J. Biol. Macromol., 15 (1), 41-50 (2015)

Note

High-yieldproduction

of

ethyl

α-D-glucosideinshochubrewing and evaluation of its functionality
Ta k a y u k i B o g a k i , 1 , 3 , *Kenji Ozeki1,2College of Bioscience and Chemistry,Department of Applied
Bioscience,

Kanazawa

InstituteofTe c h n o l o g y,

7-1

Ohgigaoka, Nonoichi, Ishikawa 921-8501, Japan,1 Genome
B i o t e c h n o l o g y l a b o r a t o r y , K a n a z a w a I n s t i t u t e o f Te c h n o l o g y,2

3-1 Yatsukaho, Hakusan, Ishikawa 924-0838, JapanandG e n e r a l R e s e a r c h L a b o r a t o r y, O z e k i C o r p o r a t i o n , 4 - 9 I m a z u ,Dezaike, Nishinomiya, Hyogo 663-8227, Japan,3Received May 25, 2015; Accepted June 10, 2015

We

developedabrewingshochus, the main mash of
10-times

more

ethyl

method

for

rice

and

barleywhich contains 3-times and

α-D-glucoside(α-EG),

re s p e c t i v e l y,compared with that from conventional method s. The 2.5%α-EG extract prepared from a distillation residue of rice
shochu was found to have a moisture -retaining effect.

Key words: ethyl α-D-glucoside (α-EG); moisturizing effect;functional food; skin care

* To whom correspondence should be

Alpha-EG is a glycoside produced byaddressed: E-mail:transglycosylationofstarch-derived

takayuki.bougaki@ozeki.co.jp

maltose or maltooligosaccharides by

α-EG production in shochu mash and its moisturizing effect42

α-glucosidase of koji-mold using ethanol

compared with a control (0.5% Triton

as an acceptor in brewing of sake

X-150) and that 0.05% α-EG reagent(Japanese rice wine) (1). Since Imanarialso showed a similar effect (6).

et al. discovered the presence of α-EG in

Moreover, orally ingested α-EG has

sake (2), influences of α-EG on thealso

flavor of sake and its production

moisture-retaining effect. Hirotsune et al.

mechanism have been studied (1,3,4)

found that α-EG and organic acids,

because α-EG is contained in sake atwhich are components of sake, reduceabout 0.1–0.7% (5), which is the fourth

TEWL levels of UVB-irradiated hairless

highest following water, ethanol, andmice (7). As a result of their subsequent

glucose

test

in

this

order

among

the

been

of

reporteddose

todependence

have

of

a

these

ingredients of sake. Alpha-EG has

substances, they confirmed that α-EG

instantaneous sweetness as well asshows efficacy. Thus, α-EG is anslow-acting and mild bitterness.

ingredient expected to be useful for skin

Alpha-EG,deeplybeauty through oral ingestion as well as

involved in the taste of sake, is also

skin application. However, no effective

reported to have a moisture-retaining

manufacturing method of α-EG produced

effect and roughness-improving whenby various brewing methods has been

applied on skin. Kitamura et al. applied areported.

sake concentrate (approximately 2-foldFor

or 0.2-fold concentrated sake) to the skincoexistenceof hairless mice for 5 days and thenmaltooligosaccharides,

irradiated the mice with ultraviolet B

α-glucosidase that catalyzes the reaction(UVB), followed by further application

is

of

the

an

ingredientconcentrate

for

the

productionofnecessaryof

ethanol,

in

the

α-EG,
maltose,andprocess

of

3

days.

fermentation. Sake brewing, a parallelthat

double-fermentation process in which

Consequently,

they

found

Transepidermal

water

loss

(TEWL)

saccharificationlevelssignificantly

reducedprogress simultaneously, satisfies these

was

and

fermentation

Bogaki and Ozeki

43requirements. On the other hand, α-EG is

distillation

not produced enzymatically in wine

exploited a manufacturing method of

brewing, which has no saccharification

shochu in which a material with aprocess, and in beer brewing, in which

moisture-retaining effect that can be used

α-glucosidase is inactivated by thein cosmetics, dietary supplements, andboiling following saccharification (8).

drinks was produced from the residue.

Sato et al suggested that a small amountFor this purpose, we aimed a high-yieldof α-EG, which is included in wine, isproduction of α-EG in shochu’s main

formed by a nonenzymatic condensation

mash and addition of an extra effect, thereaction of glucose and ethanol (5).

moisture-retaining effect of α-EG, to the

Although not detected in shochu, a typeresidue. First, we checked whether α-EGof spirit, α-EG is likely to be produced in

was produced in a general manufacturing

its main mash because it is produced bymethod of shochu and then examined

parallel double-fermentation. On thebrewing methods leading to a high α-EGother hand, the distillation residue ofcontent in the distillation residue, whileshochu is a waste liquid containing amaintaining the quality of the distillationhigh concentration of organic matters

product (shochu).

generated in the process of shochu

residueofshochu,we

The brewing was conducted with the

manufacturing. Because this residue

following

contains abundant moisture and nutrients,

Briefly, koji-mold (Aspergillus awamori),

including protein, fat, and saccharides, ayeast

variety

NBRC2373 for shochu), and barleyofmethodsforeffective

materials

and(Saccharomyces(Bordenstudied, such as feed, food, and human

enzymes used were “Sumizyme” (Shinconsumption related toits specificNihon Kagakukogyo Co. Aichi, Japan)

the hepatopathy

for α-amylase and “α-glucosidase Amano”

inhibitory effect (9). In the present study,(Amano Enzyme Inc., Nagoya, Japan)

infor α-glucosidase. In addition, the water

order

to

effectively

utilizethewere

cerevisiae

utilization of this residue have beenfunctions, such as

variety)conditions.

used.

The

44

α-EG production in shochu mash and its moisturizing effectfor brewing was used after its pH wassame. A preliminary experiment that

adjusted to 3.5 with lactic acid. For rice

investigated enzyme quantities added

shochu, 12 g of koji (rice covered with aindicated that α-EG productivity was

growth of koji fungus) was mixed withhigher when enzymes were added to the

21 mL of water and 5.3  106 cells/mL of

secondary main mash compared with thatyeast and was subjected to primaryobserved when enzymes were added tofermentation

30C,

the primary main mash and that α-EG

followed by addition of 40 g of steamed

productivity was higher when 4140 units

rice and 96 mL of water, and secondaryof α-glucosidase and 2520 units offermentation for 8 days at 20C or 30C.

α-amylase were added compared withFor barley shochu, 16 g of koji wasthat when 8280 units of α-glucosidase

mixed with 28 mL of water andand 5040 units of α-amylase were added.

subjected to primary fermentation for 5

Based on these data, 4140 units ofdays in the same conditions as those of

α-glucosidase

rice shochu, followed by the addition of

α-amylase were added to the secondary

40 g of steamed barley and 66 mL of

main mash in koji-reduced brewing towater, and secondary fermentation for 8

determine the amount of koji to be used.

days at 20C or 30C. In the brewing in

The analysis of α-EG was performed

which koji was reduced and enzymes

using

were added, the amount of koji waschromatography

reduced to 1/2 (6 g), 1/4 (4 g), and 1/8 (3HPLC system, Waters Co., MA, USA)g) of the normal amount in the case ofwith the conditions described in Fig. 1.

for

5

days

at

High

and

2520

performance(HPLC)

unitsofliquid(Alliance

rice shochu and 1/8 (2 g), 1/10 (1.6 g),

The rice shochu and barley shochu

and 1/12 (1.3 g) in the case of barley

showed similar tendencies. Alpha-EGshochu. Dried pregelatinized rice of thewas produced at a higher yield in theamount equivalent to the reduced amountcooler (20C) brewing condition than in

was added so that the total amount of

the brewing condition at 30C, as well asrice used in the brewing remained thein the brewing condition in which koji

Bogaki and Ozeki

45

FIG. 1. Alpha-EG concentration in the main mash and distillation residue
preparations of rice and barley shochus brewed with various koji contents.
Alpha-EG was analyzed using the following conditions. HPLC system; Alliance
HPLC system (Waters Co., MA, USA), detector; 2414 RI detector, (Waters Co.,
MA, USA), column; Shodex SPO 810 (Showa Denko, Tokyo, Japan), mobile
phase; H 2 O, flow rate; 400μl/min, column temperature; 65C. The temperatures
represent those of the secondary main mash. The numbers represent the amount ofkoji used, wherein 1, 1/2, 1/3, 1/4, 1/8, 1/10, and 1/12 denote 12 g, 6.0 g, 4.0 g, 3.0g, 2.0 g, 1.6 g and 1.3 g, respectively. Open bars, fermentation broth, closed bars,
shochu residue. Data represent mean values of three experiments.

content was reduced and α-amylase andbrewing of rice shochu at 20C with

α-glucosidase were added.

enzymesupplementation

was

2.1%,

We successfully developed a brewing

which was more than 3-times higher thanmethod in which the α-EG content in the

0.6% in the brewing at 30C, and theα-EG production in shochu mash and its moisturizing effect46

content in the brewing of barley shochu

preparations have glucoamylase activity

in the same conditions at 20C was 2.2%,

which

10-times higher than 0.2% of thesubstrates of α-glucosidase but does notbrewing at 30C. This was likely due tocontribute to α-EG production (10), thethe better transglycosylation efficiency

amount of enzyme preparations is aof α-glucosidase at 20C at which

future subject of examination.

degradation of starch in the raw material
byglucoamylase

becomesslowerdegrademaltose

and

other

To check the quality of shochu
brewed in the conditions of high yieldcompared with the brewing at 30C at

α-EG production, rice shochu and barley

which glucose supply from the starch byshochu containing 30% alcohol were

glucoamylase

produced by vacuum distillation, diluted

activity

becomes

predominant. On the other hand, becausetheα-amylase

and

α-glucosidase

to 25% with water, and subjected totasting separately (Table 1). A panel ofTable 1. Sensory evaluation of shochu brewed under high α-EG producing
conditions.
rice shochubarley shochu

1

1/4

1

1/12

fermentation temp.

30 C

20 C

30 C

20 C

odor

2.9±0.3

3.1±0.5

2.5±0.5

2.7±.5taste

3.1±0.5

3.1±0.5

2.7±0.5

2.7±0.5

overall quality

3.1±0.5

3.0±0.4

2.7±0.5

2.6±0.5

koji ratio

The shochu samples were evaluated by a 5-point scale, in which 1 is the best
and 5 is the worst. Flavor is scored for its acceptability and intensity. Taste isscored for its overall impression of whether sample is light or heavy, the extent ofa sweet or acid taste, presence or absence of an abnormal taste, and the balance
between the taste-components. Overall quality is evaluated with flavor, taste, andcolor. Values are the mean ±SD (n=8).

Bogaki and Ozeki

47

8 specialists judged that there were no2.5% concentration. After cleaning anddifferences in both flavor and taste andmoisture removal of the inner upper arm

no issues in quality between the shochus

of subjects in the early 20s, followed byobtained by conventional brewing and

20 min of acclimation, a patch sheet

brewing with a high content of α-EG. In

containing 100 μL of the sample wasaddition, because no change in the total

attached. After being left to stand for 2

amount of α-EG was observed before

min, the sheet was removed and theand after distillation as a result of the

moisture contained mainly in the horny

α-EGconcentration

layer of the skin surface was measured

between the distillation residue and theevery 15 min until 120 min using a skinmain mash, α-EG was confirmed tomoisture meter. The results showed thatneither react with any other ingredient in

the extraction residue of rice shochu andthe main mash nor evaporate and be

α-EG reagent caused a significantly

recovered in shochu during distillation.

different skin moisture content after 2 hcomparisonofFig. 2 shows the result of the pach testin the skin test compared with thatof the skin moisture-retaining effect ofobserved in the RO water. On the otherthe α-EG-containing extract prepared

hand, because the moisture content wasfrom the shochu residue. The samples

not significantly different despite theused in this test were prepared as

2.5% α-EG content in the extraction

follows: The residue of rice and barley

residue of barley shochu, a substanceshochus brewed with 1/4 and 1/12

negatively

amount of koji, respectively, compared

retention might have been present.

with normal was completely dried toinfluencing

themoisture

We have successfully developed aremove water and alcohol, mixed with an

brewing

methodthat

increasestheequal volume of reverse osmosis watercontent of α-EG, which has been

(RO water), and centrifuged to extract

confirmed to have a moisture-retaining

α-EG, which was then prepared at aeffect, in the distillation residue of rice

α-EG production in shochu mash and its moisturizing effect48

FIG. 2. The rate of change of skin moisture after application of theα-EG-containing shochu residue extract. The relative value of the skin moisture
content 120 min after application of each sample is shown. Skin moisture content
before treatment with sample (0 min) was set at 100%. Fourteen subjects werestudied, and each sample was tested on 3 sites per subject. Rice; rice shochu residue
extract, Barely; barely shochu residue extract, α-EG; 2.5% α-EG reagent. Results aremean ± SD (n=14). **p<0.05, significant difference in comparison to the RO water(t test).

and barley shochus, thus adding extra

α-EG concentration) of the distillation

value to the residue, which is mainly

residue of rice shochu was applied todiscarded a waste material. We consider

subjects in their early 20s, the extractthat this technical development can leadshowed a moisture-retaining effect as

to

the

indicated by a remarkable difference indistillation residue of rice shochu based

the retained moisture content 120 min

on the following results: In the moisture

after the application compared with that

retention test in which the extract (2.5%

observed in the control. Based on thetheeffective

utilizationofBogaki and Ozeki

49

result that no remarkable difference in

we did not find the moisture-retaining

the

was

effect with the barley shochu residue.

observed between the α-EG solution

Therefore, we would like to examine

extracted from the distillation residue of

whether we can improve this technique

rice shochu and the α-EG reagent, α-EGby further modifying the fermentation

is likely to be significantly involved inmethod.

moisture-retaining

effectthe moisture-retaining function. In the

Also in the past, development of

test to assess the effect of α-EG tomaterials from the shochu residue for

improve UVB-caused rough skin in mice,cosmetics and drinks has been conducted

α-EG has been reported to improve thefor its effective utilization. In our case,balancewebetweengrowthandtodevelopanewcornification of epidermal cells (6).

moisture-retaining

material

thatisHowever, because this is the result of theeffective for beauty purposes throughthird day after UVB irradiation in the test,

both oral ingestion and skin application

the moisture-retaining effect observed inusing the α-EG-enriched shochu residue

our study, which was measured in athat we developed in this study and apply

period as short as 120 min, was likely tothis to foods or cosmetics.

have been generated by a mechanism
different from the growth/cornification
balancing of epidermal cells.
Because the present study used 2.5%,
a relatively high concentration of α-EG,
we intend to examine whether the extract
is also effective at lower concentrations.
In addition, we expected a synergistic
effect of α-EG and sphingolipids (11), a
moisture-retaining substance found in thebarley shochu residue, but unfortunately,

intendα-EG production in shochu mash and its moisturizing effect

50

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