et al., 2013). The white core of
GOM is larger than that of the sake rice YAM, which has been used widely for Daiginjoshu brewing in Japan.
In 2004, to improve the polishing property of GOM, the sake rice Koshitanrei (KOS)
was originally developed in Niigata Prefecture by genetically crossing GOM with YAM(Kobayashi et al., 2006). Recently, KOS has been used widely for high-quality sakebrewing

inNiigata

Prefecture.

However,comprehensive

analysisofthecomponents/metabolites in sake prepared from KOS has not been reported yet.
The metabolomics technique is an analysis method examining a number of intravital
metabolites. Recently, this type of analysis has been applied in various fields withdiversification (Putri et al., 2013a; Putri et al., 2013b). Using these techniques for the
analysis of sake, Sugimoto et al. (2010) and Mimura et al. (2014) reported the relationshipbetween the sake component/metabolites and the taste of sake. Recently, studies examining
the relationship between sake-making parameters and sake metabolites (Tamada et al.,2017; Yazawa et al., 2019) have appeared. Although, a number of methods for analyzing
sake compounds/metabolites have been established/developed, the metabolites of sakemade from KOS have not been analyzed yet. Furthermore, the effect of the koji starter on
the characteristics of KOS rice koji and sake metabolites also has not been examined,
especially by the use of metabolomics techniques.
5

In this doctoral thesis, I studied the brewing characteristics of sake rice KOS,
especially those for the production of high-quality sake. In Chapter I, in order to clarify thecharacteristic components/ metabolites of sake made from KOS, I examined the effect of
kake-rice (rice for kake-mai) and total rice (rice for both kake-mai and koji) variation on thesake components/metabolites by using 3 different rice cultivars: KOS, YAM, and GOM.
In Chapter II, in order to investigate the effect of the koji starter on the rice-kojicharacteristics and sake metabolites, I performed combined examination using 3 different
koji starters and sake rice KOS to assess their effect on the sake components/metabolites.

6

CHAPTER ,Analysis of metabolites in Japanese alcoholic beverage sake made from the sake rice
Koshitanrei.

7

1.1.

Abstract

In sake brewing, the steamed rice is used in 2 ways, added to sake-mash (as kake-mai) andmaking koji. The rice is an important determinant for the quality of sake, as the metabolites
in sake affect its taste/aroma. The sake rice Koshitanrei (KOS) was developed in Niigata
Prefecture by genetically crossing 2 sake rice, Gohyakumangoku and Yamadanishiki.
However, the metabolites in sake from KOS have not been analyzed. Here, to investigate
the characteristic metabolites in sake from KOS, I performed 2 types of small-scale sakefermentation tests changing only the rice used for kake-mai or total rice (both kake-mai andkoji) by these 3 rice cultivars and examined the effect of KOS on sake metabolites by themetabolome analysis method using UPLC-QTOF-MS. I identified the peaks/metabolites,
whose intensity in sake from KOS was higher/lower than those from the other cultivars.
The brewing properties of KOS were partially characterized by this analysis.
1.2.

Introduction

The Japanese traditional alcoholic beverage sake is made from rice and water by a uniqueprocess using 2 microorganisms, a fungus (Aspergillus oryzae, called koji-mold) and yeast(Saccharomyces cerevisiae) (Yoshizawa, 1999). The main raw material for sake brewing
is the rice, and the characteristic features of this rice is one of the important factors
determining the quality of the final product, sake. In sake brewing, steamed rice is used in
2 ways, directly added to sake mash (as kake-mai) and used to make koji. The taste andaroma of sake are related to a variety of components. Various rice cultivars have been used
for sake brewing in order to characterize the specific taste of the sake. Therefore, theselection of the rice cultivar is one of the important concerns for brewing high-quality sake.
In general, 2 kinds of rice (Oryza sativa L), sake rice and cooking rice, have beenused for sake brewing. The grain size of sake rice is larger than that of cooking rice, andsake rice has a white core (called shinpaku). Breeding of the sake rice cultivars has been
done in many areas of Japan (Maeshige and Kobayashi, 2000). In Niigata Prefecture, theoriginally developed sake rice Gohyakumangoku (GOM) has been used widely. GOM hascontributed to high-quality sake having a clear taste, although the hardness of its grains is
insufficient for high polishing (especially to less than the 50% polishing ratio for Daiginjoshu). This disadvantage of GOM is due to the shape and size of its white core (Anzawa etal.,. 2013). The white core of GOM is larger than that of the sake rice Yamadanishiki(YAM), which was developed in Hyogo Prefecture and has been used widely for Daiginjo8

shu brewing in Japan.
To improve the polishing property of GOM, the sake rice Koshitanrei (KOS) was
originally developed in Niigata Prefecture by genetically crossing GOM with YAM(Kobayashi et al., 2006). Recently, KOS has been used widely for high-quality sakebrewing in Niigata Prefecture. Previously, Kobayashi et al. (Kobayashi et al., 2006)
characterized KOS, based on the results of examining the brewing properties of KOS in asake brewing test using 120 kg (total rice, 70% polishing ratio). Further, Anzawa et al.
(Anzawa et al.,. 2013) examined the polishing/brewing properties of KOS using a wide
range of polished rice and reported the differences in the general properties of the sakebetween that made from KOS and that from GOM. However, comprehensive analysis of
the components/metabolites in sake from KOS has not been reported yet.
The metabolomics technique is an analysis method examining cyclopaedically/coinstantaneously a number of intravital metabolites. Recently, this type of analysis has been
applied in various fields with diversification (Putri et al., 2013a; Putri et al., 2013b).
Previously, in the analysis of sake done by using the metabolomics technique CE-TOF MS,
Sugimoto et al. (Sugimoto et al., 2010) reported a correlation between sensory evaluation
scores of sake and metabolome profiles, as well as a difference in the charged metabolites
between pasteurized and unpasteurized sake during storage (Sugimoto et al., 2012). In thesake analysis using GC/MS, Mimura et al. (Mimura et al., 2014) examined therelationship between the features of sake and the component profile; and Tamada et al.
(Tamada et al., 2017) reported the characteristics of sake according to the sake-making
parameters (sake yeast, rice cultivars) by GC/MS and analyzed the correlation between thecomponent profiles of GC/MS and the intensity of “Oshi-aji” (Tamada et al., 2018).
Further, in the sake analysis using 2-dimensional GC with TOF-MS, Takahashi et al.
(Takahashi et al., 2016) examined the correlation between sake components andorganoleptic properties; and Tokuoka et al. (Tokuoka et al., 2017) used HILIC-TOF-MS
to analyze the oligosaccharide composition of sake. Recently, Yazawa et al. (Yazawa etal., 2019) conducted sake metabolome analysis using UPLC-QTOF-MS (ultraperformance liquid chromatography/time-of-flight mass spectrometry) and examined therelationship between sake-making parameters and sake metabolites. Although, as describedabove, a number of methods for analyzing sake compounds/metabolites have been
established/developed, the metabolites of sake made from KOS have not been examinedby using these methods.
9

In this study, in order to clarify the characteristic components/metabolites of sakemade from KOS, I performed 2 types of small-scale sake fermentation tests using 3 ricecultivars: KOS, YAM, and GOM. At first, to investigate the effect of kake-rice (rice for
kake-mai) variation on the sake components/metabolites, I performed a small-scale
fermentation test changing only the type of rice cultivar used for kake-rice. Then, toinvestigate the effect of total rice variation (rice for both kake-mai and koji) on the sakecomponents/metabolites, I performed another small-scale fermentation test using the same
3 rice cultivars. I analyzed the sake metabolome data obtained from the above 2 types ofsmall-scale sake fermentation tests by using the recently developed sake metabolome
analysis method; (Yazawa et al., 2019) and by it I identified the specific peaks/metabolites,
whose intensity in sake from KOS was higher/lower than those from the other 2 cultivars,
YAM and GOM. The results demonstrated that certain components/metabolites in sakefrom KOS were different from those found for YAM or GOM and that the brewing
properties of KOS could be at least partially characterized by performing this analysis.
1.3.

Materials and Methods

1.3.1. Rice cultivars
Rice cultivars used for the small-scale sake fermentation test in which only the kake-rice(rice for kake-mai), referred to as the “kake-rice sake-making test,” was changed were as
follow (Fig. 1(a), Nos. 1-6): YAM, GOM, and KOS. The rice cultivar used as koji-rice (ricefor koji) in this test was Yamadanishiki (YAM), harvested in 2015 (grow in Hiroshima
Prefecture); and the 3 rice cultivars used as kake-mai, YAM (grow in Hiroshima Prefecture),
Gohyakumangoku (GOM, grow in Niigata Prefecture), and Koshitanrei (KOS, grow in
Niigata Prefecture), were harvested in 2014, 2016, and 2016, respectively. Rice cultivars
used for the small-scale sake fermentation test in which both the kake-rice and koji-ricewere changed (referred as “kake+koji-rice sake-making test”) were as follow (Fig. 1(a),Nos. 7-12): YAM (grow in Hiroshima Prefecture), GOM (grow in Niigata Prefecture), and
KOS (grow in Niigata Prefecture), all harvested in 2016. The rice grains were polished topolishing ratios of 50% and 70% by using a milling machine (RP-5 and NF-26FA, ShinNakano Industry, Japan).
1.3.2. A model sake sample
A model sake sample was prepared as a mixture of 68 standard compounds (model sake,marked “1” in Table S1 (Chapter I Table S1.xlsx)) in 15% ethanol. Then, the prepared sake10was aliquoted into 1-mL samples in Eppendorf tubes and stored at −80 °C.
1.3.3. Small-scale koji making
Rice with a polishing ratio of 50% or 70% was soaked in tap water to 130% water
absorption and then steamed for 50 min. The steamed rice was dried to 130% water
absorption, and cooled down to room temperature. Conidia spores (1 x 106 conidia/g kojirice) of Aspergillus oryzae (Fig.1A, koji starter, Byakuya, Hishiroku-Moyashi, Japan) were
inoculated to the steamed rice, and the inoculated rice was incubated in an incubator(KCL2000, EYRA, Japan) for 46 h, during which time the temperature and moisture wereregulated. Initially, the temperature in the incubator was maintained at 32°C for 22 h, andthen it was gradually increased as follows: to 34 °C for 8 h, to 37 °C for 3 h, to 40 °C for 3
h, and to 42 °C for 10 h. Humidity in the incubator was initially maintained at 90% for 22
h and then gradually decreased as follows: to 80% for 8 h, to 70% for 3 h, and to 60% for
13 h. After 46 h following inoculation, the humidity was controlled at less than 90%, andthe koji was cooled down to slightly less than room temperature. The koji was stored at 30°C until used for sake making.
1.3.4. Small-scale sake-making test
To examine the effects of kake-rice and koji-rice on sake metabolites, I performed 2 typesof small-scale sake-making tests (2 independent experiments each), “kake-rice sakemaking test” (Fig. 1(a) Nos. 1-6) and “kake+koji-rice sake-making test” (Fig. 1(a) Nos. 712). In the first test, referred to as the “kake-rice sake-making test,” the rice cultivar YAM(40% polishing ratio) was used for the koji-rice, and 3 rice cultivars (YAM, GOM, and
KOS) each of 50% or 70% polishing ratio, were used for the kake-rice (Fig. 1, sample
Nos.1-6, referred to as “kake-rice sake making test”). In the second test, designated as the“kake+koji-rice sake-making test,” the total rice of both kake-rice and koji-rice werechanged, and 2 polishing ratios (50% and 70%) of 3 rice cultivars (YAM, GOM, and KOS)
were used for both kake-rice and koji-rice (Fig. 1, sample Nos.7-12). In the 2 types of thesake-making test, 80 g of total rice (16 g for koji-rice and 64 g for kake-rice) was used forone-step addition to the sake mash; and the sake yeast strain K701 was used. The kakemai for the sake-making test was prepared as follows: The rice (64 g) was washed at 10 °C
for 30 sec with tap water, drained for 15 sec, and washed for 30 sec again with more tap
water. After the washing step, the rice was drained for 15 sec and then soaked to reach
130% water absorption. Next, the soaked rice was drained at 10 °C for 30 min andsubsequently steamed for 50 min. The water absorption of the steamed rice was adjusted to11

130%, assuming that the water content of the original white rice was 13.5%. The steamed
rice was added to 103 mL of water with 1 mL (108 cells) of the sake yeast K701, 16 g (aspolished rice) of koji, and 72 PL of 50% lactic acid. The sake yeast strain K701 for the
sake-making test was cultured as follows: The sake yeast cells were pre-cultured overnightat 30 °C in the rice koji extract medium (Baume scale 10). The pre-cultured cells were theninoculated into fresh medium at 100-fold dilutions and incubated for 2 days at 30 °C instanding culture. The cultured cells were washed twice with sterilized water and thereafter
added to the sake mash (final concentration, approx. 1 x 106 cells/mL). Sake fermentation
was performed at 15 °C (in a water bath) for 20 days, and was monitored by measuring theweight reduction of the sake mash, corresponding to CO2 evolution. After fermentation, theclear sake sample was obtained by centrifugation (5000 rpm, 10 min, 4 °C).
1.3.5. Analysis of sake
General properties (Sm, sake meter; Alc, sake alcohol; TA, total acidity; AA, amino acidity),
and flavor components (EtOAc, ethyl acetate; iAmOH, isoamyl alcohol; iAmOAc, isoamyl
acetate; EtCap, ethyl caproate) of the sake were determined by the standard method
established by the National Tax Agency of Japan.
1.3.6. Enzyme activity analysis of rice koji
Enzyme activity of koji was analyzed by the standard method established by the National
Tax Agency of Japan. The activities of 3 enzymes, i.e., D-amylase, glucoamylase, and acidcarboxypeptidase, were measured by using the respective assay kits (Kikkoman, Japan)(Imai et al., 1996; Shirokane et al., 1996).
1.3.7. Sake metabolome analysis method
Sake samples were filtered through an Amicon Ultra 0.5 3K (Merk Millipore, Germany)and diluted 10-fold with MS-grade water. UPLC-QTOF-MS analysis was performed witha UPLC Xevo Q/TOF-MS system (Waters, Milford, USA). The method for sakemetabolome analysis established by Yazawa et al. (Yazawa et al., 2019) was followed.
Each sample from 2 independent small-scale sake-making tests was analyzed 3 times.
1.3.8. Data processing of sake metabolome
Data processing by Yazawa et al. (Yazawa et al., 2019) was basically followed. Peakdetection, alignment peak data, and calculation of peak area were conducted with Peaklynx
XS Rev. 1.0 software (Waters, Milford, USA). Mass range and retention time range forpeak detection were determined by the error range of 14 selected peaks (precursor ions of
14 compounds) from the model sake samples (Table S1 (Chapter I Table S1.xlsx)). To
12

evaluate the accuracy of metabolome data, I analyzed model sake samples 7 times by using
6 to 12 samples per interval. The error range of each mass spectrum was calculated by 5fold standard deviation of each of the 14 selected peaks. The largest value among thesevalues was used as the error range for the peak-picking method of Peaklynx. The error
range of the retention time was also calculated as the mass spectrum error. Other settingsof the peak picking-method for metabolome data were as follows: initial and final retention
times of monitoring, 0 to 15 min; peak width at 5% height, 4 sec; intensity threshold ofpeak picking, 70; mass window, 0.01 Da; and retention time window, 0.3 min. Other
settings followed the manufacturer’s instruction. After making a peak table, preliminary
data processing was performed. Three-fold coefficient of variation (CV) was calculated
from the intensities of each peak top of the selected 14 peaks, and the maximum value of
3-fold CV among 14 peaks was used as the standard value. Then, the intensity of each peak
was used to calculate CV in each sake sample, and the maximum value (Max CV) was used
as CV of each peak. The peak(s) whose maximum CV was greater than the standard value
was eliminated further analysis. Metabolites were identified, depending on 496 standard
metabolite lists (Table S1 (Chapter I Table S1.xlsx)). The threshold of mass and retentiontime error for identification was 0.05 Da and 0.5 min, respectively.
1.3.9. Multiple comparison technique using metabolome data
The software JMP 13.1 (commercially available) was used throughout the analysis in thisstudy. To examine the difference in sake metabolites by rice cultivars in the small-scale
sake-making tests, Tukey's Honest Significant Difference (HDS) test was performed byusing the intensity of each peak. The P value was calculated by using the average of theintensity of each sample, and a value of less than 0.05 was regarded as indicating asignificant difference between rice cultivars.
1.4.

Results and Discussion

1.4.1. Effect of Koshitanrei on the sake metabolites in kake-rice sake-making tests
To investigate the effect of kake-rice (rice for kake-mai) on sake metabolites, I performed
the small-scale sake fermentation test using 3 rice cultivars (YAM, GOM, and KOS) of 2
polishing ratios (70% and 50%) as kake-rice and 1 rice cultivar (YAM of 40% polishingratio) as the koji-rice (rice for koji; Fig. 1(a, b), sample Nos.1-6).
In this small-scale sake-making test, the enzyme activities of the koji used,fermentation rates (CO2 decrement) during the test, and the general properties and flavor
13components of the obtained sake samples are shown in Table S2A (Chapter I Table S2.xlsx),
Fig. S1 (A and B) (Chapter I Fig S1.pptx), and Fig. 1(d), respectively. Fermentation rates(CO2 decrement) of the sake-making test using KOS as the kake-rice tended to be slower
than those obtained for the other 2 rice cultivars, YAM and GOM (Fig. S1, A and B(Chapter I Fig S1.pptx)). Sake meter (Sm) and alcohol (Alc) of the sake samples from KOSof both polishing ratios showed a lower value than those for the other 2 rice cultivars (Fig.
1(d)). In the sake sample from the rice of the 70% polishing ratio, the total acidity (Ta) andamino acidity (Aa) for KOS were higher and lower, respectively, than those for the other 2rice cultivars (Fig. 1(d), sample Nos. 1-3). In the sake samples from the rice of the 50%
polishing ratio, the concentrations of the flavor components ethyl acetate (EtOAc) andisoamyl acetate (iAmOAc) from KOS were lower than those from the other 2 rice cultivars(Fig. 1(d), sample Nos. 4-6).
Next, to investigate the effect of the kake-rice on sake metabolites, I constructed apeak table (Table S3 (Chapter I Table S3.xlsx)) containing all metabolome data of 1215
peaks, whose intensity was higher than the threshold of peak picking, as described inMaterials and Methods (Data processing of sake metabolome). Among 1215 peaks, I
identified 153 peaks (counting “number of ino(s) from one compound”), categorized 201
peaks, and among the identified 153 peaks, detected 84 compounds (counting “1” = “morethan one ion from one compound”) in 496 compounds (Table S1 (Chapter I Table S1.xlsx)),
based on the retention time and m/z of the ion(s) (P, precursor/parent ion; fx, in-source
fragment ion; derived from the compounds) in Table S1. I used this peak table (Table S3(Chapter I Table S3.xlsx)) for further analysis. At first, to investigate the character of eachsake sample, I performed principal component analysis (PCA) by using this peak table (Fig.
2(a)). As the result of PCA score plotting of the metabolome data from the “kake-rice sakemaking test,” the contribution ratios of PC1 and PC2 were 41.8% and 19.6%, respectively(Fig. 2(a)). On the PC1 axis, the sake samples from YAM and GOM of the “kake-rice sakemaking test” (sample Nos. 1, 2, 4, and 5) were clearly separated by the polishing ratio, withthe samples of 50% and 70% polishing ratio separated in a positive and negative direction,
respectively. On the other hand, unlike the samples of the other 2 rice cultivars of the 70%
polishing ratio (No.1 for YAM and No.2 for GOM), the sake sample from KOS of the 70%
polishing ratio (No. 3) was separated to the same area (in a positive direction on the PC1axis) as the samples of the 50% polishing ratio. These results suggested that, in this “kakerice sake-making test”, the components of sake samples from KOS were less affected by
14

the difference in the polishing ratio (70% or 50%).
Next, to explore the peak(s) affecting the classification on the axis in Fig.2(a), I
performed Tukey’s HSD (honest significant difference) test by using the constructed peaktable (Table S3 (Chapter I Table S3.xlsx)). In this analysis, I eliminated 121 peaks (theintensity 0 in some of sample Nos.1-6). As the result of this Tukey HSD test, by focusing
on the peaks in sake samples from KOS, I selected the peaks whose intensities were higher(p < 0.05; KOS > YAM, GOM) and lower (p < 0.05; KOS < YAM, GOM) than those forthe others (YAM and GOM). Regarding the selected peaks, Venn diagrams of the higher
peaks (KOS > YAM, GOM) and the lower peaks (KOS < YAM, GOM) are shown in Fig.
2(b) and Fig.2(c), respectively.
In the Venn diagrams of the higher peaks (KOS > YAM, GOM; Fig. 2(b)), thenumber of the higher peaks for the 70% polishing ratio only was 202 (I in Fig. 2(b)), thenumber of the higher peaks for the 50% polishing ratio only was 110 (III), and the numberof the higher peaks for both polishing ratios was 107 (II). The number of the peaks affectedby the 70% polishing ratio only (202/I in Fig. 2(b)) was about two-fold that of those affectedby the kake-rice of 50% polishing ratio only (110/III).
     In the Venn diagrams of the lower peaks (KOS < YAM, GOM; Fig. 2(c)), the numberof the lower peaks for the 70% polishing ratio only was 143 (I in Fig. 2(c)); that for the
50% polishing ratio only, 20 (III); and that for both polishing ratios was 53 (II). Thus, thenumber of the peaks affected by the 70% polishing ratio only (143/I in Fig. 2(c)) was about
7-fold that of the peaks affected by the kake-rice of 50% polishing ratio only (20/III). These
results suggested that, in the “kake-rice sake-making test,” the difference in sakecomponents between KOS and the others at the 70% polishing ratio was bigger than that insake components between KOS and the others at the 50% polishing ratio.
Further, I categorized or identified the metabolites of the KOS-affected peaks byusing the metabolite list (Table S1 (Chapter I Table S1.xlsx)). The results (Fig. 2(b))
showed that the numbers of the identified/categorized higher peaks for KOS were 16/25
among 202 for the 70% polishing ratio only, 27/38 among 110 for the 50% polishing ratio
only, and 8/16 among 107 for both polishing ratios. On the other hand, the numbers of theidentified/categorized lower peaks for KOS were 28/38 among 143 for the 70% polishingratio only, 4/4 among 20 for the 50% polishing ratio only, and 9/9 among 53 for bothpolishing ratios (Fig. 2(c)). The metabolites (ID No. Compound_detected ion, in Table S1(Chapter I Table S1.xlsx)) of the identified peaks (Fig. 2(b) and 2(c)) are indicated in Tables
15

1 and 2. The contents of alpha-etylglucoside (D-EG) in the sake samples from KOS of bothpolishing ratios were lower than those in the samples from the other 2 rice cultivars (Table
2, II, 8 peaks). It should be noted that 1 peak (D-EG_f1, Table 1, I) of D-EG for the 70%
polishing ratio only showed behavior opposite to that of the other 8 peaks (Table 2, II).
1.4.2. Effect of Koshitanrei on the sake metabolites in total rice (kake+koji-rice)sake-making tests
To investigate the effect of the total rice variation on sake metabolites, I performed thesmall-scale sake fermentation test using 3 rice cultivars (YAM, GOM, and KOS) of 2
polishing ratios (70% and 50%) as kake- and koji-rice (Fig. 1(a, c), sample Nos. 7-12).
In this small-scale test, the enzyme activities of the used koji, fermentation rates (CO2decrement) during the test, and the general properties and flavor components of theobtained sake samples are shown in Table S2B (Chapter I Table S2.xlsx), Fig. S1 (C andD) (Chapter I Fig S1.pptx), and Fig. 1(d), respectively. In the rice of the 70% polishingratio, the 4 enzyme activities examined in the koji from KOS were lower than those in kojifrom the other rice cultivars (Table S2B (Chapter I Table S2.xlsx), sample Nos. 7-9). In therice of the 50% polishing ratio, glucoamylase of koji from KOS tended to be lower thanthose from the other rice cultivars (Table S2B (Chapter I Table S2.xlsx), sample Nos. 1012). Fermentation rates (CO2 decrement) in the sake fermentation test using KOS of the 2polishing ratios were the intermediate between those for GOM and YAM (Fig. S1, C and
D (Chapter I Fig S1.pptx)). In the sake sample from the rice of both polishing ratios, thesake meter (Sm) and total acidity (Ta) or amino acidity (Aa) values for KOS were higher
and lower, respectively, than those from the other rice cultivars (Fig. 1(d), sample Nos. 712). The concentrations of flavor components ethyl acetate (EtOAc) and isoamyl acetate(iAmOAc) in sake from KOS of the 50% polishing ratio were higher than those from YAM
or GOM (Fig. 1(d), sample Nos. 10-12).
Next, to investigate the effect of the total rice variation on sake metabolites, as in
the case of the “kake-rice sake-making test”, I constructed a peak table (Table S3 (Chapter
I Table S3.xlsx)) containing all metabolome data of 1215 peaks and used it for further
analysis. At first, to investigate the character of each sake sample, I performed the PCA byusing this peak table (Fig. 3(a)). The results of PCA score plotting of the metabolome datafrom the “kake+koji-rice sake-making test,” the contribution ratios of PC1 and PC2 were
37.9% and 35.0%, respectively (Fig. 3(a)). On the PC1 axis, the sake sample Nos. 7-12
were clearly separated by the polishing ratio; and the samples of 70% polishing ratio
16

(Nos.7-9) and those of the 50% one (Nos.10-12) were separated in a positive and negative
direction, respectively (Fig. 3(a)). On the other hand, on the PC2 axis, unlike the other 2rice cultivars, the samples from KOS (Nos. 9 and 12) were concentrated in a negative
direction (Fig. 3(a)). A comparison of the results of the “kake+koji-rice sake-making test”
with those of the “kake-rice sake-making test” suggested that the difference in the effect ofthe polishing ratio on the sake components from total rice of KOS (Nos. 9 and 12, in Fig.
3(a)) was bigger than that on those from kake-rice of KOS (Nos. 3 and 6, in Fig. 2(a)).
Next, as was the case for the kake-rice test, to explore the peaks affecting the
distribution on the axis in Fig.3(a), I performed Tukey’s HSD test using the constructedpeak table (Table S3 (Chapter I Table S3.xlsx)). In this analysis, I eliminated 92 peaks (theintensity 0 in some of sample Nos.7-12). By focusing on the peaks in sake samples from
KOS, I selected the peaks whose intensities were higher (p < 0.05; KOS > YAM, GOM)
or lower (p < 0.05; KOS < YAM, GOM) than those for the other rice cultivars. Using dataon the selected peaks, I prepared Venn diagrams of the higher peaks (KOS > YAM, GOM)
and lower peaks (KOS < YAM, GOM), Fig. 3(b) and Fig. 3(c), respectively.
In the Venn diagrams of the higher peaks (KOS > YAM, GOM; Fig. 3(b)), thenumber of the higher peaks for the 70% polishing ratio only was 23 (I in Fig. 3(b)); that for
the 50% polishing ratio only, 237 (III); and that for both ratios, 78 (II). The number of thepeaks affected by 50% polishing ratio only (237/III in Fig. 3(b)) was about 10-fold that ofthose affected by kake-rice of the 70% polishing ratio only (23/I in Fig. 3(b)). In the Venn
diagrams of lower peaks (KOS < YAM, GOM; Fig. 3(c)), the number of the lower peaks
for the 70% polishing ratio only was 70 (I in Fig. 3(c)); that for the 50% polishing ratio
only, 118 (III); and that for both polishing ratios, 289 (II).
The number of the peaks affected by the 50% polishing ratio only (118/III in Fig.
3(c)) was 1.7-fold that of those affected by the kake-rice of the 70% polishing ratio only
(70/I in Fig.3(c)). In the case of the sake components from KOS of the “kake+koji sakemaking test,” the number of peaks (higher and lower) affected by rice cultivars of the 50%
polishing ratio only was higher than that of those of the 70% polishing ratio only (Fig. 3(b),I-23 vs. III-237; Fig. 3(c), I-70 vs. III-118). These results suggested that, in the “kake+kojirice (total rice) sake-making test,” the difference in the effect on sake components between
KOS and the others at the 50% polishing ratio was bigger than that in the effect on sakecomponents between KOS and others at the 70% polishing ratio. When the results of the“kake-rice sake-making test” were compared with those of the “kake+koji-rice sake-making
17

test,” the number of lower peaks for KOS of both 70% and 50% polishing ratios was 5.5fold higher in the latter than in the former (Fig. 2(c)-II/53 vs. Fig. 3(c)-II/289).
Further, I categorized or identified the metabolites of the KOS-affected peaks byusing the metabolite list (Table S1 (Chapter I Table S1.xlsx)). As shown in the results (Fig.
3(b)), the numbers of the higher identified/categorized peaks for KOS were 1/1 among 23
for the 70% polishing ratio only, 23/26 among 237 for the 50% polishing ratio only, and13/17 among 78 for both polishing ratios. On the other hand, the numbers of the lower
identified/categorized peaks for KOS were 3/5 among 70 for the 70% polishing ratio only,
14/17 among 118 for the 50% polishing ratio only, and 64/90 among 289 for both polishingratios (Fig. 3(c)). The metabolites (ID No. Compound_detected ion, in Table S1 (Chapter
I Table S1.xlsx)) of the identified peaks (Fig. 3(b) and 3(c)) are indicated in Tables 3 and4. The higher peaks in the sake sample from KOS were D-EG and citric acid for both 70%
and 50% polishing ratios, and malic acid for the 50% polishing ratio (Table 3). On the other
hand, the lower peaks in the sake sample from KOS were amino acids and dipeptides (Table4). In the “kake-rice sake-making test”, the D-EG concentration of the sake sample from
KOS was lower than that for the other rice cultivars (Table 2, II), but in the “kake+koji-ricesake making test,” the α-EG concentration of the sake sample from KOS was higher thanthat for the other rice cultivars (Table 3, II). It should be noted that 1 peak (D-EG_f1, Table
4, III) of D-EG for the 50% polishing ratio only showed a behavior opposite to that of theother 6 peaks (Table 3, II).
1.4.3. Taste- and aroma-related sake metabolites affected by KOS
Various components including carbohydrates, organic acids, amino acids, and inorganic
ones are related to the taste and aroma of sake (Brewing Society of Japan, 1999). Therefore,
I focused on the components related to the taste and aroma of sake among the identified
metabolites/components affected by KOS (Tables 1-4). Previously it was reported that theamino acids arginine, alanine, glutamic acid, and aspartic acid, are related to the taste ofsake (Iwano et al., 2004). Among these 4 amino acids, in the 2 types of small-scale sakefermentation test (“kake-rice sake-making test” and “kake+koji-rice sake-making test”), 2
amino acids (glutamic acid and aspartic acid) showed characteristic behavior in the sakefrom KOS compared to that of those from the other 2 rice cultivars. In the “kake+koji-rice(total rice) sake-making test” using the rice of both 70% and 50% polishing ratios, the levelsof these 2 amino acids, glutamic acid and aspartic acid, in the sake from KOS were lower
than those in that from the other rice cultivars (Nos. 9 and 12, in Fig. 4(a) and 4(b)).
18

Similarly, in the “kake-rice sake-making test” using the rice of the 70% polishing ratio,these 2 amino acids in the sake from KOS were lower in concentration than in the sakemade from the other rice cultivars (No. 3 in Fig. 4(a) and 4(b)). The clear taste of the sakefrom KOS may be attributed to the lower content of these 2 amino acids.
It has been reported that the major component of “hineka”, one of the unpalatableflavors in sake, is dimethyl tri-sulfide (DMTS) (Isogai et al., 2006) and that the productionof 1,2-dihydroxy-5-(methylsulfinyl)prentan-3-one (DMTS-P1), the precursor of DMTS, isrelated to the methionine salvage pathway genes in Saccharomyces cerevisiae(Wakabayashi et al., 2013). Therefore, since sulfur-containing amino acids are related tothe methionine salvage pathway, I next focused on methionine and cysteine to determine ifthey would be affected by KOS (Fig. 4 (c) and 4(d)). The content of methionine in the sakefrom KOS in the 2 types of small-scale sake fermentation test (“kake-rice sake-making test”
and “kake+koji-rice sake-making test”) tended to be lower than that in sake from the otherrice cultivars (Nos.3, 6, 9, and 12 in Fig. 4(c)). Also, the cysteine content in the sake from
KOS in the “kake+koji-rice sake-making test” was lower than that in the sake made fromthe other rice cultivars (Nos.9 and 12 in Fig. 4(d)). These results suggested that the lower
contents of sulfur-containing amino acids in the sake from KOS would prevent
deterioration of the sake’s quality.
Next, as carbohydrate is also related to the taste of sake, I focused on the D-EG
content in the sake from KOS. In the “kake-rice sake- making test,” the content of D-EG,
responsible for the bitterness following the sweet taste (Oka and Sato, 1976), in the sakefrom KOS at either the 70% or 50 % polishing level was lower than that in the sake madefrom the other rice cultivars (Nos.3 and 6 in Fig. 4(e)). In contrast, in the “kake+koji-ricesake-making test” at both 70% and 50% polishing ratios, the D-EG contents in sake from
KOS were higher than those from the other rice cultivars (Nos.9 and 12 in Fig. 4(e)). These
results suggest that the D-EG content of sake from KOS was influenced by the koji andsupport the PCA results, which showed that the use of KOS as the koji-rice caused a greater
diversity of the sake peaks/metabolites (compare between “sample Nos. 9 and 12 in Fig.
3(a)” and “sample Nos. 3 and 6 in Fig. 2(a)”).
In the PCA results from the “kake-rice sake-making test” (Fig. 2(a)), the sake samples
from KOS of the 70% polishing ratio (sample No. 3) were located near the sake samples
from the other rice cultivars of the 50% polishing ratio (sample Nos.4-6). Further, in the
PCA results obtained from the “kake+koji-rice sake-making test” (Fig. 3(a)), the sake19

samples from KOS of both 70% and 50% polishing ratios (No. 9 and 12) were separated ina negative direction on the PC2 axis, unlike those from the other rice cultivars, which wereseparated in a positive direction on this axis. These results were also supported by cluster
analysis (data not shown).
Currently, although the number of identified peaks was just a few, I detected many
unknown peaks affected by KOS in this study. Indeed, 3 unknown peaks (RT, m/z: 1.254,
62.0608; 2.539, 287.1495; 1.252, 431.1168) were detected only in the sake from KOS(intensity 0 in the sake from others) in the total rice sake-making tests (sample Nos. 7-12).
For future clarification of the characteristics of KOS, it will be important to identify
unknown peaks affected by KOS.

20

(a)
Sample
No.
1
2
3
4
5
6
7
8
9
10
11
12

Rice cultivar (Polishing ratio %)kake-ricekoji-rice
YAM (70)
GOM (70)
KOS (70)
YAM (40)
YAM (50)
GOM (50)
KOS (50)
YAM (70)
YAM (70)
GOM (70)
GOM (70)
KOS (70)
KOS (70)
YAM (50)
YAM (50)
GOM (50)
GOM (50)
KOS (50)
KOS (50)

Koji starter

Yeast strain

Byakuya

K701

ByakuyaK701

YAM (Yamadanishiki), GOM (Gohyakumangoku), KOS (Koshitanrei)
Total rice 80g

kake-rice(b)

Sample No.

YAM

GOM

KOS

Koji 40%

Koji 40%

Koji 40%

Koji 40%

Koji 40%

Koji 40%

kake 70%

kake 70%

kake 70%

kake 50%

kake 50%

kake 50%

1

2

3

4

5

6

(c)

Sample No.

kake+koji-ricekoji 70%koji 70%koji 70%koji 50%

koji 50%

koji 50%

kake 70%

kake 70%

kake 70%

kake 50%

kake 50%

kake 50%

9

10

11

12

7

8

(d)
General properties

Sample
No.

Sm

Alc
(%)

Ta
(ml)

1
2
3
4
5
6
7
8
9
10
11
12

-6
-4
-13
-16
-9
-24
-19
-9
-7
-34
-23
-19

19.0
19.1
17.6
18.1
17.8
16.0
18.3
20.8
19.9
16.4
17.6
18.0

2.5
2.7
3.3
3.1
3.3
3.3
3.9
3.4
3.1
3.9
3.7
3.4

Flavor components (ppm)
Aa
(%)
2.6
2.5
2.0
1.7
1.7
1.7
2.7
2.7
2.0
2.1
2.0
1.4

21

EtOAc iAmOAc iAmOH
110
149
120
138
130
97
89
127
112
75
93
109

7.4
10.7
7.4
11.0
10.0
5.5
3.9
6.9
5.1
3.9
4.5
7.6

206
199
170
171
196
149
143
169
143
132
144
149

EtOCap
1.7
1.8
1.2
1.7
1.3
0.9
1.1
1.5
1.4
1.0
0.9
1.5

Fig. 1. Combination of sake-making parameters and properties of sake samples analyzed
in the small-scale sake fermentation test.
(a), Sake-making parameters, such as rice cultivar (YAM, Yamadanishiki; GOM,
Gohyakumangoku; and KOS, Koshitanrei) and polishing ratio (%) of kake- and koji-rice,koji starter, and yeast strain, are indicated. (b), Three rice cultivars (YAM/blue, GOM/green,
and KOS/red) of 2 polishing ratios (70% and 50%) for kake-rice and one common ricecultivar (YAM, 40% polishing ratio) for koji-rice were used in this test (total rice, 80 g; 16
g for koji-rice and 64 g for kake-rice). This test (sample Nos. 1-6) was referred to as the“kake-rice sake-making test” in this study. (c), Three rice cultivars (YAM, GOM, and KOS)of 2 polishing ratios (70% and 50%) were used for both kake- and koji-rice in this smallscale sake fermentation test (total rice, 80 g; 16 g for koji-rice and 64 g for kake-rice). This
test (sample Nos. 7-12) was referred to as the “kake+koji-rice sake-making test”. (d),General properties and flavor components of sake samples. General properties (Sm, sakemeter; Alc, alcohol content; TA, total acidity; AA, amino acidity) and flavor components(EtOAc, ethyl acetate; iAmOH, isoamyl alcohol; iAmOAc, isoamyl acetate; EtCap, ethyl
caproate) of sake samples were analyzed by the standard method established by the
National Tax Agency of Japan. All data are indicated as the averages for 2 independent
tests.

22

(a)

YAM
GOM

Polishing ratio (Sample No.)
70%(1), 50%(4)70%(2),
50%(5)

KOS

70%(3),

50%(6)

50
40

PC2 (19.6%)

30
20
10
-50

-40

-30

-20

0
-10-10 0

10

20

30

40

50

-20
-30
-40

-50
PC1 (41.8%)(b)

KOS > YAM , GOM( Table 1 )kake-rice 70%(1, 2, 3)

I

II

III

202
(16/25)107(8/16)

110(27/38)kake-rice 50%(4, 5, 6)

No. of peak
( Identified / Categorized )KOS < YAM , GOM(c)( Table 2 )kake-rice 70%(1, 2, 3)

I

II

III

143
(28/38)

53
(9/9)

20
(4/4)

kake-rice 50%(4, 5, 6)

Fig. 2. Analysis of sake metabolites in the kake-rice sake-making test.
(a), PCA score plots of metabolome data (3 times analyses of each sample) from 2
independent kake-rice sake-making tests (sample Nos. 1-6 in Fig. 1). Blue-triangle, greencircle, and red-square marks indicate 3 rice cultivars, YAM, GOM, and KOS, respectively.
Filled and open marks indicate sample Nos. 1-3 (kake-rice of 70% polishing ratio) and23

sample Nos. 4-6 (kake-rice of 50% polishing ratio), respectively. The dotted circle indicatesthe located area for the samples from KOS. (b), Venn diagrams of the peaks (KOS > YAM,
GOM), whose intensity in sake made from KOS (kake-rice of 70% and 50% polishingratios) was higher (p < 0.05) than that in sake made from the other 2 rice cultivars, YAM
and GOM (kake-rice of 70% and 50% polishing ratios). The numbers in the Venn diagrams
indicate the numbers of peaks (number of the identified/categorized peaks). The
metabolites (ID No. Components) of the identified peaks are shown in Table 1. (c), Venn
diagrams of the markers (KOS < YAM, GOM), whose intensity in sake made from KOS(kake-rice of 70% and 50% polishing ratios) was lower (p < 0.05) than that in sake madefrom the other 2 rice cultivars, YAM and GOM (kake-rice of 70% and 50% polishing ratio).
The metabolites (ID No. Components) of the identified peaks are shown in Table 2.

24

Table 1. The metabolites (ID No. component) of the identified peaks in Fig. 2B (KOS >YAM, GOM).

CategoryI
nitrogen compound 258 Agmatine_f1
385 L-DOPA _P

KOS > YAM, GOM
IIIII

224 L-Cysteine_f1
232 L-Histidine_(2; P, f1)
233 L-Phenylalanine_f1
234 L-Arginine_f1
236 L-Tyrosine_(4; P, f1, f3, f4)
257 beta-Phenylethylamine_(2; P, f1)
dipeptide

470 Ala-Phe_P
474 Leu-Val_P
519 Gln-Tyr_P
523 Ala-Gln_P
526 Val-Phe _P
564 Glu-Ala_P
567 Phe-Asp_P
503 Gln-Gln_P
533 Asp-Glu_P
572 Glu-Glu_P
483 Gly-Thr_(2; P, f2)
484 Gly-Trp_P
490 Gly-Phe_P
491 Gly-Pro_P
494 Gly-Tyr_P
525 Val-Gln_P
527 Leu-Phe_P
530 Pro-Gln_P
536 Gln-Leu_(2; P, f1)

carbohydrate

12 alpha-Ethylglucoside_f1
21 Raffinose_f2
367 Melibiose_f16
21 Raffinose_(2; f3, f10)organic acid

195 n-butyric acid_f4
209 Succinic acid_(3; P, f1, f2)
179 Gluconolactone_f4
157 Vanillic acid_P

vitamineester

318 Inositol_f5
67 Ethyl 3-hydroxybutyrate_f7
463 Ethyl-glucopyranoside_f6

carbohydrate alcohol 2 Erythritol_P
nucleic acidother
456 Phosphoric acid_P

273 Inosine_f3

The intensity of the peaks in sake made from KOS (kake-rice of 70% and 50% polishingratio) was higher (p < 0.05) than that of it for the sake made from the other 2 rice cultivars,
YAM and GOM (kake-rice of 70% and 50% polishing ratio). Identified peaks (ID No.
Compound_detected ion, Table S1) at 70% polishing ratio only (I), at both 70% and 50%
polishing ratios (II), and at 50% polishing ratio only (III) are indicated. The detected ions
are indicated as P, precursor/parent ion; fx, in-source fragment ion; more than one (No. of
detected ions; kind of ions).
25

Table 2. The metabolites (ID No. Compound_detected ion) of the identified peaks in Fig.
2(c) (KOS < YAM, GOM).

KOS < YAM, GOM

Category

I

II

III

nitrogen compound 218 L-Alanine_P
220 L-Serine_f2
223 L-Threonine_f2
226 L-Leucine_(2; P, f1)
227 L-Ornithine_f2
228 L-Aspartic acid_(3; f1-3)
229 L-Lysine_f1
230 L-Glutamic acid_P
231 L-Methionine_(2; f1, f2)
232 L-Histidine_f1
dipeptide
502 Leu-Leu_P
513 Val-Val_(2; P, f1)
522 Ser-Val_f2
531 Ala-Thr_P
574 His-Leu_P
carbohydrateorganic acid522 Ser-Val_f1
12 alpha-Ethylglucoside_(8; P, f4, f6-11)
174 Citramalic acid_(3; P, f6, f7)
206 Pyroglutamic acid_(2; P, f2)
190 Malic acid_(4; P, f2-4)

ester
nucleic acid

414 N-(2-phenylethyl)acetamide_f3
263 Uracil_P
266 Hypoxanthine_P

The intensity of the peaks in sake made from KOS (kake-rice of 70% and 50% polishingratio) was lower (p < 0.05) than that in sake made from the other 2 rice cultivars, YAM andGOM (kake-rice of 70% and 50% polishing ratio). Identified compounds (ID No.
Compound_detected ion, Table S1) at 70% polishing ratio only (I), at both 70% and 50%
polishing ratios (II), and at 50% polishing ratio only (III) are indicated. The detected ions
are indicated as Table 1.

26

(a)

YAM
GOM
KOS

Polishing ratio (Sample No.)
70%(7), 50%(10)70%(8),
50%(11)
70%(9),
50%(12)40

PC2 (35.0%)

30

20
10
0
-40

-30

-20

-10

0

-10

10

20

30

40

-20
-30

-40
PC1 (37.9%)(b)

KOS > YAM , GOM( Table 3)

kake+koji-rice 70%(7, 8, 9)

I

II

III

23
(1/1)

78
(13/17)237(23/26)

kake+koji-rice 50%(10, 11, 12)

No. of peak
( Identified / Categorized )(c)

KOS < YAM , GOM( Table 4 )kake+koji-rice 70%(7, 8, 9)

I

II

III

70
(3/5)

289
(64/90)118(14/17)

kake+koji-rice 50%(10, 11, 12)

Fig. 3. Analysis of kake+koji-rice sake-making test.
(a), PCA score plots of metabolome data (3 times analyses of each sample) from 2
independent kake+koji-rice sake-making tests (sample Nos. 7-12 in Fig. 1). Blue-triangle,
green-circle, and red-square marks indicate 3 rice cultivars, YAM, GOM, and KOS,
respectively. Filled and open marks indicate sample Nos. 7-9 (total rice of 70% polishing27

ratio) and sample Nos. 10-12 (total rice of 50% polishing ratio), respectively. The reddotted ellipse indicates the located area for the samples from KOS. (b), Venn diagrams of
the peaks (KOS > YAM, GOM), whose intensity in sake made from KOS (total rice of
70% and 50% polishing ratios) was higher (p < 0.05) than that in sake made from the other2 rice cultivars, YAM and GOM (total rice of 70% and 50% polishing ratios). The numbers
in Venn diagrams indicate the numbers of peaks (number of the identified/categorized
peaks). The metabolites (ID No. Components) of the identified peaks are shown in Table
3. (c), Venn diagrams of the peaks (KOS < YAM, GOM), whose intensity in sake madefrom KOS (total rice of 70% and 50% polishing ratio) was lower (p < 0.05) than that insake made from the other 2 rice cultivars, YAM and GOM (total rice of 70% and 50%
polishing ratio). The metabolites (ID No. Components) of the identified peaks are shownin Table 4.

28

Table 3. The metabolites (ID No. Compound_detected ion) of the identified peaks in Fig.
3(b) (KOS > YAM, GOM).

Category
dipeptidecarbohydrate

KOS > YAM, GOM
I

II

III
572 Glu-Glu_P

12 alpha-Ethylglucoside_f10
12 alpha-Ethylglucoside_(6; P, f6-9, f11)
19 Panose_f3
366 D-Maltotriose_f1
12 alpha-Ethylglucoside_f4
21 Raffinose_(2; f5, f10)organic acid

157 Vanillic acid_P
175 Citric acid_(4; f1, f2, f4, f5)
172 Adipic acid_f5
174 Citramalic acid_(4; P, f5-7)
175 Citric acid_f3
190 Malic acid_(4; P, f2-4)
195 n-butyric acid_f4
67 Ethyl 3-hydroxybutyrate_(4; P, f2, f5, f6)
77 Ethyl lactate_P
149 p-Hydroxybenzoic acid_P
269 Thymidine_f5
53 Tryptophol_f1

ester
phenol compound
nucleic acidalcohol

The intensity of the peaks in sake made from KOS (total rice of 70% and 50% polishingratios) was higher (p < 0.05) than that in sake made from the other 2 rice cultivars, YAM
and GOM (total rice of 70% and 50% polishing ratios). Identified compounds (ID No.
Compound_detected ion, Table S1) at 70% polishing ratio only (I), at both 70% and 50%
polishing ratios (II), and at 50% polishing ratio only (III) are indicated. The detected ions
are indicated as Table 1.

29

Table 4. The metabolites (ID No. Compound_detected ion) of the identified peaks in Fig.
3(c) (KOS < YAM, GOM).

Category

KOS < YAM, GOM
Initrogen compoundII
220 L-Serine_f2
223 L-Threonine_f2
224 L-Cysteine_f1
226 L-Leucine_(2; P, f1)
228 L-Aspartic acid_(3; f1-3)
229 L-Lysine_(2; P, f1)
230 L-Glutamic acid_P
231 L-Methionine_(2; f1, f2)
232 L-Histidine_(2; P, f1)
233 L-Phenylalanine_f1
236 L-Tyrosine_(4; P, f1, f3, f4)
257 beta-Phenylethylamine_(2; P, f1)
258 Agmatine_f3

III

227 L-Ornithine_f2
dipeptide

553 Phe-Pro_P
566 Lys-Asn_(2; f1, f4)
466 Ala-Trp_P
468 Ala-Leu_f2
470 Ala-Phe_P
474 Leu-Val_P
483 Gly-Thr_f2
484 Gly-Trp_P
490 Gly-Phe_(2; P, f3)
491 Gly-Pro_P
494 Gly-Tyr_P
498 Leu-Tyr_P
502 Leu-Leu_P
503 Gln-Gln_P
513 Val-Val_(2; P, f1)
523 Ala-Gln_P
525 Val-Gln_P
526 Val-Phe_P
527 Leu-Phe_P
531 Ala-Thr_P
532 Leu-Lys_f1
533 Asp-Glu_P
536 Gln-Leu_P
542 Asn-Ile_P
558 Arg-Val_(3; P, f2, f3)
564 Glu-Ala_P
565 Asp-Ser_P
566 Lys-Asn_(2; P, f2)
574 His-Leu_P
526 Val-Phe_f2
530 Pro-Gln_P
532 Leu-Lys_f2
536 Gln-Leu_f1
566 Lys-Asn_f3
12 alpha-Ethylglucoside_f1
367 Melibiose_f16

carbohydrateorganic acid

209 Succinic acid_f1
179 Gluconolactone_f4
209 Succinic acid_(3; P, f2, f3)

ester

414 N-(2-phenylethyl)acetamide_(2; P, f3)
463 Ethyl-glucopyranoside_f6
67 Ethyl 3-hydroxybutyrate_f7

carbohydrate alcohol
nucleic acid

2 Erythritol_P
271 Uridine_(2; P, f6)
273 Inosine_f3

other

456 Phosphoric acid_P

266 Hypoxanthine_P30

The intensity of the peaks in sake made from KOS (total rice of 70% and 50% polishingratios) was lower (p < 0.05) than that in sake made from the other 2 rice cultivars, YAM
and GOM (total rice of 70% and 50% polishing ratios). Identified compounds(ID No.
Compound_detected ion, Table S1) at 70% polishing ratio only (I), at both 70% and 50%
polishing ratios (II), and at 50% polishing ratio only (III) are indicated. The detected ions
are indicated as Table 1.

31

(b) 230 L-Glutamic acid_P
60000

2500

50000

2000

40000

Intensity

Intensity(a) 228 L-Aspartic acid_f 1
3000

1500

1000

30000
20000

10000

500

0

0
1

2

3

4

5

7 8
6
Sample No.

91

10 11 12

4000

Intensity

Intensity

5000

3000
2000
1000

0
2

3

4

5

6
7 8
Sample No.

3

4

5

6
7 8
Sample No.

9

10 11 129

10 11 12(d) 224 L-Cysteine_f 1(c) 231 L-Methionine_f 1
6000

1

2

9

10 11 129

10 11 12

1800
1600
1400
1200
1000
800
600
400
200
0
1

2

3

4

5

6
7 8
Sample No.

Intensity(e) 12 alpha-Ethylglucoside_f 8
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
1

2

3

4

5

6
7 8
Sample No.

Fig. 4. Typical metabolites (ID No. Compound_detected ion) affected by Koshitanrei.
Intensity of (a) L-Aspartic acid_f1 (RT, 1.2439; m/z, 116.0346), (b) L-Glutamic acid_P(RT, 1.3348; m/z, 148.0609), (c) L-Methionine_f1 (RT, 2.6324; m/z, 133.0306), (d) LCysteine_f1 (RT, 1.2018; m/z, 241.0325), and (e) alpha-Ethylglucoside_f8 (RT, 5.1417;
m/z, 145.0499) are indicated.

32

CHAPTER ,,Effect of koji starter on metabolites in Japanese alcoholic beverage sake made fromthe sake rice Koshitanrei

33

2.1.

Abstract

In sake brewing, the steamed rice is used in 2 ways, added to sake-mash and making ricekoji. Rice-koji is made from the steamed rice by using koji starter, and its quality is an
important determinant of the aroma/taste of sake. The sake rice Koshitanrei (KOS) was
developed in Niigata Prefecture by crossing 2 sake rice varieties, Gohyakumangoku andYamadanishiki. Recently, we reported the characteristic components/metabolites in sakemade from KOS by conducting metabolome analysis using UPLC-QTOF-MS. In this study,
to investigate the effect of koji starter and sake rice cultivars on the sake metabolites, I
performed small-scale sake-making tests using the above 3 rice cultivars and 3 koji starters.
Finally, I demonstrated that some of the characteristic components/metabolites of sakefrom KOS are affected by the koji starter. Thus, in addition to rice cultivar, koji starter plays
an important role for establishment/maintenance of the quality of the final product.
2.2.

Introduction

The Japanese alcoholic beverage sake is made from rice and water by using 2
microorganisms, Aspergillus oryzae (called koji-mold) and the yeast Saccharomyces
cerevisiae (Yoshizawa, 1999). The main raw material for sake brewing is the rice, and thesteamed rice is used in 2 ways, directly added to sake mash (as kake-rice) and used to makekoji. In order to produce the specific taste of the sake, various rice cultivars have been used
for sake brewing. Breeding of the sake rice cultivars has been done in many areas of Japan(Maeshige and Kobayashi, 2000). In Niigata Prefecture, the originally developed sake rice
Gohyakumangoku (GOM) has been used widely. GOM has contributed to high-quality sakehaving a clear taste, although the hardness of its grains is insufficient for high polishing(especially to less than the 50% polishing ratio for Daiginjo-shu), due to the shape and sizeof its white core (Anzawa et al.,. 2013). To improve this polishing property of GOM, thesake rice Koshitanrei (KOS) was originally developed in Niigata Prefecture by crossing
GOM with Yamadanishiki (YAM) (Kobayashi et al., 2006). Anzawa et al. (Anzawa etal.,. 2013) reported the polishing/brewing properties of KOS determined by using a wide
range of polished rice and showed differences in the general properties of the sake between
that made from KOS and that from GOM.
In sake brewing, koji making is one of the important processes, because koji is adeterminant of the taste and flavor component in sake. The significance aspects of koji are
its being the source of diastatic enzyme, the nutrient source for yeast cell growth, and the34source of the component(s) affecting sake quality. The quality of koji is determined by the
production process, rice cultivar, and koji starter.Ito et al. (Ito et al.,. 2013) reported thatamino acid in koji is affected by the rice cultivar used to make koji and koji starters. Also,
Takahashi et al. (Takahashi et al.,. 2008) reported that the activity of each enzyme isaffected by the rice cultivar used to make koji and koji starters. Further, Yanagiuchi et al.
(Yanagiuchi et al.,. 1993) reported that enzyme activity and enzyme balance(glucoamylase and acid carboxypeptidase) are also affected by the rice cultivar used tomake koji and koji starters and that the fatty acid composition of koji and amino acidcomposition of sake are affected by koji starters. Therefore, the selection of the koji starterfor the rice cultivar is important.
The metabolomics technique is an analysis method examining cyclopaedically/coinstantaneously a number of intravital metabolites. Recently, this type of analysis has been
applied in various fields with diversification (Putri et al., 2013a; Putri et al., 2013b).
Previously, in the analysis of sake done by using the metabolomics technique CE-TOF MS,
Sugimoto et al. (Sugimoto et al., 2010) reported a correlation between sensory evaluation
scores of sake and metabolome profiles, as well as a difference in the charged metabolites
between pasteurized and unpasteurized sake during storage (Sugimoto et al., 2012). In thesake analysis using GC/MS, Mimura et al. (Mimura et al., 2014) examined therelationship between the features of sake and the component profile; and Tamada et al.
(Tamada et al., 2017) reported the characteristics of sake according to the sake-making
parameters (sake yeast, rice cultivars) assessed by GC/MS and analyzed the correlation
between the component profiles of GC/MS and the intensity of “Oshi-aji (Tamada et al.,2018). Further, in the sake analysis using 2-dimensional GC with TOF-MS, Takahashi etal. (Takahashi et al., 2016) examined the correlation between sake components andorganoleptic properties; and Tokuoka et al. (Tokuoka et al., 2017) used HILIC-TOF-MS
to analyze the oligosaccharide composition of sake. Recently, Yazawa et al. (Yazawa etal., 2019) conducted sake metabolome analysis using UPLC-QTOF-MS (ultraperformance liquid chromatography/time-of-flight mass spectrometry) and examined therelationship between sake-making parameters and sake metabolites. In their study, theinfluence of the rice cultivar used as rice-koji was also analyzed and found to have asignificant

effectonsakequality.

Recently,I

reportedthecharacteristic

components/metabolites in sake made from KOS by using the method of Yazawa et al.
(Yazawa et al., 2019) (Ichikawa et al. (Ichikawa et al., 2019)). However, the effect of35

the koji starter on the metabolites in the sake made from KOS was not analyzed at that time.
In this present study, in order to investigate the effect of the koji starter on the sakemetabolites in the sake made from KOS, I performed small-scale sake fermentation tests
using 3 rice cultivars: KOS, YAM, and GOM, and 3 koji starters (Yoikaori/Y, High G/H,
and Byakuya/B). I analyzed the sake metabolome data obtained from small-scale sakefermentation tests by using the recently developed sake metabolome analysis method;(Yazawa et al., 2019) and I also investigated the effect of koji starters on the characteristic
sake metabolites whose contents in sake made from KOS were higher/lower than thosefrom the other 2 cultivars, YAM and GOM. Finally, I demonstrated that the characteristic
components/metabolites of sake made from KOS were affected by the koji starter.
2.3.

Materials and Methods

2.3.1. Rice
Rice cultivars used for the small-scale sake fermentation test were the following (Table 5):
YAM (grown in Hiroshima Prefecture), GOM (grown in Niigata Prefecture), and KOS(grown in Niigata Prefecture), all harvested in 2016. These rice cultivars were the same as
those used in a previous study (Ichikawa et al., 2019). The rice grains were polished to apolishing ratio of 50% by using a milling machine (RP-5 and NF-26FA, Shin-Nakano
Industry, Japan).
2.3.2. Small-scale koji making
Small-scale koji making was done as previously reported (Ichikawa et al., 2019). The
koji starters used for the small-scale koji making were as follow (Table 5); Yoikaori/Y (Bioc,
Japan), High G/H (Higuchi Matsunosuke Shoten, Japan), and Byakuya/B (Hishiroku,
Japan).
2.3.3. Small-scale sake-making test
Three rice cultivars (YAM, GOM, and KOS) each with a 50% polishing ratio were used
for both kake-rice and koji-rice (Table 5). In the sake-making test, 500 g of total rice (100g for koji-rice and 400 g for kake-rice) was used for three-step addition to the sake mash as
follows.
As the first-step, the steamed rice (kake-rice) of 56 g was added to 87 mL of waterwith 1 mL (108 cells) of the sake yeast K701, 24 g of koji, and 450 PL of 50% lactic acid.
As the second-step, the steamed rice (kake-rice) of 117g was added to the sake mash with180 mL of water, and 33 g of koji. As the third-step, the steamed rice (kake-rice) of 227g
36was added to the sake mash with 382 mL of water, and 43 g of koji.
The kake-rice for the sake-making test was prepared as follows: The rice was washedat 15 °C for 30 sec with tap water, drained for 15 sec, and washed for 30 sec again withmore tap water. After the washing step, the rice was drained for 15 sec and then soaked toreach 130% water absorption. Next, the soaked rice was drained at 15 °C for 30 min andsubsequently steamed for 60 min. The water absorption of the steamed rice was adjusted to130%, assuming that the water content of the original white rice was 13.5%.
The sake yeast strain K701 for the sake-making test was cultured as follows: The
sake yeast cells were pre-cultured overnight at 30 °C in the rice koji extract medium (Baumescale 10). The pre-cultured cells were then inoculated into fresh medium at  100-folddilution and incubated for 2 days at 30 °C as a standing culture. The cultured cells were
washed twice with sterilized water and thereafter added to the sake mash (final
concentration, approx. 1 x 106 cells/mL).
Sake fermentation was performed at 15 °C (in a water bath) for 20 days, and was monitored
by measuring the weight reduction of the sake mash, corresponding to CO2 evolution. After
fermentation, the clear sake sample was obtained by centrifugation (5000 rpm, 10 min,4 °C).
2.3.4. Analysis of sake
General properties (Sm, sake meter; Alc, sake alcohol; TA, total acidity; AA, amino acidity),
and flavor components (EtOAc, ethyl acetate; iAmOH, isoamyl alcohol; iAmOAc, isoamyl
acetate; EtCap, ethyl caproate) of the sake were determined by the standard method
established by the National Tax Agency of Japan (Brewing Society of Japan, 2017).
2.3.5. Enzyme activity analysis of rice koji
Enzyme activity of koji was analyzed by the standard method established by the National
Tax Agency of Japan (Brewing Society of Japan, 2017). The activities of 3 enzymes, i.e.,
D-amylase, glucoamylase, and acid carboxypeptidase, were measured by using therespective assay kits (Kikkoman, Japan) (Imai et al., 1996; Shirokane et al., 1996).
2.3.6. Sake metabolome analysis method
A model sake sample was prepared as in the previous report (Ichikawa et al., 2019). Sake
samples were filtered through an Amicon Ultra 0.5 3K (Merk Millipore, Germany) anddiluted 10-fold with MS-grade water. UPLC-QTOF-MS analysis was performed with a
UPLC Xevo Q/TOF-MS system (Waters, Milford, USA). The method for sake metabolome
analysis established by Yazawa et al. (Yazawa et al., 2019) was followed.
37

2.3.7. Data processing of sake metabolome
Data processing by Yazawa et al. (Yazawa et al., 2019) and Ichikawa et al. (Ichikawa etal., 2019) was basically followed. Peak detection, alignment peak data, and calculation ofpeak area were conducted with Peaklynx XS Rev. 1.0 software (Waters, Milford, USA).
Mass range and retention-time range for peak detection were determined by the error rangeof 14 selected peaks from the model sake samples (Table S4 (Chapter II Table S4.xlsx)).
To evaluate the accuracy of the metabolome data, I measured the model sake sample once
among 9 sake samples. The model sake sample analyzed total 7 times. The error range ofeach mass spectrum was calculated by 5-fold standard deviation of each of the 14 selectedpeaks. The largest value among these values was used as the error range for the peakpicking method of Peaklynx. The error range of the retention time was also calculated as
the mass spectrum error. Other settings of the peak picking-method for metabolome data
were as follow: initial and final retention times of monitoring, 0 to 15 min; peak width at
5% height, 4 sec; intensity threshold of peak picking, 40; mass window, 0.03 Da; andretention time window, 0.1 min. Other settings followed the manufacturer’s instruction.
After making a peak table, preliminary data processing was performed according to methodby Ichikawa et al (Ichikawa et al., 2019).
2.3.8. Multiple comparison technique using metabolome data
The software JMP 13.1 (commercially available) and Microsoft Excel were used forprincipal component analysis (PCA) and welch’s test (two-tailed, Bonferroni corrected),respectively. To examine the difference in sake metabolites generated by the rice cultivars
in the small-scale sake-making tests, I performed Welch’s test (two-tailed, Bonferroni
corrected) by using the intensity of each peak. The P value was calculated by using the
average of the intensity of each sample, and a value of less than 0.05 was regarded asindicating a significant difference between rice cultivars.
2.4.

Results and Discussion

2.4.1.

Effect of koji starters on the general properties of sake and the enzyme

activities of koji
In a previous report (Ichikawa et al., 2019), to investigate the characteristics of sakemetabolites in sake prepared from KOS, I performed the small-scale sake fermentation test
using 3 rice cultivars (YAM, GOM, and KOS) and 1 koji starter (Byakuya/B). Then, in the“kake+koji-rice (total rice) sake-making test,” the contents of the 2 tasterelated amino acids,38

glutamic acid and aspartic acid, were lower in the sake from KOS than in those from theother 2 rice cultivars. In the “kake-rice sake-making test,” alpha-ethylglucoside (α-EG)
content in the sake of KOS was lower than that in the sake made from the other ricecultivars; in contrast, in the “kake+koji-rice sake-making test,” the α-EG content in the sakeof KOS was higher than that in the other rice cultivars.
In this present study, to investigate whether the characteristic metabolites in sakemade from KOS varied according to the koji starters, I performed the small-scale sakefermentation test followed by the small-scale koji making, using 3 rice cultivars (YAM,
GOM, and KOS) and 3 koji starters (Yoikaori/Y, High G/H, and Byakuya/B) (Table 5).
These koji starters are commercially available. In this small-scale fermentation test, the 4
enzyme activities (α-amylase, glucoamylase, acid carboxypeptidase, and acid protease) of
the koji, fermentation rates (CO2 decrement) during the test, and the general properties andflavor components of the obtained sake samples were determined and are shown in Table
5 and Fig. 5(b), Fig. S2 a, b, and c (Chapter II Fig S2.pptx), and Table 6, respectively. The
α-amylase activity of koji from KOS was lower than that of the koji prepared from the otherrice cultivars regardless of the type of koji starter used, especially, it was significantly lower
in koji starter-Y and -H (Table 5 and Fig. 5(b)). In either koji starters, acid carboxypeptidase
activity in koji from KOS was intermediate between that from YAM and that from GOM,
and the glucoamylase activity in koji from KOS was comparable to that in YAM koji. In
the koji made by koji starter-B, the 3 enzymatic activities (α-amylase, glucoamylase, andacid protease) were higher than those in the koji made from the other 2 koji starters (Table
5 and Fig. 5(b)). In the koji made by koji starter-H, 2 enzymatic activities (α-amylase andacid carboxypeptidase) were lower than those in the koji made from the other 2 koji starters.
In the koji made by koji starter-Y, 2 enzymatic activities (glucoamylase and acid protease)were lower than those in the koji made from the other koji starters, -H and -B (Table 5 andFig. 5(b)). The G/α ratio (glucoamylase activity/α- amylase activity) for the koji made fromthe koji starter-B was higher than that for koji made from koji starter-H or -Y (Table 5andFig. 5(b)).
In the small-scale fermentation test using the koji made from either koji starter-Y or-H, fermentation rates (CO2 decrement) in the sake mash using KOS showed the sameprofiles as those using GOM. While, in the test using the koji made from koji starter-B, thefermentation profile (CO2 decrement) in the sake mash using KOS indicated anintermediate value between that using YAM and using GOM (Fig. S2a, b and c (Chapter II
39

Fig S2.pptx)). Regardless of the type of koji starter, the concentration of the flavor
component isoamyl acetate (iAmOAc) in the sake sample from KOS was lower than thatin the sake made from the other rice cultivars, GOM and YAM. The concentration of
isoamyl alcohol (iAmOH) in the sake samples using koji made from koji starter-Y or -H
from KOS was lower than that in the case of GOM or YAM (Table 6).
2.4.2. Principal component analysis of the metabolome data of the sake fermentation
test
To investigate the effect of the koji starter on the characteristic sake metabolites from KOS,
I constructed a peak table (Table S5 (Chapter II Table S5.xlsx)) containing all metabolome
data of 1116 peaks, whose intensity was higher than the threshold of peak-picking, as
described in Materials and Methods (Data processing of sake metabolome). Among these1116 peaks, I identified 130 peaks (counting “number of fragment ion(s) from one
compound”) and categorized 191 peaks. Among the identified 130 peaks, I detected 77
compounds (counting “1” = “more than 1 ion from 1 compound”) among 496 compounds(Table S4 (Chapter II Table S4.xlsx)), based on the retention time and m/z of the ion(s) (P,precursor/parent ion; fx, in-source fragment ion; derived from the compounds) in Table S4(Chapter II Table S4.xlsx). I used this peak table (Table S5 (Chapter II Table S5.xlsx)) for
further analysis.
At first, to investigate the character of each sake sample, I performed principal
component analysis (PCA) by using this peak table (Fig. 6(a,b)). As the result of PCA scoreplotting of the metabolome data from the small-scale sake fermentation test, thecontribution ratios of PC1 and PC2 were 53.5% and 20.3%, respectively (Fig. 6(a,b)).
In the classification based on the rice cultivars, on the PC2 axis, the sake samples from
KOS (sample Nos. 3, 6, and 9) were concentrated in a negative direction; whereas thosefrom YAM and GOM (sample Nos. 1, 2, 4, 5, 7 and 8) were concentrated in a positive one(Fig. 6(a)). The sake samples from YAM and GOM were closely located in almost the same
region (Fig. 6(a)). The analysis/result of loading plot of the PCA indicated that unknown
peaks contributed mainly to the distribution to a negative direction on the PC2 axis (Fig.
S3 (Chapter II Fig S3.pptx)).
Next, in the classification based on koji starters, the sake samples from koji starter-B(sample Nos. 7–9) and koji starter-Y (sample Nos. 1–3) were concentrated on the PC1 axis
in a positive and negative direction, respectively; and the sake samples from koji starter-H(sample Nos. 4–6) were located intermediate between those from koji starter-B and –Y (Fig.
40

6(b)). These results suggested that the sake components were changed by the koji starters(Fig. 6(b)). Indeed, the analysis/result of loading plot of the PCA indicated that many peaks
contributed to a distribution on the PC1/2 axis, and that as the peaks contributing to the
distribution. α-EG and nitrogen compound/dipeptide were located on the PC1 axis in anegative and positive direction, respectively (Fig. S3 (Chapter II Fig S3.pptx)). Previously,
Yazawa et al. (Yazawa et al., 2019) reported that α-EG was negatively correlated withglucose. As the glucoamylase activity of the koji made from starter-B was higher than thosefrom other koji starters, the amount of glucose in the koji made from starter-B would behigher than those from other koji starters. Therefore, it is suggested that the koji made fromstarter-B was concentrated in a positive direction on the PC1 axis, the opposite of α-EG.
2.4.3. Effect of koji starters on the sake metabolites obtained with Koshitanrei
Next, to analyze/explore in detail the peaks affecting the distribution on the axis in Fig. 6
and the characteristic peaks for KOS, I performed Welch’s test (two-tailed, Bonferroni
corrected) using the constructed peak table (Table S5 (Chapter II Table S5.xlsx)). In thisanalysis, I eliminated 75 peaks (the intensity 0 in some of the samples Nos.1–9). With either
koji starter, by focusing on the peaks in samples of sake made from KOS, I selected thepeaks whose intensities were higher (p < 0.025, Bonfferoni corrected; KOS > YAM, GOM)
or lower (p < 0.025, Bonfferoni corrected; KOS < YAM, GOM) than those for the otherrice cultivars. Further, the selected peaks were classified according to koji starters. In orderto clarify the difference of components/metabolites affected by three koji starters, I
analyzed the components/metabolites using Venn diagram. Regarding the selected peaks,
Venn diagrams of the higher peaks (KOS > YAM, GOM) and lower peaks (KOS < YAM,
GOM) are shown in Fig. 7(a,b), respectively.
In the higher peaks with KOS (KOS > YAM, GOM; Fig. 7(a)), the number of thehigher peaks for koji starter-Y was only 16 (“I” in Fig. 7(a)); that of those for koji starterH, only 20 (“III” in Fig. 7(a)); that of those for koji starter-B, only 17 (“V” in Fig. 7(a));
and that of those for either koji starter, 41 (“VII” in Fig. 7(a)). Thus, in the characteristic
sake metabolites from KOS, there were 2 peak groups, one affected by koji starter and theother unaffected by rice cultivar.
On the other hand, in the lower peaks found for KOS (KOS < YAM, GOM; Fig. 7(b)),
the number of the lower peaks for koji starter-Y was only 53 (“I” in Fig. 7(b)); that of thosefor koji starter-H, only 23 (“III” in Fig. 7(b)); that of those for koji starter-B, only 107 (“V”
in Fig. 7(b)); and that of those for either koji starter, 301 (VII in Fig. 7(b)). Further, the41

number of lower peaks for KOS in the sake using the koji made from starter-B was larger
than those from other koji starters (Fig. 7(b) 107/V vs. 53/I and 23/III), indicating that in
the sake using the koji made from starter-B the character of sake metabolites derived fromrice cultivar is likely to emerge. With either koji starter, the number of the lower peaks was
about 7-fold (301/B-VII vs. 41/A-VII) over the number of the higher peaks, suggesting thatsome metabolites in the sake from KOS tended to be lower than those from the other ricecultivars. Thus, as in the previous report (Yazawa et al., 2019; Ichikawa et al., 2019),
the sake metabolites were affected by the rice cultivar used, and, further, the characteristic
sake metabolites of the rice cultivars were changed by the koji starter used.
2.4.4. Identification of the sake metabolites affected by Koshitanrei and koji starters
Further, I categorized or identified the metabolites of the affected peaks by using themetabolite list (Table S4 (Chapter II Table S4.xlsx)). The results (Fig. 7(a) and Table 7)
showed that the numbers of the identified/categorized higher peaks for KOS were 2/2
among 16 for koji starter-Y (I), that no peaks were identified/categorized among the higher
peaks for koji starter-H (III) and -B (V), and that the numbers of identified/categorized
peaks were 9/9 among 41 for either koji starter (VII). On the other hand, theidentified/categorized lower peaks (Fig. 7 (b) and Table 8) for KOS were 2/5 among 53 forkoji starter-Y (I), 6/9 among 23 for koji starter-H (III), 10/18 among 107 for koji starter-B(V), and 41/61 among 301 for either koji starter (VII). The metabolites (ID No.
Compound_detected ion, in Table S4 (Chapter II Table S4.xlsx)) of the identified peaks(Fig. 7(a,b)) are indicated in Tables 7 and 8. With either koji starter, the contents ofalphaethylglucoside (α-EG) in the samples of sake made from KOS were higher than thosein the samples from the other 2 rice cultivars (Table 7, VII, 8 peaks). On the other hand, thelower peaks in the KOS sake samples were amino acids and dipeptides (Table 8, VII).
2.4.5. Effect of koji starter on the taste- and aroma-related sake metabolites affectedby KOS
Various components including carbohydrates, organic acids, amino acids, and inorganic
ones are related to the taste and aroma of sake (Brewing Society of Japan, 1999). A previous
report showed 5 components (aspartic acid, glutamic acid, methionine, cysteine, and alphaethylglucoside) related to the taste and aroma of sake among the identified metabolites/
components found in sake made from KOS (Ichikawa et al., 2019). Therefore, I
investigated whether these components were affected by the koji starter used.
Previously, it was shown that the concentrations of the 2 taste-related amino acids,42

glutamic acid and aspartic acid (Iwano et al., 2004), in the sake made from KOS were lower
than those in the sake from other rice cultivars. At first, I examined these 2 amino acids. As
the result (Fig. 8(a), Nos. 3, 6, 9), with any of the koji starters, the aspartic acid content in
the sake from KOS was lower than that in the sake prepared from the other rice cultivars.
In regard to aspartic acid, I make standard curve using four different concentration ofstandard material (Fig. S4a (Chapter II Fig S4.pptx)), and I confirmed that standard curve
showed good linearity in the scope of intensity of aspartic acid (Fig. 8(a)). As the lowcontent of aspartic acid in sake from KOS was found regardless of the koji starter used, thisfinding suggests that the content of aspartic acid would be affected by the rice cultivar rather
than by the koji starter.
Previously, it has been reported that the major component of “hineka,” one of theunpalatable flavors in sake, is dimethyl tri-sulfide (DMTS) (Isogai et al., 2006) and that the
production of 1,2-dihydroxy-5-(methylsulfinyl) prentan-3-one (DMTS-P1), the precursorof DMTS, is related to the methionine salvage pathway genes in Saccharomyces cerevisiae(Wakabayashi et al., 2013). In our previous study, I found that the content of methionine
in the sake from KOS was lower than that in the sake from the other rice cultivars.
Next, therefore, I examined the methionine content. In regard to methionine, as withaspartic acid, I confirmed that standard curve showed good linearity in the scope of intensityof methionine (Fig. 8(b) and Fig. S4b (Chapter II Fig S4.pptx)). As the result (Fig. 8(b),Nos. 3, 6, 9), regardless of the koji starter used, the content of methionine in the sake from
KOS was lower than that in the sake from the other rice cultivars, suggesting that the lowcontents of sulfur-containing amino acids in the sake from KOS would be unaffected by
the koji starter used.
Finally, I examined the carbohydrate alphaethylglucoside (α-EG), which is
responsible for the bitterness following the sweet taste of sake (Oka and Sato, 1976).
Furthermore, in regard to α-EG, as with aspartic acid, I confirmed that standard curve
showed good linearity in the scope of intensity of α-EG (Fig. 8(c) and Fig. S4c (Chapter II
Fig S4.pptx)). Previously, it was shown that the α-EG content was affected by rice cultivar
and that the α-EG content in sake using koji-rice made from KOS was higher than that in
the sake using koji-rice made from other rice cultivars. As the result (Fig. 8(c), Nos. 3, 6,9), with any of the koji starters, the α-EG content of sake made from KOS was higher thanthat from the other rice cultivars, suggesting that the α-EG content of sake would be affectedby both rice cultivar and the koji starter.
43

Previously, I showed that sake metabolites affected by the sake rice cultivars,
especially KOS. In this study, I further examined the effect of the koji starter used on themetabolites of sake made from KOS. For future clarification of the effect of koji starter and
KOS on the sake metabolites, it will be important to identify the unknown peaks affectedby KOS and koji starter.

44

Table 5. Combination of sake-making parameters and properties of koji-rice

Rice cultivar

SampleNo.

kake -ricekoji -rice

1
2
3
4
5
6
7
8
9

YAM
GOM
KOS
YAM
GOM
KOS
YAM
GOM
KOS

YAM
GOM
KOS
YAM
GOM
KOS
YAM
GOM
KOS

Koji
starterYeaststrain

Aaase
765
935
612
797
903
553
804
948
708

YoikaoriHigh G

K701

Byakuya

Enzyme activity (U/g koji )
G/D
Gaase
ACPase
ratio
140
0.18
2,659
169
0.18
3,804
153
0.25
3,380
204
0.26
2,532
237
0.26
4,014
196
0.35
3,096
368
0.46
2,729
474
0.50
4,418
373
0.53
3,388

Apase
1,863
2,489
2,186
1,804
2,354
2,337
2,261
2,520
2,514

Sake-making parameters, such as rice cultivar (YAM, Yamadanishiki; GOM,
Gohyakumangoku; and KOS, Koshitanrei), koji starter, yeast strain, and enzyme activities(AAase, a-amylase; GAase, glucoamylase; G/a ratio, GAase/AAase; ACPase, acidcarboxypeptidase; and APase, acid protease) of koji-rice are indicated.

45

Fig. 5.

Combination of sake-making parameters and properties of koji-rice and sakesamples analyzed in the small-scale sake fermentation tests.
(a), Conceptual diagram of cobweb chart showing 4 enzyme activities of koji-rice.
(b), Three cobweb charts of 4 enzyme activities of koji-rice prepared by using 3 koji starters(Yoikaori, High G, and Byakuya) made from 3 rice cultivars (YAM/blue, GOM/green, and
KOS/red) are indicated.

46

Table 6. sake samples analyzed in the small-scale sake fermentation tests

SampleNo.

Ricecultivar

1
2
3
4
5
6
7
8
9

YAM
GOM
KOS
YAM
GOM
KOS
YAM
GOM
KOS

Koji
starter
YoikaoriHigh G

Byakuya

Sm
+2
+10
+11
+5
+12
+15
+9
+17
+19

Gneral properties
Alc
TA
(%)
(ml)
17.3
2.0
18.6
2.2
18.5
2.1
18.6
2.4
18.7
2.6
19.3
2.4
19.0
1.9
19.8
2.2
19.6
2.1

Flavor components (ppm)
AA
(ml)
1.3
1.8
1.5
1.7
1.9
1.9
1.3
1.5
1.5

EtOAc

iAmOAciAmOH

EtOCap

106
113
119
117
116
115
124
128
117

8.7
9.7
7.4
8.1
8.6
5.9
5.2
6.1
4.6

209
222
176
189
205
166
129
141
133

3.0
2.3
2.7
3.2
2.4
2.6
3.6
2.7
2.9

General properties and flavor components of sake samples designated in “a.” All data are
indicated as the averages for 2 independent tests.

47

Fig. 6. PCA analysis of small-scale sake making tests.
(a), PCA score plots of metabolome data (3 analyses of each sample) from 2 independentsake-making tests (sample Nos. 1-9 in Table 1). Blue, green, and red symbols indicate the
3 rice cultivars, YAM, GOM, and KOS, respectively. Squares, triangles, and circles
indicate the 3 koji starters, Yoikaori, High G, and Byakuya, respectively. Blue-, green-, andred-dotted ovals indicate the located areas for the samples from the 3 rice cultivars, YAM,
GOM, and KOS.
48

(b), PCA score plots of metabolome data. All symbols are the same as in “a.” The 3 dotted
ovals indicate the located area for the samples from 3 koji starters, Yoikaori, High G, andByakuya.

49

(a)KOS > YAM , GOM( Table 1 )Yoikaori(1, 2, 3)II
20(0/ 0)

I16(2/ 2)
VI2( 1/ 1)VII
41(9/ 9)

III
20(0/ 0)High G(4, 5, 6)IV28(7/ 7)

V17( 0/ 0 )Number of peak
( Identified / Categorized )Byakuya(7, 8, 9)(b)

KOS < YAM , GOM( Table 2 )Yoikaori(1, 2, 3)

II145(8/ 17)I53(2/ 5)
VI21(5/ 5)

VII
301(41/ 61)

III23( 6/ 9)High G(4, 5, 6)IV
97(18/ 26)

V
107(10/ 18)
Byakuya(7, 8, 9)

Number of peak
( Identified / Categorized )Fig. 7. Venn diagrams of peaks affected by KOS in sake using koji from each koji starter.
(a), For the intensity of peaks in sake made from the 3 rice cultivars (YAM, GOM, and
KOS), welch’s test (two-tailed, Bonferroni corrected) were performed to compare theintensities between 2 evaluations (KOS vs. YAM, KOS vs. GOM). Venn diagrams of thepeaks (KOS > YAM, GOM), whose intensity in sake made from KOS was higher (p <0.025, Bonfferoni corrected, KOS > YAM, GOM) than that in sake made from the other 2rice cultivars, YAM and GOM. The numbers in Venn diagrams indicate the numbers ofpeaks (number of the identified/categorized peaks). The metabolites (ID No. Components)of the identified peaks are shown in Table 3.
(b), Venn diagrams of the peaks (KOS < YAM, GOM), whose intensity in sake made from50

KOS was lower (p < 0.025, Bonfferoni corrected, KOS < YAM, GOM) than that in sakemade from the other 2 rice cultivars, YAM and GOM. The metabolites (ID No.
Components) of the identified peaks are shown in Table 4.

51

Table 7. The metabolites (ID No. Compound_detected ion) of the identified peaks in Fig.
3(a) (KOS > YAM, GOM).

Category
dipeptidecarbohydrate
ester
other

I

III

V

VII
517 Gln-Glu_P
12 alpha-Ethylglucoside_(8; P, f2, f4, f6-10)

67 Ethyl 3-hydroxybutyrate_f7
456 Phosphoric acid_P

The intensity of the peaks in sake made from KOS was higher (p < 0.05) than that in sakemade from the other 2 rice cultivars, YAM and GOM. Identified compounds (ID No.
Compound_detected ion, Table S1) at yoikaori only (I), at high G only (III), at byakuya
only (V), and at common 3 koji starters (VII) are indicated. The detected ions are indicatedas P, precursor/parent ion; fx, in-source fragment ion; more than one (No. of detected ions;kind of ions).

52

Table 8. The metabolites (ID No. Compound_detected ion) of the identified peaks in
Figure. 3(b) (KOS < YAM, GOM).

Category

I

III

V

nitrogen compoundVII

224 L-Cysteine_P
232 L-Histidine_f1
237 L-Tryptophan_f2
228 L-Aspartic acid_(2; f1, f3)
229 L-Lysine_P
231 L-Methionine_(2; f1-2)
236 L-Tyrosine_(5; P, f1-4)
257 beta-Phenylethylamine_P
258 Agmatine_P

dipeptide

533 Asp-Glu_P
484 Gly-Trp_P
496 Leu-Ala_P
513 Val-Val_f1
526 Val-Phe_f2
531 Ala-Thr_P
532 Leu-Lys_(2; f1, f3)
468 Ala-Leu_(3; P, f2-3)
474 Leu-Val_P
476 Gly-Ala_P
483 Gly-Thr_f2
490 Gly-Phe_f3
502 Leu-Leu_P
510 Pro-Arg_P
511 Gly-Val_(2; P, f3)
519 Gln-Tyr_P
522 Ser-Val_P
530 Pro-Gln_P
536 Gln-Leu_f1
552 Arg-Ala_P
554 Arg-Gln_P
566 Lys-Asn_(2; P, f2)
567 Phe-Asp_P

carbohydrateorganic acid387 D-Ribose_f9
206 Pyroglutamic acid_P
190 Malic acid_(2; f3-4)
209 Succinic acid_(2; P, f3)
174 Citramalic acid_(4; P, f5-7)

phenol compoundester

54 Tyrosol_f1
67 Ethyl 3-hydroxybutyrate_(2; f5-6)
414 N-(2-phenylethyl)acetamide_(2; P, f3)
318 Inositol_f5

vitamine

The intensity of the peaks in sake made from KOS was lower (p < 0.05) than that in sakemade from the other 2 rice cultivars, YAM and GOM. Identified compounds (ID No.
Compound_detected ion, Table S1) at yoikaori only (I), at high G only (III), at byakuya
only (V) , and at common 3 koji starters (VII) are indicated. The detected ions are indicatedas Table 1.

53

(a) 228 L-Aspartic acid_f 1

Intensity

1000

800
600
400
200

2

3

4

5

6

7

8

9

6

7

8

9

8

9
KOS

Sample No. 1

GOM0(b) 231 L-Methionine_f 1
3000

Intensity

2500
2000
1500
1000

500
0

Sample No.

1

2

3

4

5

(c) 12 alpha-Ethylglucoside_f 7
12000

Intensity

10000

8000
6000
4000

2000

4

Total rice

GOM

KOS

YAM

Koji starter

Yoikaori

5

6

7
YAM

3

KOS

2

GOM

1
YAM

0

Sample No.

High G

ByakuyaFig. 8. Typical metabolites (ID No. Compound_detected ion) affected by Koshitanrei.
Intensity of (a) L-Aspartic acid_f1 (RT, 1.2516; m/z, 116.0348), (b) L-Methionine_f1 (RT,
2.5127; m/z, 133.0299), and (c) alpha-Ethylglucoside_f7 (RT, 4.6532; m/z, 163.0589) areindicated.

54

Concluding remarks
The Japanese alcoholic beverage sake is made from rice and water by using 2 kindsof microorganisms, A. oryzae (called koji-fungi) and the yeast S. cerevisiae. The main raw
material for sake brewing is the rice, and the characteristic features of rice are one of themost important factors determining the quality of the final product, sake. Therefore, theselection of the rice cultivar is one of the important concerns for brewing high-quality sake.
Moreover, in sake brewing, rice-koji making is the other important processes, because ricekoji is a determinant of the aroma/ taste component of sake. Therefore, it is necessary tochoose the proper koji starter for a rice cultivar, with taste designing of the sake in mind. In
this study, I examined the suitability of sake rice Koshitanrei (KOS), developed in Niigata
Prefecture, for the brewing of high-quality sake.
In Chapter I, to investigate the characteristic metabolites in sake from KOS, I
performed 2 types of small-scale sake-fermentation tests for comparing KOS with its
parental cultivars, YAM and GOM. These rice cultivars were examined as kake-rice (ricefor kake-mai) by using the same YAM koji or total rice (both kake-mai and koji) at 2
polishing ratios (70% and 50%). Subsequent fermentation was carried out by using thesame sake yeast strain and parameters, and then sake components were measured by thesake metabolome analysis method. As a result, in the kake-rice sake-making tests, the sakemetabolomes of YAM and GOM were significantly affected by the difference in polishingratio (70% or 50%), whereas that analysis of KOS was less affected, regardless of theprogeny of these cultivars. The crude protein content of KOS was lower than that of theother rice cultivars. For that reason, especially the KOS sake made with the 70% polishingratio had a lower amino acid content than that found for the YAM and GOM sakes. As aresult of principal component analysis (PCA), many nitrogen compounds/dipeptides
impacted the plot of the 70% YAM and GOM sakes. On the other hand, some other nitrogencompounds affected the 70% KOS sake plot. Thus KOS was the sake with the lowest aminoacid content even at the low polishing ratio of 70%.
In the total rice sake-making tests, the sake metabolome for KOS, unlike that for theother 2 rice cultivars, was affected by the difference in the polishing ratio (70% or 50%). A
comparison of “kake+koji-rice sake-making test” and “kake-rice sake-making test”
suggested that for KOS rice, the effect of the polishing ratio was greater for the total rice
KOS sake than that for the kake-rice. And, even in the kake+koji-rice sake-making test, thesake components of KOS sake showed characteristics different from those of YAM and55

GOM sakes. The amino acid content of the KOS sake was also lower than that of the otherrice cultivars at either polishing ratio. At the 70% polishing ratio, the koji from KOS
indicated low proteolytic enzyme activity. But, at the 50% polishing ratio, the acidcarboxypeptidase (ACP) activity of KOS koji was higher than that of the YAM koji, andthe acid protease (AP) activity was similar to that of the YAM koji. Thus, KOS sake may
be considered to have a lower amino acid content due to the lower crude protein content of
the rice. In the “kake-rice sake-making test,” the level of α-EG in the sake from 70% or
50% polished KOS was lower than that in the sake made from the other rice cultivars. In
contrast, in the “kake+koji-rice sake-making test” at 70% and 50% polishing ratios, the αEG content in the sake from KOS was higher than that from the other rice cultivars. Yazawa
et al. reported that α-EG was negatively correlated with glucose. In the “kake-rice sakemaking test,” fermentation rates (CO2 decrement) of KOS sake mash, and sake meter (Sm)and alcohol (Alc) of the KOS sake tended to be lower than those of the other 2 cultivars.
On the other hand, in the “kake-+koji rice sake-making test,” compared with that from theother rice cultivars, koji from KOS showed lower activity of 4 enzymes at the 70%
polishing ratio and slightly lower glucoamylase activity at the 50% polishing ratio. And
then, KOS sake mashes indicated fermentation rates (CO2 decrement) intermediate
between those of GOM and YAM sake mash. It may be considered that the enzyme activityof koji and the difference in fermentation process influenced the behavior of D-EG. In thisstudy, the sake components from KOS were different from those of the parental cultivars.
In KOS, kake-rice had little effect on the polishing ratio, and changing the total rice affected
the polishing ratio. The data revealed that KOS had components that were higher or lower
than those of the parent cultivars depending on whether it is used for the kake-rice only or
for the total rice.
In Chapter II, to investigate the effect of the koji starter on the rice-koji characteristics
and sake metabolites, I performed small-scale sake-making tests using the above 3 ricecultivars (KOS, YAM, and GOM) and 3 koji starters (Yoikaori, High G, and Byakuya).
Based on the PCA analysis of the sake metabolites, KOS sake plotted at an area different
from that of the other 2 rice cultivars in the case of all 3 koji starters. In addition, koji starters
also affected the sake components of all 3 rice cultivars, but the effect was more significant
for the parental cultivars. To reveal the difference in KOS sake metabolites in the entire
koji starter, I focused on higher/lower peaks for the KOS sake sample compared with thosepeaks for sake samples from the other rice cultivars. The KOS-specific peaks could be56

classified into 2 groups, one affected by the koji starter and the other, is unaffected by it.
With either koji starter, the number of the lower peaks was about 7-fold over the number ofthe higher peaks, suggesting that some metabolites in the sake from KOS tended to be lower
than those from the other rice cultivars.
In the classification based on the difference in koji starters, the number of lower peaks of
KOS sake prepared with starter-B was larger than that for the other koji starters, indicatingthat the appearance or disappearance of sake metabolites depended on the kind of koji
starter used conjugating with difference of rice cultivar. The α-EG content of KOS sake
was higher than that for the other rice cultivars independent of the koji starter used,suggesting that the α-EG content of sake would be more affected by the rice cultivar. As inprevious experiments, the levels of nitrogen compounds and dipeptides in sake from KOS
were lower than those in the sake from the other rice cultivars. The ACP activity of KOS
koji was intermediate between the YAM and GOM koji activities, and the AP activity wasalmost the same as that of the GOM koji with all koji starters. Thus, the lower amino acidcontent of KOS sake was also due to the low crude protein content of KOS.
In this study, I investigated the suitability of sake rice KOS for high-quality sakebrewing by evaluating the difference in sake metabolites between it and YAM and GOM
and the effect of koji starters. The data clarified that the sake made from KOS possessed aquality and characteristics different from those of the parental cultivars, YAM and GOM,
in terms of usage as kake-rice or koji-rice. In addition, by changing the koji starter, it became
clear that characteristics of the KOS sake were still observed, but the koji starter was also
observed to affect the sake components in a qualitative manner. Thus, it is necessary to
select the koji starter according to the desired sake quality.
As the number of identified metabolites was low, it will thus be important to makean effort to identify the unknown peaks affected by KOS and koji starter.

57

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60

Acknowledgments
It is my pleasure to express my sincere thanks to Visiting Professor Dai Hirata and Visiting
Professor Kazuhiro Iwashita for invaluable guidance, criticism and encouragement.
I am very grateful to Professor Akio Kuroda, and Professor Jun-ichi Kato, for helpful
suggestions and discussions.
I am very grateful to Dr. Hisashi Yazawa (National Research Institute of Brewing),
Yuko Hata (National Research Institute of Brewing), and Shogo Hirata (Hiroshima
University) for helpful experiment and advice.
I am very grateful to Dr. Hiroyasu Tamura (Asahi Sake Brewing Co., Ltd.,) and Dr.
Ryo Nakamura (Asahi Sake Brewing Co., Ltd.,) for helpful advice, and encouragement.
I also want to thank participants of the experiment for their cooperation.
I also thank all members of Asahi Sake Brewing Co., Ltd. for their support andencouragement.

61

Related publications(1) Analysis of metabolites in Japanese alcoholic beverage sake made from the sake rice
Koshitanrei.
Eri Ichikawa, Shougo Hirata, Yuko Hata, Hisashi Yazawa, Hiroyashu Tamura, Mitsuoki
Kaneoke, Kazuhiro Iwashita, and Dai Hirata
Bioscience, Biotechnology, and Biochemistry, 83(8), 1570-1582 (2019).

(2) Effect of koji starter on metabolites in Japanese alcoholic beverage sake made from the sakerice Koshitanrei.
Eri Ichikawa, Shougo Hirata, Yuko Hata, Hisashi Yazawa, Hiroyashu Tamura, Mitsuoki
Kaneoke, Kazuhiro Iwashita, and Dai Hirata
Bioscience, Biotechnology, and Biochemistry, 84(8), 1714-1723 (2020).

62

බ⾲ㄽᩥ
         (1) Analysis of metabolites in Japanese alcoholic beverage sake made from the sake rice
Koshitanrei.
Eri Ichikawa, Shougo Hirata, Yuko Hata, Hisashi Yazawa, Hiroyashu Tamura, Mitsuoki
Kaneoke, Kazuhiro Iwashita, and Dai Hirata
Bioscience, Biotechnology, and Biochemistry, 83(8), 1570-1582 (2019).

(2) Effect of koji starter on metabolites in Japanese alcoholic beverage sake made from the sakerice Koshitanrei.
Eri Ichikawa, Shougo Hirata, Yuko Hata, Hisashi Yazawa, Hiroyashu Tamura, Mitsuoki
Kaneoke, Kazuhiro Iwashita, and Dai Hirata
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