（ 1 ）163

日本食品保蔵科学会誌
３
５

VOL．

会
長
編集委員長
編集委員

４

NO．

松本 信二
上田 悦範
石田
裕
寺井 弘文

副

会

長

稲熊 隆博
津久井亜紀夫

上田

悦範

小宮山美弘

早坂

薫

井上 茂孝
藤島 廣二

内野 昌孝
松田 茂樹

竹永

章生

＜報 文＞（英文）
メタン発酵における原料生ごみの電解処理 …………………………………………………………………（１６５）／徳田宏晴・本間裕人・入江満美
牛久保明邦・中西載慶
野草抽出物のメタンチオールに対する消臭効果 ……………………………………………………………（１７１）／浦部貴美子・東 理恵・稲田多希
南野可奈子・灘本知憲・小野廣紀
フキ（Petasites japonicus）のクロロゲン酸酸化酵素の精製と性質 ………………………………………（１７９）／韓 雲哲・趙 福善・小川剛史
太田真由美・藤田修二
収穫後のエタノール蒸気処理がスダチ果実の品質に及ぼす影響 …………………………………………（１８７）／野間雄太・鈴木康生・寺井弘文・山内直樹＜技術報告＞
ナッツ類を用いたチーズ様食品の製造 ………………………………………………………………………（１９５）／本間裕人・徳田宏晴・中西載慶＜講 座＞
身近な野菜・果物〜その起源から生産・消費まで（９）コンニャク（!）−生産と利用− ………（２０
１）
／内田秀司

＜文献抄録＞ ……………………………………………………………………………………………………（２０７）＜本会記事＞ ……………………………………………………………………………………………………（２０８）＜会
告＞ ……………………………………………………………………………………………………（２１
５）

164

（ 2 ）

Food Preservation Science

CONTENTS OF VOL．
３
５ NO．
４（２
０
０
９）
＜Article＞（English）
Electric Pretreatment of Garbage for the Methane Production
TOKUDA Hiroharu, HOMMA Hiroto, IRIE Mami,
USHIKUBO Akikuni and NAKANISHI Kotoyoshi …………………………………………………………（１６
５）
Deodorizing Effects of Wild Grass Extracts against Methanethiol
URABE Kimiko, HIGASHI Rie, INADA Taki,
MINAMINO Kanako, NADAMOTO Tomonori and ONO Kouki …………………………………………（１７１）
Purification and Characterization of Polyphenol Oxidasefrom Japanese Butterbur（Petasites japonicus）
HAN Yunzhe, ZHAO Fushan, OGAWA Takeshi,
OHTA Mayumi and FUJITA Shuji ………………………………………………………………………（１７９）
Effects of Postharvest Ethanol Vapor Treatment on Qualityof Sudachi（Citrus sudachi hort. ex. Shirai）Fruit
NOMA Yuta, SUZUKI Yasuo, TERAI Hirofumi and YAMAUCHI Naoki…………………………………（１８７）＜Technical Report＞（Japanese）
Cheese-like Food Production from Various Nuts
HOMMA Hiroto，TOKUDA Hiroharu and NAKANISHI Kotoyoshi ………………………………………（１９５）＜Serialization Lecture＞（Japanese）
Konjac（Part!）
UCHIDA Shuji ………………………………………………………………………………………………（２０１）

（ 3 ）

Food Preservation Science VOL. ３
５ NO. ４ ２００
９ 〔Article〕

165

Electric Pretreatment of Garbage
for the Methane Production
TOKUDA Hiroharu＊１§，HOMMA Hiroto＊１，IRIE Mami＊２，
USHIKUBO Akikuni＊２ and NAKANISHI Kotoyoshi＊１
Department of Brewing and Fermentation, Junior College of Tokyo University of Agriculture,1−1−1, Sakuragaoka, Setagaya-ku, Tokyo 156−8502＊２ Department of International Agricultural Development, Tokyo University of Agriculture,1−1−1, Sakuragaoka, Setagaya-ku, Tokyo 156−8502＊１

In this study, we investigated the effects of the electric pretreatment of garbage on biogas（methane ）production. Both the garbage atomization, and garbage solubilization were enhanced by
pretreatment with ３
０ V of direct electric current. Moreover, the number of contaminants attached tothe surface of garbage components was decreased by electric pretreatment. Furthermore, garbage
pretreatment with direct electric current enhances biogas（methane）fermentation.
（Received Mar. ７, ２００８；Accepted Feb. ９, ２００
９）Key words：pretreatment, direct current, garbage, biogas, hydrogen
前処理，直流電流，生ごみ，バイオガス，水素In Japan, approximately ２
０ million tons of garbage
１）

is discharged annually , and approximately half of

garbage, and the many contaminants that adhere to
garbage, which inhibit fermentation３）.

this is household waste. Although approximately

In this study, we investigated the effects of the１
０％ of garbage is reused as fodder or manure,electric pretreatment of garbage on the physicalmost garbage is incinerated and ends up in landfills.

properties of and the microorganisms adhering to

One problem with this method of garbage disposal

garbage. We further investigate the possibility thatis that the very high water content of garbage

methanelowerspretreated garbage.

combustion

temperature,

leading

to

the

could

formation of dioxin. In recent years, the pollution of
offensiveodor

aroundland

reclaimedfromgarbage landfills have become serious problems. On

producedfromelectrically

Materials and Methods

soil and groundwater as well as the generation ofanbe

１．Garbage
Artificial garbage prepared according to methodsadescribed in the literature４） was used in this study.

recyclable and renewable resource since it contains

Carrots, cabbage, bananas, apples, grapefruit, horse

the

other

hand,

garbageisconsideredasa large variety and amount of biological compounds

mackerel, steamed rice, tea dregs, and egg shells

such as polysaccharides and proteins, as well as

were cut into small cubes（about ２〜３㎜ per side）,

mineral salts. These components can be converted

and mixed in a ratio ９：９：５：５：５：５：５：２：１ by

intoweight.

usefulmaterials

byfermentation

processes.

Based on this information, much research on thepossible use of garbage as a source material for

２．Microorganisms
The

methanefermentation

sludgeusedwasbiogas production has been carried out and someobtained from Hitachi Plant Construction & Services

fermentation processes for methane production fromCo., Ltd. Before use for methane fermentation fromgarbagearealready

in

２）

use .

many

garbage, the sludge was filtered by screening withproblems remain in the utilization of garbage asa ２-㎜ -size mesh and habituated according to theraw material for fermentation. These problems arise

following method.

becauseofthevariousshapesHowever,andqualityof§ Corresponding author, E-mail : E-mail tokuda@nodai.ac.jp

Sludge（３．
６!）was placed in a continuously stirred

166

Food Preservation Science

VOL．３
５ NO．
４ ２
０
０
９

（ 4 ）

tank（４#）and the air in the tank was replaced with

３
０
０- ! Erlenmeyer flask. The gas phase in thenitrogen gas. The tank was then maintained at ３
７℃

upper layer of the flask was replaced with nitrogenfor ５
２ days. During this incubation, １２
０! of sludge

gas and anaerobic culture was carried out at ３
７℃.

was substituted for the same amount of garbage

Evolved gas was collected in an inverted measuringwith or without electrical pretreatment every ２４cylinder over water and measured of its volume. As

hours. This habituated sludge was used as a starter

control,

for methane production from garbage.

pretreated

３．Electric pretreatment of garbage

insteadofacomponentofelectricallygarbage,acomponentofuntreated

continuous

methanegarbage was used.

The apparatus used for the pretreatmentofgarbage is illustrated in Fig．
１. It is composed of

５．Continuous methane production
In

order

to

measuresemipermeable

production from garbage, ３．
６# of habituated sludge

membrane. The volume of each bath is １# , andand １
２０! of electrically pretreated garbage were

two

baths

partitioned

with

a

platinum electrodes（３．
５×１２．
０㎝）were installed inplacedeach bath. Five hundred milliliters of phosphate

replacement of the gas phase in the upper layer ofinastirredreactor.

Followingthebuffer（M／３
０ KH２PO４-M／３
０ Na２HPO４；pH ７．
０）wasthe reactor with nitrogen gas, anaerobic culture wasdispensed into both baths and incubated at ３０℃ .

carriedWet garbage （３１５" ） was placed on either thecontinuous

cathode or anode side, and direct electric current

electrically pretreated garbage and

out

at ３
７℃ .
culture

After
was

the

batch

culture,byfeeding

starteddrawingthe（DC ３
０ V・０．
３A）was supplied from a power supply

fluid culture with pumps at a rate of １
２
０!／day. As（Model ２００／２．
０；Japan Bio-Rad, Japan）. Following

control,２４-helectrically pretreated garbage.

treatment

with

the

electric

current,

thesemipermeable membrane was removed, and theuntreated

garbagewas

fed

insteadof

６．Analytical methods

garbage suspension was mixed with the solution in

The dry weight of the garbage was measured at

the opposite bath. This mixture then served as theroom temperature after drying the garbage at ８
０℃

electricallyfor ２
４ h. The particle size of the garbage was measured

pretreatedgarbagepreparation.

Theelectric energy requirement（Pt）for this pretreatmentusing a testing sieve（Tokyo Screen Co., Japan）. The

was approximately ０．
７
８ MJ（!Pt＝３
０ V×０．
３ A×

apertures of the sieve mesh were １
８
０, ３
０
０, ４
２
５, and８６，４
００ s）. As control, ３
１５" of garbage soaked for

６００!. The number of contaminants in the garbage

２
４ h in the same buffer at ３
０℃ was used.

was determined by plating aliquots of suspensions

４．Methane production in batch cultureThe

methanecomponent

wasof the garbage on nutrient broth agar plates followed byproductivity

of

each

garbagea４
８-h incubation at３
０℃５）. The chemical oxygen demand

estimated

in

batch

culture.

（CODcr）of the filtrate of the garbage suspension

Habituated sludge（１４４!）and ９
６ ! of a component

was measured with a COD analyzer （ HACH Co. ,of electrically pretreated garbage were placed in a

USA, DR ２０００）. The total solid（TS）and volatile solid（VS）of the garbage were analyzed by conventional
methods６）. The composition of the generated biogas

Garbage

was determined by gas chromatography （ Hitachi

Pt electrodeCo., Tokyo, G ３
９００）under the following conditions :Waterjacketcolumn,Cathode
Buﬀer

Anode
Buﬀer

+
−
Power supply

SHINCARBON

ST ５
０／８
０； carriergas,helium ; flow rate, ３
０!／min ; column temperature,
４
０〜２
０
０℃（１
０℃／min）；detector, TCD.

Results and Discussion
１．Effect
Magnetic stirrer

Semipermeable
membrane

Pre-treated Garbageofelectric

pretreatmentongarbageproperties
We first tested the effect of electric current
application on the pH ofthesolutionsinthechambers of the apparatus to be used for garbage

Fig．
１ Apparatus for the electric pretreatment of garbage

treatment. When ３
０ V of direct electric current was

Garbage was pretreated with３
０V of direct electric current for２４h.

applied to the baths, the pHs of the solutions in the（ 5 ）〔Article〕 Electric Pretreatment of Garbage

80change in pH was observed thereafter. It waspossible to maintain the pHs of the solutions in bothbaths by applying direct electric current as low as
５ V. However, when the current application was
terminated, the pHs of the solutions in both baths
gradually returned to their initial values. Moreover,

Solubilizationrate（%）

anode and cathode gradually reached approximately
２ and １
２, respectively, after approximately ３ h ; no

167

60

40

20

the pHs of the solutions could be immediately
0

isdirectcurrentasuitablemethodforgarbage

ﬀe

that

bu

appearsinapplicationitedresults,

ak

these

So

pretreatment with an acid or alkali solution. By
using an adequate buffer solution whose pH has

Fig．
２ Effect of electric pretreatment on garbage
solubilization

been optimized for methane-producing microorganisms,
it is possible to start methane production immediately
after garbage pretreatment. As this equipment can
pretreatgarbagewithout

requiring

wastewater

recovery and neutralization, the negative environmentale）rsolutions from the two baths together. Based on

od

the

V（
an

mixing30

byDC

valuesthode）initial

V（
ca

their

30

to

DC

returned

The solubilization rate of garbage was calculated using thefollowing equation : RS（ ％ ）＝（（ Wb - Wa ）／ Wb ）×１００．
Abbreviations : RS, solubilization rate ; Wb, weight before
pretreatment ; Wa, weight after pre-treatment.

impact of this pretreatment method should be lessthan that of other chemical methods.
garbage properties was examined. First, the weightsof the garbage samples before and after the electricweremeasured

and

the

rate

of

solubilization of the garbage was calculated using
the following equation.
RS
（％）
＝（
（Wb-Wa）
／Wb）
×１
０
０

80
60
40
20
0

th
od
e）

pretreatment

Abundance ratio（%）

100

Next, the influence of electric pretreatment on

This figure reveals that the solubilization rate of the

V（
ca
30
C

Fig．
２ shows the solubilization rate of the garbage.

D

So
a

ke
d

U
nt

Wa : weight after pretreatment.

in

re
a

Wb : weight before pretreatment.

bu

te
d

ﬀe
r

RS : rate of solubilization.

Fig．
３ Effect of electric pretreatment on particle size
distribution of the garbage

garbage was enhanced by electric pretreatment,((

particularly following pretreatment with DC ３
０ V

Symbols：，２．
８㎜〜；（cathode）, after which the solubilization rate reached

，１．
４〜２．
０㎜；

about ６
０％.

，〜１．
０㎜.

，２．
０〜２．
８㎜；
，１．
０〜１．
４㎜；

Secondly, the particle size distributions of thegarbagesampleswithandwithout

electric

pretreatment were measured （ Fig ．
３）. In Fig ．
３,“untreated” indicates the particle size distribution ofthegarbage

immediately

after

garbage atomization that is set off following theswelling of garbage particles.
Finally, the CODcr values for all components ofthepreparation.

garbagesuspensions

beforeandafter

Approximately ６０％ of the electrically pretreatedpretreatment were measured. This analysis showedgarbage（DC ３０ V（cathode））had a particle size ofthat the electrical pretreatment markedly enhanced

２㎜ or less. In contrast, more than ８
０％ of thethe CODcr values for horse mackerel, tea dregs,inand bananas in the suspension（Fig．
４）. These results

buffer）had a particle size greater than ２．
８㎜. Theseshow that some organic components are released

results indicate that electric pretreatment causes

from the garbage when it is electrically pretreated.

garbagewithout

pretreatment （ i. e. ,soakedRate of change（%）

168

Food Preservation Science

VOL．３
５ NO．
４ ２
０
０
９

（ 6 ）

Table １ Effect of electric pretreatment on garbage
contaminants

75
50

Time（h）

25

Number of contaminants
Treated with （viable cells/g wet-garbage）
DC ３
０V

Stored atroom temp.

0
­25

０
２
４

­50

Ca
rr
ot
Ca
bb
ag
e
Ba
na
na
A
pp
le
Gr
Ho ape
fru
rs
e
it
m
ac
ke
St
ea
re
l
m
ed
r
i
ce
Te
a
dr
eg
s
Eg
g
sh
ell

­75

０

２×１
０５
３×１
０１０

１
２
３
６

１×１
０９
４×１
０７
６×１
０４
０

value reached ３×１
０１０（cells／$ wet-garbage）and an

Fig．
４ Effect of electric pretreatment of garbage
constituents on TS, VS and CODcr

offensive odor was generated after incubating for ２
４

Each value is the rate of change from the initial value. The rates of((）, VS（）and CODcr（）, forchanges in TS（
individual garbage components, compared with initial values, areshown.

then pretreated by the electric method and the

２．Effect of electric pretreatment on contaminants

electric current and continued to gradually decreaseattached to the surface of garbage components

thereafter. No living cells were detected within ６

To generate useful materials from raw material

hours of electric pretreatment.

h at room temperature. The garbage sample wassources using biochemical processes, a raw materialchangeinthenumberofcontaminants

wasdetermined. The number of contaminants began todecrease immediately after the application of direct

The

combined

data

electric

garbagepretreatment

reports on sterilization methods７）. However, mostbeneficial effects on ! solubilization, " the extraction

food wastes, such as garbage, are usually used as aof organic substances, and # the sterilization of

raw material without sterilization, even though there

contaminants. Further studies are needed to clarify

are many kinds of contaminants attached to thethesurface of garbage. It is considered thatconsidered that some amounts of heatmechanism

directthatis usually sterilized prior to its use. There are many

most

with

indicate

underlying

thesecurrent

effects.

has

It

is

and ／ orgarbage contaminants are aerobic bacteria, and ahydrogenlarge number of which die out during methane

reactions during the electrical pretreatment of thefermentation. Presently, it is suspected that the

garbage ;deteriorationbyenergy resource, it is also necessary to develop a

contaminants constitutes an inhibitory factor formethod for their recovery, to reduce the operating

fermentation. However, little is known about thecost of electric pretreatment.

ingarbagequalitycausedsterilization of contaminants attached to garbage or

Clearly,gas

aregeneratedbyelectrochemical

however, since they are useful as anthemethodofgarbagepretreatment

about the influence of the deterioration in garbage

presented here is very promising as a novel method

quality on fermentation productivity.

that enables efficient

We have reported that the application of directelectric current has a bacteriostatic or bactericidal
８）

methanefermentation

andreduces the amount of fermentation residues.
３．Biogas production from electrically pretreatedaction against certain microorganisms . Therefore, itgarbageisthe

Biogas production from garbage with and without

pretreatment of garbage will also be effective forelectrical pretreatment was examined in continuous

not

the

culture in the absence of pH control. Fig．
５ shows

sterilization of contaminants in garbage. To test thisthe biogas production with time. No garbage was

hypothesis, the numbers of contaminants in the

suppliedgarbage before and after electrical pretreatment

fermentationwere

expected
only

that

garbagethiselectric

methodsolubilization,

but

for
also

to

the

tank

liquid

when

the

pH

became ６．
５ or

of

the

less.

The

of

concentrations of methane and carbon dioxide in thecontaminants in the garbage was ２×１
０５（cells／$

biogas were ５
５〜６０％ and ４
０〜４
５％ , respectively.

wet-garbage）even directly after its preparation. ThisThe biogas

determined （ Table１）.

The

numberproductivity

pergarbagewithand（ 7 ）

Relative biogas productivity

Total biogas（×103mℓ）

25
20
15
10
5
0
400
300
200
100
0
6

Garbage（VS-g）

Biogas productivity（mℓ／VS-g）〔Article〕 Electric Pretreatment of Garbage

169

3

2

1

0

Cabbage Steamed rice Grapefruit

Carrot Horse mackerel

Fig．
６ Effect of electric pretreatment on the biogas
productivity of specific garbage components
The indicated garbage components were left untreated（□）or
were pretreated with DC３０V at the cathode（■）. The biogas
productivities of all samples, relative to that of the untreated

4
2
0
0

10

20
30
Time（day）

sample（value＝１）are shown.

40

Fig．
５ Time course of biogas production from garbage

effective on the biogas productivity of bananas,

Untreated（○）or pretreated（ ▲ ） garbage was subjected tocontinuous culture without pH control.

apples, tea dregs, or egg shells（data not shown）.
Except in for bananas and tea dregs, the increases
in both the solubilization rate and the amount of

without electric pretreatment was changed daily

extracted organic

substances

causedand was not necessarily higher than the literature

pretreatment were associated with the improvement

data９）. Up to ２
７ days of fermentation, higher biogas

inbiogasproductivity

productivity was obtained for the untreated garbage.

components.

From

The total amount of biogas generated from thefollowing conclusions.

from

these

by

individual

data,

we

electric
garbagedraw

theuntreated garbage was somewhat higher than that

! The improvement in total biogas production

generated from the pretreated garbage. However,from pretreated garbage mainly arises from anbiogas productivity decreased rapidly after that, andimprovement in the biogas productivity of the ５

the generation of biogas stopped on the ３９ th day.

components shown in Fig ．
６. " Some materials

On the other hand, biogas productivity from theeluted from each garbage component by electric

pretreated garbage was stable for a while andpretreatment

continuous

biogasproduction

was

successfullymay

inducecertaininteractions

inhibitory to biogas production in the prometaphaseachieved for over ４
２ days. Consequently, the totalof fermentation.

amount of biogas generated from the electricallyclarify the details．# Garbage pretreated with direct

pretreatedelectric current is suitable for a long series ofgarbagewasabout １．
２ timesthat

Further

researchisneededtobiogas fermentation.

generated from the untreated garbage.
In order to clarify the factors that enhance the

The improvement in total biogas （ methane ）total amount of biogas, the fermentation efficiencies

productivityof all components of the garbage samples with andseems to lead to an efficient and low-cost disposal

without electric pretreatment, were examined. Fig．
６of garbage in the future. This method of electric

shows

pretreatment of garbage is considered applicable tothedifferent

relativebiogasgarbagecomponents.

productivities
Each

of

the

biogasproductivity is expressed relative to the productivity

other

from

agricultural

wastespretreated

for

the

garbagepurposeofmanufacturing raw materials for fermentation.

of untreated sample. Pretreatment with DC ３０ V

Conclusions（cathode）improved the relative biogas productivityof cabbage, steamed rice, grapefruit, carrots, andelectrically

A methodfortheelectric

pretreatment

of

the

of

horse mackerel. For cabbage and steamed rice, thegarbagebiogas productivities of the pretreated samples werepermitting methane generation from garbage. The

more than twice as high as those of the untreatedmethod involves the treatment of garbage with ３０samples. However, electric pretreatment was not as

V of direct electric current

wasdevelopedforpurpose

for ２
４ h.

Garbage

170

Food Preservation Science

VOL．３
５ NO．
４ ２
０
０
９

“ Mizu（ 8 ）solubilization and the elution of organic materials

Y. :

from

method.

Japanese）”（Morikita Publishing Co., Ltd., Tokyo）

to

the

pp.１
５
１〜１
５
３（１
９
９
４）

and

the

garbage

Furthermore,garbageweretheimprovedbycontaminants

components

were

this

attachedsterilized,

KankyoKogakuno

Kiso （ in７）Toray Research Center Chosa Kenkyu Jigyobu :amount of biogas （ methane ） generated from the“ Atarashii

electrically

Youtotennkai （ in Japanese ）, ” （ Toray Research

pretreatedgarbagewasincreasedcompared with that generated from the untreatedgarbage.

Thismethod

of

using

a

low

electric

Sakkinnzai ・ Sakkinngijutu

noCenter, Tokyo）
（１
９
９
０）
８）TOKUDA, H. and NAKANISHI, K. : Application ofcurrent for garbage pretreatment to produce biogas

direct（ methane ） may

contamination, Biosci. Biotech. Biochem., ５
９（４）
,７
５
３

also

be

applicable

to

the

pretreatment of other raw materials.

current

toprotectbioreactor

against

〜７
５
５（１
９
９
５）
９）TANIKAWA, N., FURUICHI, T., ISHII, K. and NISHIGAMI,

References

K. : Study on methane generation, environmental

１）CMC Publishing Co., Ltd., Henshubu : “Realities

burdens and cost effectiveness of kitchen-waste

and View of Food Waste Recycling（in Japanese）”

biogasification facilities, Haikibutsu Gakkai Ronbunshi（CMC Publishing Co., Ltd., Tokyo）, prefatory note,（in Japanese）
,１
９（３）１
８
２〜１
９
０（２
０
０
８）

（２
０
０
２）
２）HONDA, A., OOI, S., KAMINAGA, S., SEKIKAWA, S.,
YAMADA, H., TSUGUYOSHI, O., TAKEI, N.,ODAWARA, Y.,
ISHIDA,

M. ,HOSOI,K.

and

NAKATSUKA,

メタン発酵における原料生ごみの電解処理
徳田宏晴＊１・本間裕人＊１・入江満美＊２

T. :牛久保明邦＊２・中西載慶＊１“ Haikibutsu no Methane Hakko （ in Japanese ）”（Scientist Co., Ltd., Tokyo）, pp. ６
４〜１
３
６（２
０
０
０）
３）SHIMIZU,

“ JBA

H. :

News （ Bio-engineering

Kenkyu Kohenkai）”, Baio Saiensu to Indasutori （inJapanese）
,６
５, １
０
３〜１
０
５（２
０
０
７）
４）SANKAI,

T. : Development

recycling
MeetingsystemofbyuseArchitecturalofof

Institute

５）TOKUDA,

H. ,

SATO,

TAKAHASHI,

J. :Immobilization

method,J.

東京農業大学国際食料情報学部国際農業開発学科

thegarbageAnnual

生ごみを原料としたバイオガス（メタン）発酵においJapan, （ inて，試料生ごみの電解前処理効果について検討した。生of

ごみ試料を電解処理（直流３０ V・２４ h）すると，試料
andの微粒子化が促進され，その可溶化率が向上するとともxtracellularに，有機性成分の溶出が認められた。また，電解処理に

gas

phase

より，試料生ごみに付着している雑菌数が減少した。さJapan （ in

らに，原料生ごみを電解処理するとバイオガス（メタMUKATAKA,

Brew.

＊２

disposer,ofenzymes on non-woven fabrics bycultivation

東京農業大学短期大学部醸造学科

（〒１
５
６−８
５
０
２ 東京都世田谷区桜丘１―１―１）

Japanese）Hokkaido, ５２
５〜５
２
６（１
９
９
５）
S. ,

＊１

（〒１
５
６−８
５
０
２ 東京都世田谷区桜丘１―１―１）

Soc,

S.

Japanese）
,８
６（９）６
８
９〜６
９
４（１
９
９
１）
６）NAKAJIMA, S., KANOU, M., KOJIMA, Y. and KANEKO,ン）発酵の生産性が向上することも明らかとなった。
（平成２０年３月７日受付，平成２
１年２月９日受理）

（ 9 ）

Food Preservation Science VOL. ３
５ NO. ４ ２００
９ 〔Article〕

171

Deodorizing Effects of Wild Grass Extracts
against Methanethiol
URABE Kimiko＊１§，HIGASHI Rie＊１，INADA Taki＊１，
MINAMINO Kanako＊１，NADAMOTO Tomonori＊１ and ONO Kouki＊２＊１＊２

Department of Food Science and Nutrition, School of Human Cultures, The University of Shiga Prefecture,
2500, Hassaka-cho, Hikone 522−8533
Department of Food and Nutrition, Gifu City Women’s College, 7−1, Hitoichibakita-machi, Gifu 501−0192

We investigated the deodorizing effects of hot - water extracts of samples of ２
６ species of wild
grasses native to Hikone City, Shiga Prefecture against methanethiol（CH３SH）, and compared themwith the deodorizing effects of methanol extracts of the samples. The deodorizing activities were
determined from the amounts of CH３SH remaining using headspace gas chromatography. Each of the５．
０! hot-water extracts and methanol extracts was added to CH３SH and then examined. As a result,deodorizing effects stronger than those of green tea were observed in hot-water extracts of samplesof １
２ species and methanol extracts of samples of １
１ species. Furthermore, deodorizing activities of１
００％ were found in eight species. Among them, the deodorant active substances of Glechomahederacea［２］, Artemisia capillaries［３］, Porentilla fragarioides［４］, Gnaphalium affine［５］, Stenactis annuus［６］, and Oenothera biennis［８］were more effectively extracted by hot-water extraction than methanol
extraction. These substantial deodorizing effects of hot-water extracts of wild grasses have not beenreported to data. This experiment suggests that substances originating from plants are promising forpossible deodorant applications.
（Received Jul. ９, ２００８；Accepted Mar. ９, ２００
９）Key words：dedorizing effect, methanethiol, wild grass, hot-water extract, methanol extract
消臭効果，メタンチオール，野草，熱水抽出物，メタノール抽出物

Recently, there has been much attention focused

effects than sodium copper chlorophyllin （ SCC ），on finding new usages for natural substances thatwhich is widely used as a deodorant５）. To the bestarepositiveof our knowledge, none of these plants have been

physiological effects on human beings. Plants possess

reported to have deodorizing properties. On thevarious, functional, substances, and they are consideredother hand, vegetables or wild edible plants areto be a potential source of deodorants. Edible plants,

commonly boiled for cooking and commonly used as

herbs, and herb vegetables have been used for

medicinal herbs. Therefore, the purpose of

cooking and in preservation for some time now.

present study is to

They

environmentally

have

also

safe

been

and

used

to

have

improveinvestigate

the

this

deodorizing

odor

effects of hot-water extracts of wild grasses and toproblems in the past. For example, there have been

compare them with those of methanol extracts ofmany reports concerning their suppression of odor,

wild grasses.

in other words, deodorizing effects１）〜４）. So, we aimed
at studying the deodorizing effects of wild grasses,
which are commonly around us, in our gardens andMaterials and Methods
１．Wild grasses and extraction procedureyards but are not usually used as food sources. We

The wild grasses that grow naturally in Hikone

previously reported on the deodorant effects ofCity, Shiga prefecture were gathered between themethanol extracts of wild grasses（４２ species）aroundmonths of April and October. As shown in Table １,

us including so-called “weeds” against methanethiol

we examined ２
６ species from twelve families. The（CH３SH ）. As a result, methanol extracts from １４

species of wild grass was identified on the authority

species of wild grasses showed stronger deodorizingof two pictorial books of the Japanese flora６），７）. The§ Corresponding author, E-mail：kurabe@shc.usp.ac.jp

172

Food Preservation Science

VOL．３
５ NO．
４ ２
０
０
９

（ 10 ）

Table １ List of Wild Grasses Used in the ExperimentNo.

English name or Japanese nameScientific name

Part used

Gathering date１
２

EyebaneIndian pennywort

Eurphorbia maculata
Glechoma hederacea

Aerial parts
Aerial parts

Oct．
２
０
０
４
May．
２
０
０
４

３
４

Kawarayomogi
Potentil

Artemisia capillaris
Porentilla fragarioides

Stems and leaves
Aerial parts

Aug．
２
０
０
０
May．
２
０
０
４

５

Jersey-cutweed

Gnaphalium affine

Aerial parts

May．
２
０
０
４

６
７

Daisy-fleabane
Himeyomogi

Stenactis annuus
Artemisia feddei

Aerial parts
Stems and leaves

Jun．
２
０
０
０
Oct．
２
０
０
０

８

Evening primrose

Oenothera biennis

Aerial parts

Jul．
２
０
０
０

９

Harujion

Erigeron philadephious

Aerial parts

May．
２
０
０
４

１
０
１
１

Saw-thistle
American false daisy

Sonchus oleraceus
Eclipta prostrata

Aerial parts
Aerial parts

Jun．
２
０
０
０
Oct．
２
０
０
０

１
２

Flax-leaf fleabane

Conyza bonariensis

Aerial parts

Jun．
２
０
０
０

１
３
１
４

Dokudami
BurweedHouttuynia cordata
Xanthium strumarium

Stems and leaves
Stems and leaves

Jun．
２
０
０
４
Aug．
２
０
０
０

１
５

Hananigana

Lactuca dentata

Aerial parts

May．
２
０
０
０

１
６

Kosendangusa

Bidens pilosa

Aerial parts＊

１
７

Common purslana

Portulaca oleracea

Stems and leaves

Sep．
２
０
０
４
Sep．
２
０
０
４

１
８

Dock

Rumex japonicus

Aerial parts

May．
２
０
０
０

１
９

Ibukijakousou

Thymus quinquecostatus

Aerial parts

Jun．
２
０
０
０

２
０
２
１

Karasunoendou
Dayflower

Vicia angustifolia
Commelina communis

Stems and leaves
Aerial parts

Apr．
２
０
０
０
Sep．
２
０
０
０

２
２

Dandelion

Taraxacum officinale

Stems and leaves

Apr．
２
０
０
４

２
３
２
４

Indian lettuce
Black nightshade

Lactuca indica
Solanum carolinense

Aerial parts
Aerial parts

Sep．
２
０
０
４
Aug．
２
０
０
０

２
５

YaemuguraGalium spurium

Stems and leaves

May．
２
０
０
４

２
６

Common sorrel

Rumex acetosa

Stems and leaves

Apr．
２
０
０
４

＊Aerial parts are stems, leaves and flowers.

parts of the wild grass used were the aerial parts

standard solution（１$／! in benzene）was dissolved（the leaves, flowers, and stems）, because the partsin ９
９! of ethanol and kept in a freezer（−２０℃）

are generally used for food. The wild grass was

until use. ２．
９! of ０．
１ M sodium phosphate buffer

washed in running water, and the water on the（pH ７．
５）was placed in a ３
０! glass vial and mixed

surface was wiped with a paper towel. The wildwithgrass, cut into small pieces, was placed into an

Immediately after the addition of ０．
１! of CH３SH

Erlenmeyer flask with about ten times its amountethanol solution, the vial was sealed with rubberof distilled water, and left to boil for one hour. The

and aluminum caps and stirred for ５ s with amixturewasVortex mixer. The mixture was incubated in a

reducedwater bath at ３
７℃ for １
５ min and ２ ! of thewasconcentrated

filtered,
by

an

and

the

evaporator

filtrate
under

the

indicatedamountoftestsample.

pressure at ４
０℃ , then lyophilized, and used as aheadspacehot-waterofchromatography （ model GC-１
４ B, Shimadzu Corp. ,extract

sample.

The

preparation

gas

was

then

analyzedbygasmethanol extracts was carried out as follows ： !

Japan）with a flame photometric detector. The gas

Shredded wild grass was soaked in an Erlenmeyer

chromatography conditions were the same as thoseflask with about ten times the amount of methanol ;described in our previous report５）. The amount ofand " was allowed to stand for seven days at room

CH３SH was calculated from a standard curve drawn

temperature ；# after

up in advance. Deodorizing activity for each sample

filtration,

thefiltrate

was

evaporated under reduced pressure at ４
０℃ and wasused as a methanol extract sample.

Deodorizing activity（％）＝（C-S）／C×１
０
０

２．Measurements of deodorant activities
The

methodof

TOKITAetal .８） was

was expressed as follows :slightly

modified and used in this study. １ ! of CH３SH

C：The amount of CH３SH without the sample（control）

（ 11 ）

〔Article〕 Deodorizing Effects of Wild Grass Extracts173

the values of methanol extracts were also shown in

S：The amount of CH３SH with the sample

theAll experiments were performed in triplicate. The

table,

previous

values of coefficient of variation（％）were under １
３％.

thoughpaper５）.

some

data

Green

tea,

had
well

reported
known

as

in
a

deodorizer, was used as a reference. The deodorant
３．Chemicals
p-Coumaric

activities of hot-water and methanol extracts fromICN

green tea were ７８．
６％ and ７２．
４％ , respectively.

Biochemicals Inc. , rosmarinic acid, apigenin, andTwelve out of ２
６ hot-water extracts from species ofrutin from Chromadax Inc., caffeic acid from Sigma-

wild grass had higher deodorizing activities than

Aldrich Chemicals Co. , USA, myricetin, naringenin

green tea. The following eight hot-water extracts inand（＋）catechin from Funakoshi Co., Japan, andparticular had a deodorizing activity of １００％ ：chlorogenic acid and hesperidin from Nacalai Tesque

Artemisia

Inc., Japan. A standard solution of CH３SH（１!／! inGnapalium affine［５］, Stenacis annuus ［６］, Glechoma

benzene ）and other chemicals were obtained fromhederacea ［ ２ ］, Euphorbia maculata ［ １ ］, Oenothera

Wako Pure Chemical Inc., Japan.

biennis［８］and Porentilla fragarioides［４］
.

acid

was

purchasedfromcapillaries［３］,Artemisia

feddaei［７］,In the case of methanol extracts, eleven species

Results and Discussion

had higher deodorant activities than green tea, and１．Deodorizing activity of wild grass extracts

a deodorizing activity of １００％ was found in eightwild

The extracts （５．
０! ） from ２
６ species of wild

grasses,

namely,Artemisia

capillaries［３］,activity

Artemisia feddaei［７］, Eclipta prostrata［１１］, Gnapalium

against CH３SH as shown in Table ２. For comparison,affine ［５］, Stenacis annuus ［６］, Glechoma hederacea

grasseswereexaminedfordeodorant

Table ２ Deodorizing Activity of Extracts from Wild Grasses against Methanethiol
Wild grass

Deodorizing activity（％）
Hot-water extract

Methanol extract

EyebaneIndian pennywort

１
０
０
１
０
０

１
０
０
１
０
０

Kawarayomogi
Potentil

１
０
０
１０
０

１
０
０＊
１
０
０

Jersey-cutweed

１
０
０

１
０
０

Daisy-fleabane

１
０
０

１
０
０＊

Himeyomogi

１
０
０

１
０
０＊

Evening primrose

１
０
０

５
１．
９＊

Harujion

８
５．
０

７
０．
１

Saw-thistle

８
４．
９

２
９．
９＊

American false daisy

８
３．
８

１
０
０＊

Flax-leaf fleabane

８
０．
１

７
８．
７＊

Dokudami
Burweed

６
２．
８
６
０．
４

７
０．
０
５
４．
３＊

Hananigana

６
０．
４

２
２．
３＊

Kosendangusa
Common purslana

４
６．
２
４
４．
３

５
７．
５
９
６．
４

Dock

４
２．
６

４
７．
６＊

Ibukijakousou

４
１．
１

２
５．
７＊

Karasunoendou
Dayflower

３
８．
６
３
７．
６

２
６．
６＊
３
３．
７＊

Dandelion

２
７．
２

６
３．
６

Indian lettuce
Black nightshade

２
５．
３
２
０．
８

３
９．
５
２
１．
５＊

Yaemugura

１
５．
４

７
４．
２

Common sorrel

１
４．
４

６
３．
２

Experimental procedures are described in the text.
＊Reported values（URABE et al .５）, ２
００
３）
.

174

Food Preservation Science

VOL．３
５ NO．
４ ２
０
０
９

（ 12 ）［２］, Euphorbia maculata［１］and Porentilla fragarioides

approximately the same as or twice the deodorizing［４］. Glechoma

activity as those extracted by methanol. It washederacea［２］, Euphorbia

Porentilla［１］andmaculata

fragarioides［４］, whichhad

consideredthat

hot-water

extracted

deodorizing

previous

activity substances are more effective than methanol.

report５）, had deodorizing activities of １
００％ in thisAs none of these hot-water extracts have been

experiment as well. This confirms that these wild

reportedtograsses have high deodorizing activities regardlessexpectedthatof growing time or place. As a result, almost half of

deodorizer in the future.

deodorizing

activities

of １
０
０％ in

the

have

deodorant

they

will

be

properties,
good

it

is

sourcesofthe grasses tested had higher deodorizing activities

On the other hand, the methanol extract ofthan green tea in either hot-water or methanol

Eclipta prostrata［１１］had a high deodorizing activity

extracts. However, correlation between the values ofof approximately ２
０ times that of the hot-waterhot-water and methanol extracts, excluding wild

extract, ８５．
６％ in ０．
２
５! of methanol extract andgrasses with a deodorizing activity of １００％, both in

８３．
８％ in ５．
０! of hot-water extract. On the basis

hot-water and methanol extracts, was substantiallyof this data, it was proposed that Eclipta prostrata

low（r＝０．
２２）.

［１１］possibly contains an unstable substance, obtained

２． Comparison of deodorizing effects ofwild

through the process of heating, or a hydrophobic

grass extracts

substance difficult to extract by water. Although it

In order to compare the deodorizing effects of １
６

is not clear whether Eclipta prostrata［１１］is used forextracts from wild grasses, which had a deodorizing

food, it has been used as a drug９）. It is possible toactivity of １
００％ in Table ２, we measured theeffectively

deodorizing activities of thedeodorant activity from a methanol extract.

samples,decreasing

gradually step by step from １．
０! to ０．
２
５!. Theresults are shown in Table ３. For six species ofasubstancewith

substantial

３．Deodorizing Activity of Polyphenols
The

constituentsofplantswith

deodorizing

capillaries ［３］,activities have been isolated are various, including

Gnapalium affine［５］, Stenacis annuus ［６］, Glechoma

polyphenols２），４），１０）, diterpenes３）, biphenyl compounds１１）

hederacea ［２］, Oenothera biennis ［８］and Porentilla

etc. Among them, polyphenols are considered tofragarioides ［４］, those extracted by hot water had

contribute materially to the deodorizing effects ofwild

grasses,

namely,Artemisia

obtainTable ３ Comparison of Deodorizing Activities of １
６ Extracts of Wild Grasses against Methanethiol.
Amounts of addition（mg）
Wild grass

Extract
１．
０

０．
５

０．
２
５

EyebaneHot-water
Methanol

８
６．
２（％）
１
０
０

−（％）
７
５．
３

−（％）
−

Indian pennywort

Hot-water
Methanol

６
６．
８
５
８．
２

−
−

−
−

Kawarayomogi

Hot-water
Methanol

１
０
０
１
０
０

１
０
０
６
８．
３

７
２．
８
−

Potentil

Hot-water
Methanol

４
７．
４
３
７．
０

−
−

−
−

Jersey-cutweed

Hot-water
Methanol

４
１．
７
３
０．
８

−
−

−
−

Daisy-fleabane

Hot-water
Methanol

１
０
０
４
３．
５

３
７．
４
−

−
−

Himeyomogi

Hot-water
Methanol

６
５．
９
−

−
−

−
−

Evening primrose

Hot-water

１
０
０

６
６．
６

−

American false daisy

Methanol

１
０
０

１
０
０

−；Not tested８５．
６

（ 13 ）

〔Article〕 Deodorizing Effects of Wild Grass Extracts

175

wild grasses. In this study, the amounts and species

scavenging activities of ５-CQA and ３ , ５-diCQA,of polyphenols included, in some wild grasses withwhich are CQA derivatives, have been reported１５）.

highAccording to the report, ５-CQA of ２ µM（０．
７１!）

deodorizing

activities,

have

not

yet

been

reported in detail. However, from pharmacologic andand ３, ５-diCQA of １ µM（０．
５２!）had very low

physiologic

highactivities of ４％ and ０％, respectively. However, indeodorizing activities, including polyphenol in wild

our experiment, １．
０! of ５-CQA（２．
８ µM） had agrasses, have been reported１２），１３）. TOKITA et al .１４）

much greater activity of ５２．
７％, which is substantiallyreported the deodorant activities of some polyphenols,

differentbutdeodorizing

Judging from the activity of ５-CQA in this report,

activity were different from ours in this study. So,

we suppose that the deodorizing activity depends

we once again measured the deodorizing activities

upon the amount of ５-CQA included in Artemisia

of some polyphenols. Table ４ shows our results. In

capillaries［３］or Artemisia feddaei［７］
.

their

actions,somemeasurement

substances

results

for

with

TOKITA’s report, the deodorizing activities of thepolyphenols were as followed ; catechol（９５％）, gallic

from

the

value

reportedbyNegishi.

Nishikisou （Euphorbia humifusa ）, which is a plantclosely

relatedtoEuphorbia

maculata［１］
,

is

１
６）

acid（１００％）, protocatechuic acid（７％）, caffeic acidconfirmed to contain gallic acid and hydrolyzable（３０％）, apigenin （１５％ ）, kaempferol （ ０％ ）, rutintannins１７）. On the basis of this fact, it can be（２％）, myricetin（９０％）, and naringenin（０％）. Someassumeddifferences in deodorizing activity were observed

contain these two substances. Gallic acid has a

Euphorbia

thatmaculata［１］must

also

but the same tendency was observed in the extent

deodorizing activity of ７
０．
３％ with the addition ofof deodorizing effects. Below, we examine the wild

０．
０
１! ,

grasses that have substantial deodorizing activities

Consequently, gallic acid is considered to greatly

for the possibility or even the slightest possibility ofcontribute to the deodorizing activity of Euphorbia

having polyphenol and then we will look into themaculata［１］, varying

deodorant applications of these wild grasses.

abundance.

Ooyomogi （ Artemisia Montana ） and yomogi （ A.
princeps ） oftheasteraceae

family

which

is

a

markedlyinhighaccordance

activity.

with

its

It is reported that the leaf of Glechoma hederacea

contain［２］contains １．
４
８％ rosmarinic acid, which is adicaffeoylquinic acids （ DiCQA ） in large amounts.

distinctive substance in the plants of the mint

Artemisia capillaries ［３］, a closely related plant, isfamily１８）. Furthermore, because it is reported to be

confirmed to contain a substantial amount of DiCQA

stable against heating in the process of infusion,１
２）（７％ in the extract） and Artemisia feddaei ［７］,there presumably exists a considerable amount ofalso

rosmarinic acid in the hot-water extract that we

possibly

contains

DiCQA.

Recently,

theTable ４ Deodorizing Activities of Polyphenols against Methanethiol
Polyphenols

Amount of addition（mg）

Deodorizing activity（％）

１．
０
１．
０

２
４．
８
１
０
０

０．
１
０．
０
１

１
０
０
７
０．
３

caffeic acid

１．
０

７
３．
６

chlorogenic acid（５−CQA）
rosmarinic acid

１．
０
１．
０

５
２．
７
１
０
０

０．
１

４
８．
１

protocatechuic acidgallic acidapigenin

１．
０

５．
４

kaempferol
quercetin

１．
０
１．
０

０．
０
５
８．
１

quercetin-３-O -rutinoside（rutin）

１．
０

０．
０

myricetinnaringenin

１．
０
１．
０

６
９．
３
１．
２

catechol

１．
０

１
０
０

（＋）catechin

１．
０

２
０．
９

176

Food Preservation Scienceused. We obtained ７．
９" of the hot-water extract
from

the

fresh

aerialof １
００" Glechomapartshederacea［２］in this experiment. If rosmarinic acid

VOL．３
５ NO．
４ ２
０
０
９

（ 14 ）

Jpn. Soc. Nutr. Food Sci., ５
６, ２
３〜２
７（２
０
０
３）
６）KITAMURA, S., MURATA, G. and HORI, M. : Colored
Illustrationsof

Herbaceous

Plants

of

Japan

（１．
４
８"）is contained in this extract（７．
９"）, the ratio（Hoikusya, Higashiosaka）
（１
９
９
８）

of rosmarinic acid to the extract is １
８．
７％ . The

７）MAKINO, T. : Revised Makino’s Illustrated Floradeodorizing activity of rosmarinic acid was ４８．
１％with the addition of ０．
１! in our result. Consideringthecontent

andthedeodorizing

activityofin Colour（Hokuryukan, Tokyo）
（１
９
８
９）
８）TOKITA, F., ISHIKAWA, M., SHIBUYA, K., KOSHIMIZU,
M. and ABE, R. :Deodorizing activity of somerosmarinic acid, rosmarinic acid is presumed toplant extracts against methyl mercaptan, Nippon

significantlyNōgeikagaku Kaishi, ５
８, ５
８
５〜５
８
９（１
９
８
４）

enhancethedeodorizing

effectof

Glechoma hederacea［２］.

９）MORS, W.B., DO, NASCIMENTO, M.C., PARENTE, J.P.,

In this study, we proved that high deodorizing
activity

substances

of

some

wild

grasses

DA SILVA, M. H., MELO, P.A. and SUAREZ-KURITZ,

were

G. : Neutralization of lethal and myotoxic activities

extracted by hot-water, rather than by methanol. In

of South American rattlesnake venom by extracts

particular, hot-water extracts of Gnapaliumaffine［５ ］, Glechoma hederacea ［ ２ ］, Artemisia capillaries［３］, Stenacis

annuus［６］andOenothera

biennis［８］are known to be edible. Some wild grasses may

and constituents of the plant Eclipta

prostrata（Asteraceae）, Toxicon, ２
７, １
０
０
３〜１
０
０
９（１
９
８
９）
１
０） YASUDA, H. and ARAKAWA, T. :Deodorizing

mechanism of（−）-epigallocatechin gallate against

contain polyphenol with high deodorizing activity.

methyl mercaptan, Biosci. Biotech. Biochem. , ５
９,But a correlation between the deodorizing activities

１
２
３
２〜１
２
３
６（１
９
９
５）

and

polyphenol

contents

wasnotobserved.

１１） NAKATANI, N. , MIURA, K. and INAGAKI, T. :Fractionation of potential extracts is essential for

Structure of new deodorant biphenyl compounds

determining the phenolic or nonphenolic compounds

from thyme（Thymus vulgaris L.）and their activity

responsible for the deodorizing activities. We areagainst methyl mercaptan, Agric. Biol. Chem., ５
３,

now analyzing the constituents contained in some

１
３
７
５〜１
３
８
１（１
９
８
９）

wild grasses with high deodorizing activities.

１２）OKUDA, T., HATANO, T., AGATA, I., NISHIBE, S. and
KIMURA, K. :References
１ ） YASUDA, H. and UI, M. :Tannins in Artemisia Montana, A.

princeps and Related Species of Plant, Yakugaku

Deodorant effect ofplant extracts of the family Rosaceae against
methyl mercaptan, Nippon Nōgeikagaku Kaishi, ６６,Zasshi, １
０
６, ８
９
４〜８
９
９（１
９
８
６）
１
３）IROBI, O.N., MOO-YOUNG, M., ANDERSON, W.A. andDARAMOLA, S. O. :Antimicrobial activity of bark

extracts of Bridelia ferruginea （Euphorbiaceae）, J.

１
４
７
５〜１
４
７
９（１
９
９
２）
K. :

Ethnopharmacol ., ４
３, １
８
５〜１
９
０（１
９
９
４）

Deodorizing activity of diterpenes from the leaves

１４） TOKITA, F. and FUNAYAMA, T. :２）YASUDA,H. ,SHIMIZU,

K.

andOSAWA,“ Saishin noof Rabdosia trichocarpa and of several α, β, -syōsyūzai to syōsyūgijyutsu ” , （ Kogyogijyutsukai,

unsaturated

Tokyo）, p. １
３
９（１
９
８
９）

carbonylcompounds

againstmethanethiol, Biosci. Biotechnol. Biochem., ６
０, ８
８〜９
１
（１
９
９
６）

SUGAHARA,

３）SHIMIZU, K., MAEDA, Y., OSAWA, K., SHIMURA, S.
and TSUNODA, M. :Deodorizing effect of cacao

polyphenols against methyl
ShokuhinKagakuKogakumercaptan,

Nippon,Kaishi, ４
８, ２
３
８〜２
４
５

T. :

O.

and

deodorization

with

NEGISHI,raw

fruits,Y. :Enzymatic

vegetables

and

（２
０
０
４）
１
６）KITAMURA, S. , TSUKAMOTO, Y. and KIJIMA, M. :K. :Deodorant

effectofextracts from wild grasses against methanethiol, J.

Vol.

２

（ Dohosya,

１７）YOSHIDA, T., AMAKURA, Y., LIU, Y. and OKUDA,
Euphorniaceous

YASUMOTO,,

Kyoto）, pp. ８
０
１〜８
０
２（１
９
８
８）

（１
９
９
９）
and

ku-ding-cha

derivatives, J. Agric. Food Chem. , ５
２, ５
５
１
３〜５
５
１
８

T. :

５）URABE, K., NADAMOTO, T., HURUTANI, M., TANAKA,

with

containing a large amount of caffeoyl quinic acidmushrooms, Food Sci. Technol. Res. , ５ , １
７
６〜１
８
０

Y.

Deodorization“ Honzouzufusogokaisetsu

（２
０
０
１）
４）NEGISHI,

１５） NEGISHI, O. , NEGISHI, Y. , YAMAGUCHI, F. andTanninsandrelatedplants.

XI.

polyphenols

of

Three

new

hydrolyzable tannins and a polyphenol glucosidefrom Euphorbia humifusa, Chem. Pharm. Bull . , ４
２,
１
８
０
３〜１
８
０
７（１
９
９
４）

（ 15 ）

〔Article〕 Deodorizing Effects of Wild Grass Extracts

１８）OKUDA, T., HATANO, T., AGATA, I. and NISHIBE,
S. :

The

componentsoftannicactivities

in

177

野草抽出物のメタンチオールに対する消臭効果

Labiatae plants. I. Rosmarinic acid from Labiatae

浦部貴美子＊１・東plants in Japan, Yakugaku Zasshi, １
０
６, １
１
０
８〜１
１
１
１

南野可奈子＊１・灘本知憲＊１・小野廣紀＊２

（１
９
８
６）

＊１

理恵＊１・稲田多希＊１

滋賀県立大学人間文化学部生活栄養学科

（〒５
２
２―８
５
３
３ 滋賀県彦根市八坂町２
５
０
０）
＊２

岐阜市立女子短期大学食物栄養学科

（〒５
０
１―０
１
９
２ 岐阜県岐阜市一日市場北町７―１）
滋賀県彦根市内に自生している野草（２６種類）の熱水
抽出物について，悪臭物質のひとつであるメタンチオー
ル（CH３SH）に対する消臭率を測定し，さらにメタノール抽出物の消臭率と比較検討した。消臭率は，ヘッドスペースガスを用いたガスクロマトグラフ法により残存するCH３SH量から求めた。その結果，熱水抽出物およびメタノール抽出物５．
０!添加により，それぞれ１２種と１１種の試料に，緑茶（熱水抽出物７８．
６％，メタノール抽
出物７２．
４％）よりも高い消臭効果が認められた。さらに，１００％の消臭率を示した抽出物はそれぞれ８種類であった。このうち，カキドオシ（Glechoma hederacea），
カ ワ ラ ヨ モ ギ（Artemisia capollaris），キ ジ ム シ ロ（Porentilla fragarioides），ハ ハ コ グ サ（Gnaphaliumaffine），ヒメジョン（Stenactis annuus），メマツヨイグサ（Oenothera biennis）に含まれる消臭活性成分は，メ
タノールよりも熱水によって効率よく抽出された。これ
ら野草熱水抽出物の高い消臭性は，これまでのところ報告されていない。消臭効果の期待できる新しい植物由来の素材であることが示唆された。
（平成２０年７月９日受付，平成２
１年３月９日受理）

（ 17 ）

Food Preservation Science VOL. ３
５ NO. ４ ２００
９ 〔Article〕

179

Purification and Characterization of Polyphenol Oxidasefrom Japanese Butterbur（Petasites japonicus）
HAN Yunzhe＊１，ZHAO Fushan＊１，OGAWA Takeshi＊２，
OHTA Mayumi＊２ and FUJITA Shuji＊２§＊１ Yanbian Institute of Agricultural Science, Longjing Jilin 133400, China＊２ Laboratory of Food Science, Faculty of Agriculture, Saga University,
1, Honjoumachi, Saga-shi, Saga 840−8502, Japan
Polyphenol oxidase（o-diphenol : oxygen oxidoreductase, EC １．
１０．
３．
１, PPO）in Japanese butterbur was
purified about 〜１５
１-fold with a recovery rate of １
１．
３％ by acetone fractionation, ion exchange
chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme appeared as asingle band on PAGE and SDS-PAGE. The molecular weight of the enzyme was estimated to beabout ２６，
０
００ and ２５，０
００ by gel filtration and SDS-PAGE, respectively. The purified enzyme quickly
oxidized chlorogenic acid and（−）-epicatechin. The Km values of the enzyme were ０．
１
４ mM forchlorogenic acid（pH ５．
０, ３０℃）and ０．
７ mM for（−）-epicatechin（pH ８．
０, ３０℃）. The optimum pHs
were ５．
０ and ８．
０ for chlorogenic acid oxidizing（ChO）and（−）-epicatechin oxidizing（EpO）activities,
respectively. In the pH range from ４．
０ to ９．
０, both ChO and EpO activities were stable at ４℃ for２２ h. The optimum temperature of both activities was found at ３
０℃ . Both activities were ５
０％ inactivated after heat treatment at ６
０℃ for １
０ min. Both activities were strongly inhibited by Lascorbic acid and L-cysteine at ５ mM
（Received Jan. ８, ２００９；Accepted Mar. １
１, ２
０
０
９）
Key words：polyphenol oxidase, Japanese butterbur（Petasites japonicus）, chlorogenic acid oxidase,（−）-epicatechin oxidase, purification and characterization
ポリフェノール酸化酵素，フキ（Petasites japonicus），クロロゲン酸酸化酵素活性，
エピカテキン酸化酵素活性，精製と性質

Enzymatic browning in raw fruits and vegetables
is important in their preservation and processing,and is generally considered to be an undesirable

differences in the substrate specificity and someproperties

of

PPO

from

various

fruits

and

４）
〜１
１）
，
１
３）
〜１
８）

vegetables.

reaction because it leads to unpleasant appearance

Japanese butterbur（Petasites japonicus）is one of theand the concomitant development of off - flavors.

most common vegetables in Japan, and a brown

Polyphenol oxidase（o-diphenol : oxygen oxidoreductase,scum caused by PPO always forms during its

EC １．
１
０．
３．
１，PPO）is found in most higher plants

processing and／or cooking. However, details of theandpurification and properties of Japanese butterbur

isresponsiblefor

enzymatic

browning.

It

catalyzes the oxidation of polyphenols in plants to o-

PPO

have

not

hitherto

been

published.

Toquinones, which react with themselves or other

determine the properties of PPO, pyrocatechol andphenolics to form a brown pigment. To prevent the

４-methylcatechol

aregenerally

used

as

１
１）
，
１
３）
，
１
４）

browning that results in decreased marketability of

substrate

the agricultural products, this enzyme has been

quinic acid）seems to be the normal substrate as it

characterized in many fruits and vegetables.１）〜１１）

is widely distributed in plant tissues and is strongly

Many researchers have demonstrated that PPO in

oxidizedplanttissuesislocalizedinthe；however, chlorogenic acid（５-caffeoyl

by

plant

PPO６），７），９），１５）〜１８） .

In

Japanese１９）chloroplast,butterbur , the presence of a few chlorogenic acidparticularly in the thylakoid membrane, or in theanalogues has been reported, and the changes in

２）
，
３）
，
１
２）

nongreen plastid-type component. In the field offood chemistry, many researchers have also shown§ Corresponding author, E-mail : fujitas@cc.saga-u.ac.jp

absorption

anddifference

spectraduringtheoxidation of Japanese butterbur extracts as well as180

Food Preservation Science

VOL．３
５ NO．
４ ２
０
０
９

（ 18 ）of edible burdock extracts have indicated that theby the colorimetric method１０）. The reaction mixture

browning is caused by chlorogenic acid oxidation１９），２０）.

containing ０．
５! of １０ mM aqueous solution of

Therefore,various polyphenols（see Table ２）, ４! of McIlvaine

theenzymatic

browningofJapanesebutterbur seems to be mainly due to the oxidationbuffer （ pH ５．
０）, and ０．
５ ! of enzyme solution.

of chlorogenic acid analogues by endogeneous PPO.

After ５ min of incubating the mixture at ３
０℃, theIn our laboratory, it has been clarified that theincrease

purified PPOs of composite vegetables, such asmeasured

１
５）

１
６）

in

at ４
２
０ ! （ ∆A４２０ ） was

absorbance
using

a

Shimadzu

MPS- ２０００garland chrysanthemum , leaf lettuceand edible

spectrophotometer. One unit of the enzyme activity

burdock１７） ,oxidizeand （ − ）-was defined as ∆A４２０ of ０．
１ per minute and per

epicatechin,however,pH

milliliter of enzyme solution（１㎝ light path）.

chlorogenic
the

acid

optimumoftheenzyme differs depending on the type of substrateused. It is interesting that the same difference in

３．Protein determination
Proteinwas２２）determined

by

the

methodofoptimum pH was observed in the purified PPO of

HARTREE

JapaneseJapanese

Katayama Chemical Co., Osaka, Japan）as standard.

butterbur PPO was purified using chlorogenic acid

In the chromatography, protein was determined byas substrate, and some properties of the purified

measuring the absorbance at ２
８０ !.

enzyme were determined.

４．Enzyme purification

butterbur.

In

this

study,All steps of purification were carried out at ４℃.

Materials and Methods

The edible part（leafstalk）of Japanese butterbur（３１．Materials
Japanesebutterbur

waspurchasedusing bovine serum albumin（fraction V,

at

local

# ） washomogenized

in １，
５
０
０ !

potassium

phosphate ／ ０．
１ M

of ０．
１ M

sodiumphosphatemarkets in Saga Prefecture, Japan. DEAE-Cellulofine

buffer （０．
１ M phosphate buffer, PB, pH ７．
０）

AL

containing ２％ NaCl, １％ L-ascorbic acid, and １％

waspurchasedfrom

Chisso,Tokyo,

Japan.

Butyl-Toyopearl ６
５０ M, Phenyl-Toyopearl ６
５０ M andpolyvinyl

Toyopearl HW ５
５-superfine（HW ５５-S）were purchased

homogenate through cotton cloth, the filtrate waspolypyrrolidone.

Afterfilteringthefrom Toso Co. , Tokyo, Japan. The other reagentscentrifuged at １０，
３
００×" for ２
０ min, and acetone

used were purchased from Wako Pure Chemical Co.,wasOsaka, Japan and Katayama Chemical Co. , Osaka,

precipitate obtained from the ８０％ acetone fraction

Japan.

was collected by centrifugation at １０，
３
００×" for ２
０

added

to

the

supernatant.

The

protein

２．Assay of enzyme activity

min, dissolved in a small volume of ０．
０
１ M PB（pH（１）Chlorogenic acid oxidase（ChO）activity The

７．
０）, and then dialyzed against the same buffer forspectrophotometric method developed by TONO et al .２１）

３
６ h with four or more changes of the dialysis

was

The

medium. The dialyzed solution（crude enzyme）was

mixture to be tested consisted of ０．
５! of ０．
４ mM

applied to a DEAE-Cellulofine AL column（４×１４．
５

aqueous solution of chlorogenic acid, １! of ０．
１M㎝ ） equilibrated with ０．
０
１ M PB （ pH ７．
０）, andcitrate／０．
２ M potassium phosphate buffer（McIlvaine

eluted with a linear gradient of sodium chloride（０employedtomeasureChO

activity.

buffer, pH ５．
０）and ０．
５! of the enzyme solution ;to １．
０ M NaCl in ０．
０
１ M PB, pH ７．
０）. Enzyme

it was incubated at ３
０℃ for ５ min. After incubation,

activity

the reaction was stopped by adding ３ ! of ２％
metaphosphoric acid solution. For the control, ０．
５!
of the enzyme solution was added to a mixture ofammonium０．
５! of ０．
４ mM aqueous solution of chlorogenic

Butyl-Toyopearl column （１．
６×１１㎝ ） equilibrated

waselutedthebuffercontaining ０．
５ to ０．
８ M

NaCl.

The

enzymefractionsbrought

to

a １ M

were

with

collected,

sulfateconcentrationand

solution

appliedtoaacid, １! of McIlvaine buffer（pH ５．
０）, and ３! ofwith ０．
０
１ M PB（pH ７．
０）containing １ M ammonium

２％ metaphosphoric acid solution. The difference insulfateandelutedwithalineargradientofabsorbance at ３
２
５!（∆A３２５）between the control andammonium sulfate（１ to ０ M in ０．
０
１ M PB, pH

test solutions was measured using a Shimadzu MPS-

７．
０）. The enzyme was eluted with the buffer

２０００ spectrophotometer. One unit of the enzyme

solution containing ０．
６ to ０ M ammonium sulfate.

activity was expressed as ∆A３２５ of ０．
１ per minuteThe

per milliliter of the enzyme solution（１㎝ light path）.

dialyzed against ０．
０
１ M PB（pH ７．
０）with four or（２）PPO activity

more changes of the dialyzed medium. The dialyzed

PPO activity was measured

enzymeactivefractionswerepooledand（ 19 ）

enzyme〔Article〕 Characterization of Japanese Butterbur PPO

solution

ammoniumwasbrought

sulfateconcentrationand

to

a １．
５ M

applied

to

a

181

buffers at ４℃ for ２
２ h. Residual ChO and EpO
activities were determined under standard conditions

Phenyl-Toyopearl column （１．
６×１１㎝ ） equilibrated（ChO : pH ５．
０, ３
０℃；EpO : pH ８．
０, ３
０℃）
.

with ０．
０
１ M（３）Optimum temperature

PB （ pH ７．
０ ） containing １．
５ M

ChO and EpO

ammonium sulfate and eluted with a linear gradientactivities were determined at various temperaturesof ammonium sulfate（１．
５ to ０ M in ０．
０
１ M PB,（２
０ to ６０℃）in McIlvaine buffer（ChO : pH ５．
０；

pH ７．
０）. The enzyme was eluted with the buffer

EpO : pH ８．
０）.

solution containing ０．
８ to ０．
５ M ammonium sulfate.

（４）Thermal stability

The

and

heated at various temperatures between ３
０ to ８０℃

dialyzed against ０．
０
１ M PB（pH ７．
０）with four orfor １
０ min and residual ChO and EpO activities

more changes of the dialyzed medium. The dialyzed

were determined under standard conditions（ChO :enzyme solution was applied to a Toyopearl HW ５
５-

pH ５．
０, ３
０℃；EpO : pH ８．
０, ３
０℃）
.

enzymeactivefractionswerepooledThe enzyme solution was

S（１．
６×８
９㎝）column equilibrated with ０．
１ M PB（５）Effect of various compounds（pH ７．
０）and eluted with the same buffer. The PPO

EpO activities were measured in the presence（final

active fractions were pooled and used for enzyme

concentration, ５ mM or １
０ mM ） and absence ofcharacterization.

various compounds under standard conditions（ChO :gelelectrophoresis （ native

pH ５．
０, ３
０℃；EpO : pH ８．
０, ３
０℃）
.

PAGE）

Results and Discussion

The purified enzyme was electrophoresed by themethod of DAVIS ２３）, using ７．
５％ polyacrylamide gelat pH ９．
０.

Enzyme６．Molecular weight determinationbygelfiltration

and

was

purifiedfromhomogenates

SDS-PAGE.

DEAE-Cellulofine AL, Butyl-Toyopearl ６
５０ M, Phenyl-

Gel

0.5

filtration was conducted using a Toyopearl HW ５
５-S60

eluted with ０．
１ M PB（pH ７．
０）, by the method of
ANDREWS２４）. Chymotrypsinogen A（MW ２５，０００）, egg
albumin（MW ４５，０００）, bovine serum albumin（MW６５，０００）, and γ-globulin（MW １２５，０００）were used as
marker proteins at a flow rate of ２
０! ／ h. SDSPAGE was carried out by the method of LAEMMLI２５）,
using an SDS marker protein kit （ Oriental Yeast
Co., Tokyo, Japan）as standard.

Protein（OD 280 nm）

column （１．
６×８９ ㎝ ）, which was equilibrated and

50

0.4

40
0.3
30
0.2
20
0.1

10

７．Assay of enzyme properties（１）Optimum pH

The effects of pH on ChO

and EpO activities were determined at ３
０℃ in

0

buffers.
The enzyme was preincubatedin McIlvaine（pH ３．
０ to ８．
０）and Atkins & Pantin

00McIlvaine （ pH ３．
０ to ８．
０） and Atkins & Pantin（２）pH stabilityofJapanese butterbur by acetone fractionation, and

The molecular weight of the purified enzyme wasestimated

１．Enzyme purification

Enzyme activity（unit/mℓ）

５． Polyacrylamide

ChO and

20
40
60
80
100
Fraction number（2.7 mℓ/tube）

Fig．
１ Elution pattern of enzyme on Toyopearl HW５５-S○：Protein，●：Enzyme activity, ― : Pooled fraction

Table １ Purification of PPO from Japanese butterbur
Volume（!）

Total activity（unit）

８０％ acetone sulfate
DEAE-Cellulofine AL

２
５
０
５６
３

１
５，
９
０
０
９，
１
８
８

Butyl-Toyopearl ６
５０M
Phenyl-Toyopearl ６
５
０M

５
６
３
５
６

２，
４
３
２
２，
３
６
０

ToyopearlHW ５
５-S

７
０
０

１，
７
９
７

Step

Purification（−fold）

Yield
（％）

７
２
４
８

１
３
５

１
０
０
５
８

４．
６
３．
７

５
２
８
６
３
８

７
５
９
１

１
５
１
４．
８

１．
７

１，
０
５
７

１
５
１

１
１．
３

Total protein
Specific activity（mg）（unit／mg protein）
２，
２
８
８
３
７

182

Food Preservation Science

VOL．３
５ NO．
４ ２
０
０
９

（ 20 ）

HW ５
５ -S

butterbur PPO quickly oxidized not only chlorogenic

chromatographies. Fig ．
１ shows the typical elution

acid but also（−）-epicatechin. The enzyme had apatternlow

Toyopearl ６
５
０ M
of

Toyopearl

the

and

ToyopearlenzymeHW ５
５ -S

activitycolumn.

ontheEnzymefinal

activity

activity

towardo-diphenols

other

such

as

pyrocatechol, catechin and caffeic acid, but had noshowed one peak and the peak fractions from

activity toward dopamine orseveral columns were pooled as the purified PPO.

substrate specificity was observed in the PPOs of

DL-dopa.

A similar

A summary of the typical purification of thecomposite vegetables such as head lettuce７）, garland

enzyme is given in Table １. Finally, the enzyme

chrysanthemum１５）, leaf lettuce１６） and edible burdock１７）,

was purified 〜１
５
１-fold with a recovery rate ofand of Japanese pears５）. The enzyme also had no１
１．
３％, relative to that of the crude enzyme.

activity

２．Characterization of Japanese butterbur PPO

pyrogallol, gallic acid,

The purified enzyme produced a single band on

towarddiphenolssuch

trihydroxybenzenes
as

and

such

phloroglucinol,

resorcinol.

Km

The

or

as
m-

valuesnative PAGE and SDS-PAGE（Fig．
２）. These results
suggest that Japanese butterbur PPO was purified

（A）

（B）

to a homogeneous state. The molecular weight ofM.W.

the enzyme was estimated to be about ２
６，
０
０
０ and

P.M

E

２
５，
０
０
０ by gel filtration and SDS-PAGE, respectively

Origin（ Fig ．
３）. These results indicate that the purified
PPO is a monomer protein. The molecular weight issmaller

than

those

of

the

PPOs

of

←

composite

74,400７）
vegetables such as head lettuce （ MW ５
６，
０
０
０）
,

49,600

１
５）garland chrysanthemum（MW ４
５，
０
０
０）
, leaf lettuce

37,200

１
６）
１
７）
（MW ４
６，
０
０
０）
, and edible burdock（MW ４
０，
０
０
０）
,

all of which mainly oxidize chlorogenic acid. The

Front

←

24,800molecular weight is also smaller than those of the９）
PPOs of apples（MW ６
５，
０
０
０）
, banana pulp（MW
１
０）
１
５）
４
１，
０
０
０）
, and Japanese pears（MW ５
６，
０
０
０）
, but isalmost the same as that of another edible burdock
２
７）
PPO（MW ２５，０００）which mainly oxidize pyrogaroll

Fig．
２ SDS-PAGE（A）and native PAGE（B）of purified
enzymeand phloroglucinol.
As shown in Table ２, the purified

M.W : molecular weight, P.M : protein marker,←：purified enzyme.

Japanese

5.4

4.9
1

（A）

5
4.8

2

4.6

3

4.2
1.5

1.6

1.7

Ve/Vo

2

4.6

3

4.4

E
1.4

4.7

（B）

4.5

4

4.4

1

4.8

Log M.W.

Log M.W.

5.2

1.8

1.9

E

4

4.3
0.2

0.4

0.6

0.8

1

Relative Mobility

Fig．
３ Estimation of molecular weight of the enzyme by gel filtration on Toyopearl HW５５-S（A）and SDS-PAGE（B）（A）
M.W : molecular weight, Vo : void volume of the column, Ve : elution volume of the substance, １：r-globulin；
（１
６
０，０
０
０）
, ２：bovine
serum albumin（６５，０００）, ３：egg albumin（４５，０００）, ４：chymotrypsinogen A（２５，０００）, E : purified enzyme.
（B）
１：Cytochrome c hexamer（M.W：７
４，
４
０
０）
, ２：Cytochrome c tetramer（４９，０００）, ３：Cytochrome c trimer（３７，２００）, ４：Cytochromec dimmer（２４，８００）, E : purified enzyme.

（ 21 ）

〔Article〕 Characterization of Japanese Butterbur PPO

Table ２ Substrate specificities of enzyme
Substrate（Michaelis constant）of the enzyme, measured using
chlorogenic acid（pH ５．
０, ３０℃）and（−）-epicatechin

Relative activity（％）

Chlorogenic acid（−）
−EpicatechinCaffeic acid
Catechol
Catchin
Dopamin
DL-dopa
Resorcinolcinol
Phloroglucinol
Gallic acid
Pyrogallol

183（pH ８．
０, ３０℃）as substrates, were ０．
１
４ and ０．
７ mM,

１
０
０
９４
４
２
３
９
３
７
９
２
０
０
０
０

respectively（Fig．
４）. These values were lower thanthose of leaf lettuce PPO１６） and edible burdock
PPO１７）.
Several PPOs show different optimum pHs fordifferent substrates : for example, the optimum pHsof garland chrysanthemum PPO１５） for chlorogenic
acid and epicatechin were ４．
０ and ８．
０, respectively.
The optimum pHs of sweet pepper PPO２７） for
chlorogenic acid and pyrocatechol oxidation were
４．
０ and ７．
０, respectively. Fig．
５（A）shows the effectsof pH on the ChO and EpO activities of thepurified Japanese butterburPPO. The optimum pHs
for ChO and EpO were ５．
０ and ８．
０, respectively.
These optimum pH differences were also observed

20
（A）

40

−1
1/V（unit/mℓ/min）−1
1/V（unit/mℓ/min）

50

30
20
10

15
10
5
0

0
­20

（B）

0

20
40
1/S（mM−1）

­2

60

0

2
4
1/S（mM−1）

6Fig．
４ Lineweaver-Burk plots of chlorogenic acid（A）and（−）−epicatechin（B）oxidation by the enzyme.
V : Rate of the enzyme reaction, S : substrate concentration.

120
ChO

100

Relative activity（%）

Relative activity（%）

120
EpO

80
60
40

ChO

100

EpO

80
60
40
（B）

（A）
20

20

0

0
0

2

4

6
pH

8

10

0

2

4

6

8

10

pH

Fig．
５ Effects of pH on the activity（A）and stability（B）of enzyme○：ChO, chlorogenic acid oxidase activity；●：EpO，（−）-epicatechin oxidase activity.
（Ａ）PPO activity was measured at３０℃ in McIlvaine（pHs３．
０to８．
０）and Atkins & Pantin（pH ９）buffers.
（Ｂ）PPO was preincubated in McIlvaine（pHs３．
０to８．
０）or Atkins & Pantin（pH ９）buffers at ４℃ for２
２h. Residual ChO and EpO
activities were measured under standard conditions（ChO : pH５．
０，３０℃；EpO : pH８．
０，３
０℃）
.

184

Food Preservation Science

VOL．３
５ NO．
４ ２
０
０
９

120

120
ChO

100

ChO

100

EpO

Relative activity（%）

Relative activity（%）（ 22 ）

80
60
40
（A）

EpO

80
60
40
（B）
20

20

0

0
0

20
40
60
Temperature（℃）

20

80

40

60

80

100

Temperature（℃）

Fig．
６ Effects of temperature on the activity（A）and stability（B）of the enzyme○：ChO, chlorogenic acid oxidase activity；●：EpO，（−）-epicatechin oxidase activity.
（Ａ）PPO activity was assayed at various temperatures（２０〜６０℃）in McIlvaine buffer（ChO : pH５．
０，EpO : pH８．
０）.
（Ｂ）PPO solution was incubated at various temperatures（３０〜８０℃）for１
０min before measuring PPO activity. Residual ChO and EpO
activities were measured under standard conditions（ChO : pH５．
０,３０℃；EpO : pH８．
０,３０℃）

in composite vegetables such as head lettuce７）, leaf
１
６）

lettuce１
７）

６）

thermal stabilities（Fig．
６（B））. After heat treatment

and edible burdock , and eggplants . On

at ６
０℃, ７
０％ of the EpO activity, but only about５）

the other hand, the purified Japanese pear PPO

５０％ of the ChO activity remained. The thermal

had an optimum pH of ４．
２ for the two substrates.

stabilities of both ChO and EpO activities were

The acidic optimum pHs for chlorogenic acid haverelatively

also been reported for PPOs of eggplants６）, apples９）

lettuce７）, garland chrysanthemum１５） and leaf lettuce１６）.

approximated,asobserved

inhead

and leaf lettuce１６）, which were found in the pH

The effects of various compounds on the purified

range of ４．
０ to ５．
０, for chlorogenic acid oxidation.

enzyme activity are listed in Table ３. The ChO andIn comparison with this, the optimum pHs of the

EpO activities were markedly inhibited by sodium

PPOs of banana pulp１０）, banana peel２８）, and guava２９）diethyldithiocarbamate, KCN, and NaF at ５ and １０were found to be near neutrality using （ − ）-mM. The presence of metal ions（Zn２＋ , Cu２＋ , Ba２＋ ,epicatechin,andand Mn２＋）at １
０ mM reduced ChO activity by ７０％.

dopamine as substrates. Therefore, the ChOs in

The complete inhibition of ChO and EpO activities

these plants were assumed to be acidic PPOs. Fig．
５

was induced by L-ascorbic acid and L-cysteine at ５（B）shows the pH stabilities of the ChO and EpO

and １
０ mM, respectively. Similar effects of these

activities of the purified PPO. Both activities werecompounds were found for the PPOs of Japanesestable in the pH range of ４ to ９；〜７
０％ of enzyme

pears５）, head lettuce７）, garland chrysanthemum１５）, leaf

activity remained after incubation in solutions oflettuce１６） and edible burdock１７）. Sodium chloride also

various pHs from ３ to ９ for ２
２ h. The pH stability

markedly inhibited the ChO and EpO activities.

pyrocatechol,

４ -methylcatechol,of the purified enzyme was different from that ofConsidering the above-mentioned different effects

garland chrysanthemumPPO１３）, the purified enzyme

of pH, temperature and various compounds on the

being stable in the pH range from ５ to １
１. The pH

ChO and EpO activities of the purified enzymes ofstability of the purified enzyme was also slightly

composite vegetables and Japanese pear PPO, it１７）different from that of edible burdock PPO , theappears that these enzymes may have separate

enzyme being stable in the pH range from ５ to ７.

sites for the two activities.

As shown in Fig．
６（A）,the purified enzymeshowed aConclusion

single optimum temperature with a maximum activity
at ３
０℃, which is similar to the optimum temperatures
７）of the PPOs of head lettuce

１６）

and leaf lettuce , but

The PPO of Japanese butterbur was purified
approximately １５
１-fold witharecoveryrateofis slightly different from that of edible burdock１
１．
３％ using chlorogenic acid as a substrate. The

PPO１７）（２０℃）. ChO and EpO had slightly different

molecular

weightofthepurified

PPO （ MW（ 23 ）

〔Article〕 Characterization of Japanese Butterbur PPO

185

Table ３ Effects of various compounds on the enzyme activity
Relative activity（％）
aCompound

（−）
−EpOb

ChO
５ mMc

１
０ mMc

５ mMc

１
０ mMc

None
Sodium diethyldithiocarbamate

１
０
０
６

１
０
０
０

１
０
０
２

１
０
０
０

KCN
EDTA

１
８
１

０
６
２

０
１
０
０

０
９
４

ZnSO４

８
３

２
６

―d

―d

CuSO４
BaCl２

６
７
７
８

１
９
２
８

１
０
０
―d

―d
―d

MnCl２

６
１

２
５

―d

―d

NaF

３

０

１
０
０

１
０
０

NaCl

９
３

９
３

１
０
０

１
０
０

L-ascorbic acid

０

０

６

０

L-cysteine

０

０

０

０

a

ChO : chlorogenic acid oxidase. The enzyme activity was determined at pH ５ using Mcllvaine buffer.

b

EpO：（−）−epicatechin oxidase. The enzyme activity was determined at pH ８ using Mcllvaine buffer.
Final concentration of the compound.

cd

Not determined.

２
５，
０
０
０） is smaller than those of the PPOs ofcomposite vegetables such as head lettuce （ MW
７）
５
６，
０
０
０）
, garland chrysanthemum（MW ４５，０００）１５）,
１
６）
leaf lettuce（MW ４
６，
０
０
０）
, and edible burdock（MW

４
０，
０
０
０）１７）；however, the substrate specificity of thepurified PPO, and the effects of pH, temperature,and various compounds on the purified enzyme

Nogei Kagaku Kaishi, ６
０, ７
０
５〜７
１
２（１
９
８
６）（ inJapanese）
６）FUJITA, S. and TONO, T. : Purification and someproperties

ofpolyphenol

oxidasein

eggplant（Solanum melongena ）, J. Sci. Food Agric., ４
６, １
１
５〜
１
２
３（１
９
８
８）
７）FUJITA,

S. ,

TONO,

T.

and

KAWAHARA,

H. :activity were similar to those on the enzymes of

Purification and properties of polyphenol oxidase

the composite vegetables. These results suggest thatin head lettuce（Lactuca satiua）, J. Sci. Food Agric.,

the browning of Japanese butterbur is caused by
the oxidation of chlorogenic acid and its analogues,

５
５, ６
４
３〜６
５
１（１
９
９
１）
８）FUJITA, S., SAARI, N., MAEGAWA, M., TETSUKA, T.,

and that L-ascorbic acid and L-cysteine are effective

HAYASHI,inhibitors of such enzymatic browning.

properties of polyphenol oxidase from cabbage

N.

and

TONO,

T. :

Purificationand（ Brassica oleracea L. ）, J. Agric. Food Chem. , ４
３,References
１）MAYER, A. E. :

１
１
３
８〜１
１
４
２（１
９
９
５）

Polyphenol oxidase in plants-

Recent progress, Phytochemistry, ２
６, １
１〜２
０（１
９
８
７）
２）MAYER,

A.

E.

and

HAREL,E. :Polyphenol

oxidases in plants, Phytochemistry, １
８, １
９
３〜２
１
５
（１
９
８
７）
３）VAUGHN, K. C. , LAX, A. R. and DUKE, S. O. :lyphenol oxidase : the chloroplast oxidase with no９）MURATA, M. , KUROKAMI, C. and Homma, S. :Purification and properties of chlorogenic

acid

apple （ Malus

Biosci.

oxidasefrom

pumula ）,Biotechnol. Biochem., ５
６, １
７
０
５〜１
７
１
０（１
９
９
２）
１
０） YANG, C. -P. , FUJITA, S. , ASHURAFUZAMAN, Md. ,NAKAMURA, N. and HAYASHI, N. : Purification andcharacterizationofpolyphenol

oxidasefromestablished function, Plant Physiol . , ７
２, ６
５
９〜６
６
５

banana （ Musa sapientum L. ） pulp, J. Agric. Food

（１
９
８
８）

Chem., ４
８, ２
７
３
２〜２
７
３
５（２
０
０
０）

４）VAUGHN, K. C. and DUKE, S. O. : Function ofpolyphenol oxidase in higher plants, Plant Physiol .,
６
０, １
０
６〜１
１
２（１
９
８
４）

１
１）KAHN,V. :Polyphenol

oxidasein

avocado,Phytochemistry., １
５, ２
６
７〜２
７
２（１
９
７
６）
１
２）PARISH, R. W. :The intracellular location of

５）TONO, T., FUJITA, S., KAWASAKI, H. and LI, C-F. :phenol oxidases, peroxidase and phosphatases in

Purification and high L-epicatechin oxidase activity

the leaves of spinach beet （ Beta vulgarisL. ssp,

in polyphenol oxidase of Japanese pear, Nippon

vulgaris）, Eur. J. Biochem., ３
１, ４
４
６〜４
５
５（１
９
７
２）

186

Food Preservation Science

VOL．３
５ NO．
４ ２
０
０
９

（ 24 ）

１３）LUH, B. S. and PHITHAKPOL, B. : Characteristics of２６）YANG, C.-P., FUJITA, S., KOHNO, K., KUSUBAYASHI,

polyphenoloxidase related to browning in cling

A., ASHURAFUZZAMAN, Md. and HAYASHI, N. : Partial

peaches, J. Food Sci., ２
７, ２
６
４〜２
６
８（１
９
７
２）

purification

１４）RIVAS, N. J. and WHITAKER, J. R. : Purificationand some properties of two polyphenol oxidases
from Bartlettpears, Plant Physiol . , ５
２, ５
０
１〜５
０
７
（１
９
７
３）

and

characterizationofpolyphenol

oxidase from banana（Musa sapientum L.）peel, J.
Agric. Food Chem., ４
９, １
１
４
６〜１
１
４
９（２
０
０
１）
２
７）MURAO, S., OYAMA, H., NOMURA, Y., TONO, T. and
SHIN,

T. :

Purification

andcharacterizationof１５）NKYA, E., KOUNO, C., LI, Y.-J., YANG, C-P., HAYASHI,

Arctium lappa L. （ Eddible burdock ） polyphenol

N. and FUJITA, S. : Purification and characterization ofoxidase, Biosci. Biotechnol. Biochem. , ５
７, １
７
７〜１
８
０

polyphenol oxidase from garland chrysanthemum

（１
９
９
３）

（Chrysanthemum coronarium L.）, J. Agric. Food Chem.,
５
１, ５
４
６
７〜５
４
７
１（２
０
０
３）

KAWASAKI, H. : Occurrence of enzymatic browning

１６）HAN, Y., HAYASHI, N. and FUJITA, S. : Purificationand properties of polyphenol oxidase from leaf
lettuce（Lactuca sativa ）, Food Preservation Science.,
３
１, ２
９
５〜３
０
１（２
０
０
５）
（in Japanese）
S. : Purification

chlorogenic

acid

and

oxidasecharacterizationfromof sweet pepper

seedduringlow-temperaturestorage, Bull. Fac. Agric. Saga Univ. , ６
３, １
５〜２
４
（１
９
８
７）
（in Japanese）
２
９）AUGUSUTIN, M. A., GHAZALI, H. M. and HASHIM,

１７）HAN, Y., MAMIYA, A., NKYA, E., HAYASHI, N. and
FUJITA,２８）LI, Z.-F., TONO, T., KOJIMA, T., FUJITA, S. andedibleofburdock

H. :Polyphenoloxidasefrom

guava （ Psidium

guajavaL ）, J. Sci. Food Agric. , ３
６, １
２
５
９〜１
２
６
５
（１
９
８
５）

（Arctium lappa L.）, Food Preservation Science., ３
２,
２
７
５〜２
８
１（２
０
０
６）
１
８）FUJITA, S. and TONO, T. : Enzymatic browning ofimmature fruits of Satsuma mandarin and its
chlorogenic acid oxidase activity, Nippon Shokuhin
Kogyo Gakkaishi.,２
８,６
０
０〜６
０
５（１
９
８
１）
（in Japanese）
１
９）NAKABAYASHI, T. : Studies on the tannin of fruits
and vegetables. Part III. Polyphenolic compounds
and phenol oxidative enzymes of edible burdock,Nippon Shokuhin Kogyo Gakkaishi. , １
５, １
９
９〜２
０
６

フキ（Petasites japonicus）のクロロゲン酸
酸化酵素の精製と性質
韓雲哲＊１・趙福善＊１・小川剛史＊２＊２

太田真由美 ・藤田修二＊２＊１

延辺農業科学研究院

（〒１
３
３
４
０
０ 中国：吉林省龍井市）＊２

佐賀大学農学部

（〒８
４
０―８
５
０
２ 佐賀県佐賀市本庄町１番地）

（１
９
６
８）
（in Japanese）
２０）TONO, T., KAWASAKI, H., FUJITA, S. and Li, C.-F. :Determination of chlorogenic acid

in

food

フキのポリフェノール酸化酵素をアセトン沈殿，イオby

ン交換クロマトグラフィー，疎水クロマトグラフィーおdifference spectra spectrophotometry, Nippon Nogei

よびゲル濾過により精製した。これらの操作により，本Kagaku Kaishi, ６
１, １
４
４
１〜１
４
４
７（１
９
８
７）

酵素はアセトン分画後の粗酵素液の約１１５倍に精製され，

２１）TONO, T. and FUJITA, S. : Assay of chlorogenic

回収率は１
１．
３％であった。本酵素はPAGEおよびSDS-

acid oxidase by difference spectra spectrophotometry,

PAGE上で単一のバンドを示し，電気泳動的に均一であ

Agric. Biol. Chem., ５
１, ２
８
４
３〜２
８
４
４（１
９
８
７）

った。精製酵素の分子量はゲル濾過およびSDS-PAGEに

２
２）HARTREE, E. F. :Determination of protein :aよりそれぞれ２
６，
０
０
０および２
５，
０
０
０と推定された。本酵素

modification of Lowry method that gives a linear

はクロロゲン酸および（−）-エピカテキンを強く酸化し，

photometric response, Anal. Biochem., ４
８, ４
２
２〜４
２
７

クロロゲン酸（pH ４．
０，３０℃で測 定）お よ び（−）-エ
ピカテキン（pH ８．
０，３０℃で測定）の酸化反応時のミ

（１
９
７
２）
２
３）DAVIS, B. J. : Disc gel electrophoresis. ２. Method

カエリス定数はそれぞれ，０．
１
４ mMおよび０．
７ mMで

and application to human serum proteins, Ann. N.

あった。本酵素のクロロゲン酸酸化活性（ChO）およびY. Acad. Sci., １
２
１, ４
０
４〜４
２
７（１
９
６
４）

（−）
-エピカテキン酸化活性（EpO）の最適pHはそれぞof

れ，４．
０および８．
０であった。ChO，EpOの両活性ともpH

proteins related to their molecular weight over a

４〜９の範囲で，４℃，２２時間安定であった。両活性とwide range, Biochem. J ., ９
６, ５
９
５〜６
０
５（１
９
６
５）

も最適温度は３０℃であり，６０℃で１０分間の加熱処理に対２４）ANDREWS,

P. :

The

gel-filtration

behavior

２５）LAEMMILI, U. K. : Cleavage of structural proteinsduring the assembly of the heat of bacteriophage
T ４, Nature, ２
２
７, ６
８
０〜６
８
５（１
９
７
０）

して安定であった。両活性とも５ mMのL-アスコルビン
酸およびL-システインにより強く阻害された。
（平成２１年１月８日受付，平成２
１年３月１
１日受理）

（ 25 ）

Food Preservation Science VOL. ３
５ NO. ４ ２００
９ 〔Article〕

187

Effects of Postharvest Ethanol Vapor Treatment on Qualityof Sudachi（Citrus sudachi hort. ex. Shirai）Fruit
NOMA Yuta＊１，SUZUKI Yasuo＊１§，TERAI Hirofumi＊１ and YAMAUCHI Naoki＊２＊１Graduate School of Agricultural Science, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657−8501＊２ Faculty of Agriculture, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753−8515

Sudachi（Citrus sudachi hort. ex. Shirai）fruit is a kind of acid citrus. It rapidly degreens at room
temperature. Ethanol treatment retards ripening and senescence of harvested horticultural products.
We investigated the effects of postharvest ethanol vapor treatment with ethanol pads on the qualityof sudachi fruit to prolong the storage period. Mature green sudachi fruit were packed in a perforated
polyethylene bag without（control）or with a ０．
３, ０．
６, １, ３ or ６" ethanol pad as the ethanol vapor
treatment and stored at ２
０℃ in darkness. The chlorophyll contents of fruit treated with a １ "ethanol pad decreased more slowly than those of the control fruit, accompanying suppression of loss offresh weight. There were no significant differences in the internal quality between the control andethanol vapor-treated fruit, including total soluble solids（TSS）, titrable acidity（TA）, TSS ／ TA ratio,
pH, L-ascorbic acid content, and L-dehydroascorbic acid content in the pulp juice. However, peelbrowning of fruit treated with ３ and ６" ethanol pads occurred. From these results, it isdemonstrated that ethanol vapor treatment of sudachi fruit could retard degreening and suggest thepossibility that treatment could prolong the storage period without negative impacts on internal
quality.
（Received Jan. １９, ２００９；Accepted Mar. ２
６, ２
０
０
９）
Key words：chlorophyll degradation, degreening, ethanol vapor treatment, quality, sudachi
クロロフィル分解，脱緑，エタノール蒸気処理，品質，スダチ

Sudachi（Citrus sudachi hort. ex. Shirai）fruit is awhich shows no change in its low CO２ and ethylene

kind of acid citrus similar to lime and yuzu fruit.

production rates during ripening. However, ethylene

Green sudachi fruit is preferred by consumers. It

has been reported to be necessary for the ripening

has a characteristic aroma, to which limonene, α-of citrus fruit, accompanying degreening of the peel

pinene, decanal, citronellal and others contribute. It

color,is harvested at the green stage because favorable

degreening of the peel３） andchangeinaroma

occursatthattime１） .

The

becauseinhibitors,

exogenous

ethylene

inducesethylene

including ２，５ -norbornadiene,

４）

the

responsesilver５）〜７）

characteristic aroma disappears as the fruit turns

nitrate , and １-methylcyclopropene（１-MCP） , inhibit

yellow. Sudachi fruit rapidly changes from green to

degreening.

Ethylene

can

be

used

for

the

yellow at room temperature . Therefore, the green

degreening

of

whose

green

color

is

color is an important index of fruit quality, and it isundesirable, such as lemon, tangerine, and orange.

necessary to maintain the green of the peel during

On the other hand, in the case of citrus whosemarketing and storage for consumer acceptance. A

green color is desirable, such as lime, sudachi, andlong-term storage method for green sudachi fruitkabosu, ethylene must be removed or kept away

has been reported to be a combination of low

from it.

２）

temperature, sealed packaging with ０．
０
２! thick low

citrusEthylene promotes the ripening and senescence ofThis

harvested horticultural products, which affect their

combination keeps sudachi fruit green for up to ４

quality. Ethanol treatment retards ethylene-induced

to ６ months without loss ofripening-densitypolyethylene

film,

and
the

curing.

characteristic２）

aroma .

Citrus is categorized as a non-climacteric fruit,§ Corresponding author, E-mail : ysuzuki@kobe-u.ac.jp

and

８）

senescenceof
９）

products

tomato , avocado, muskmelon , broccoli
１２）including

１
０）
，
１
１）

, and cutcarnation . In this research, we investigated the188

Food Preservation Science

VOL．３
５ NO．
４ ２
０
０
９

（ 26 ）

effects of postharvest ethanol vapor treatment withbrowning, and １
０＝９
１〜１
０
０％ browning. The higher

ethanol pads, which is a simple and cost-effectivescores indicate a change of color from green tomethod, on the quality of sudachi fruit to easebrown.

postharvest handling and prolong the storage period,

３．Chemical analyses
Total soluble solids（TSS）, titrable acidity（TA）,

emphasizing the green color of the peel.

juice pH, and ascorbic acid contents in the pulp

Materials and Methods

juice of sudachi fruit were measured. Fruit juice

１． Plant materials and ethanol vapor treatment

extracted by hand - pressing using two layers ofwith an ethanol pad

cheesecloth from peeled fruit was applied. TSS was

Mature green sudachi fruit（Citrus Sudachi Hort. ex.

measured with a portable Brix meter RA-２
５０（KyotoShirai）were purchased from the Japan Agricultural

Electronics Manufacturing Co. , Ltd. , Japan ） andCo-operatives in Tokushima Prefecture. They wereexpressed

classified according

as

%

Brix

in

the

juice.

TAwascolor,

determined by titrating a mixture of １．
０! juiceshape, and size, and injured or yellowing fruit wereand ９．
０! distilled water with ０．
１ N NaOH and aremoved. After measured fresh weight, four fruit

phenolphthalein indicator. TA was calculated as thewere placed in a polyethylene bag（１
０
０"×１
４
０",

percentage of citric acid equivalents. Juice pH wastouniformity

in

peel

thickness ０．
０４"）with two holes（diameter ５"）on

measured with a twin pH meter B-２
１
２（Horiba, Ltd.,

each side in the presence or absence（control）of a

Japan）. L-ascorbic acid（AsA）and L-dehydroascorbic

０．
３, ０．
６, １, ３, or ６# ethanol pad（Antimold Mild!,acid（DHA）contents were assayed according to the

Freund Industrial Co., Ltd., Japan）as the ethanol

method of ROE１４）.

vapor treatment. The bags were stored at ２０℃ in

４．Statistical analysis

darkness until analyses. An

allowsGram was used as units for statistical analysis ofethanol vapor to diffuse gradually. A ０．
３, ０．
６, １, ３,ethanolpadloss of fresh weight. In comparison of loss of theor ６# ethanol pad contains ０．
１
８, ０．
３
６, ０．
６, １．
８, or

fresh weight（g）of control fruit and fruit treated

３．
６# of ethanol, respectively１１）. All of the experimentswith a １# ethanol pad, examination of significant

were conducted immediately after removing thedifferences

fruit from the bags after ０, １, ３, ４, ５, ７, and ９

analysis of variance first and then by Tukey’s

wasperformed

for

both

indicesbydays in storage（DIS）. Because the degreening rate

multiple comparison test（P ＜０．
０５）, using a softwarevaries in sudachi fruit, two of four fruit from eachpackage（Statcel-The Usefull Addin Forms on Excel-

bag that received an average score regarding color

２nd ed., Japan）.

were selected for analysis. All experiments were

Results

conducted in triplicate ; each two fruit from threedifferent bags were used for each treatment in theexperiments.
２．Measurement of peel color

１． Effectsofethanolvapor

treatmentondegreening of sudachi fruit
Changes in the chlorophyll a and chlorophyll b

Changes of peel color were measured with thecontent in the peel of the sudachi fruit treated withchlorophyll content and peel browning. Chlorophyll

ethanol vapor at various concentrations are shown

was assayed according to the method of MORAN１３）.

in Fig．
１. The change in chlorophyll content in the

Fruit peel （１．
０ g ） was immersed in ２
０! N , N -peel corresponded to the visual change from green

dimethylformamide and stored at ５℃ in darkness

to yellow. The chlorophyll a content in control fruit

for ２
４ h. The chlorophyll content was determineddecreased linearly from ０ to ７ DIS. The contents

by measuring the absorbance of the solvent at ６４
７

in fruit treated with ０．
３ and ０．
６# ethanol pads

and ６
６
４ nm. Peel browning was visually assessedalso decreased in a manner similar to that of theaccording to the ratio of the browning area to thecontrol. The decrease in fruit treated with a １#

surface area of the fruit peel. The scale ranged

ethanol pad was slower ;from ０ to １
０： ０ ＝ ０％ browning, １ ＝ １ 〜１
０％treated with a １ # ethanol pad was significantly

thecontent

infruit

browning, ２ ＝１
１〜２
０％ browning, ３ ＝２
１〜３０％

more than that in control fruit at ４ and ５ DIS.

browning, ４ ＝３
１〜４０％ browning, ５ ＝４
１〜５０％

Changes in the chlorophyll b content were almost

browning, ６ ＝５１〜６０％ browning, ７ ＝６
１〜７０％

the same as those in chlorophyll a content. These

browning, ８ ＝７１〜８０％ browning, ９ ＝８１〜９０％

results indicate that ethanol vapor treatment of

（ 27 ）

〔Article〕 Postharvest Ethanol Treatment of Sudachi

5.0
control
0.3 g
0.6 g
1g

20
15

Weight loss（%）

Chlorophyll content（mg 100g−1）

25

10
5

（A）

0
0
Chlorophyll content（mg 100g−1）

189

1

2

8
7
6
5
4
3
2
1 （B）
0
0 1 2

3

4

5

6

7

8

control

4.0

0.3g
0.6g

3.0

1g

2.0
1.0

9

0.0
0

1

2

3

4

5

6

7

8

9

Days in storage
Fig．
２ Effects of ethanol vapor treatment on weight loss（％）in sudachi fruit during storage at２
０℃

3 4 5 6 7
Days in storage

8

9

Fig．
１ Effects of ethanol vapor treatment on chlorophyll a（A）and chlorophyll b （B）content in the peel of sudachi
fruit during storage at２
０℃
The sudachi fruit treated without（control）（●）an ethanol pador with a０．
３!（□）
，０．
６!（∆），or１!（○）ethanol pad werestored at２０℃. Measurements were conducted in triplicate. The
results presented are the means ± S. D. Some error bars andsymbols are hidden by symbols. No data on fruit treated with a ３! or６! ethanol pad are shown in this figure because the peel ofat least one sudachi fruit in each treatment turned brown at ３
and １ DIS, respectively.

The sudachi fruit treated without（control）（●）an ethanol pador with a０．
３!（□）
，０．
６!（∆）
，or １!（○）ethanol pad werestored at２０℃. Measurements were conducted in triplicate. The
results presented are the means ± S. D. Some error bars andsymbols are hidden by symbols. No data on fruit treated with a ３! or６! ethanol pad are shown in this figure because the peel ofat least one sudachi fruit in each treatment turned brown at ３
and １ DIS, respectively.

analyzed in order to better understand the effects.
Gram instead of % was used as units for statisticalanalysis. There was significantly difference between
the weight loss（g）of the control fruit and that offruit treated with a １! ethanol pad at P ＜０．
０５（data not shown）.
３．Effects of ethanol vapor treatment on internalsudachi

fruit

with

a １!

ethanol

pad

could

quality of sudachi fruit

suppress the degradation of chlorophyll in fruit peel.

Changes in the internal quality of sudachi fruit,

The chlorophyll a and b contents in fruit treatedincluding those in the total soluble solids （ TSS ）,with ３ and ６ ! ethanol pads and other quality

titrable acidity（TA）, TSS ／ TA ratio（Fig．
３）, pHdescribed below were not measured after ３ DIS（ Fig ．
４）, L-ascorbic acid （ AsA ） content, and L-

because peel browning was remarkably ;that ofdehydroascorbic acid（DHA）content（Fig．
５）, in the

fruit treated with ３ and ６! ethanol pads started

pulp juice of the fruit treated with various ethanol

to occur at ３ DIS and １ DIS, respectively（datapads were measured. There were no significant

not shown）.

differences in

２．Effects of ethanol vapor treatment on loss ofcontrol and ethanol vapor-treated fruit. In both the

fresh weight of sudachi fruit

theinternal

quality

betweenthecontrol and ethanol-treated fruit, the TSS, TA, TSS

Fig．
２ shows changes in the loss of fresh weight,／ TA ratio, and juice pH were stable throughout

which is described as %, of the sudachi fruit treatedthe storage period. The AsA contents in the control

with various ethanol pads. The loss of fresh weight

and（％）of fruit treated with a １! ethanol pad tended

throughout storage, while the DHA contents were

to be less than that of the control fruit. On theslightly increased in the control and ethanol-treatedother hand, the loss of fruit treated with ０．
３ andfruit.

０．
６! ethanol pads tended to decrease similarly tothat in the control. Furthermore, the effect of
ethanol vapor treatment on the weight loss（g）offruit treated with a １! ethanol pad was statisticallyethanol-treatedfruit

remained

constant

190

Food Preservation Science

VOL．３
５ NO．
４ ２
０
０
９

（ 28 ）

3.0

2.5

TSS（% Brix）

pH

10.0

2.0

control

8.0

0.3g

6.0
control
0.3g
0.6g
1g

4.0
2.0

（A）

1.5

0.6g
1g

1.0
0

1

2

3

0.0

5

6

7

8

9

Days in storage
0

1

2

3

4

5

6

7

8

9
Fig．
４ Effects of ethanol vapor treatment on pH in the pulp
juice of sudachi fruit during storage at２
０℃

6.0
TA（%）

4

The sudachi fruit treated without（control）（●）an ethanol pador with a０．
３!（□）
,０．
６!（∆）
, or１!（○）ethanol pad were stored
at２０℃. Measurements were conducted in triplicate. The resultspresented are the means±S.D. Some error bars and symbols are
hidden by symbols. No data on fruit treated with a ３! or ６!
ethanol pad are shown in this figure because the peel of at least
one sudachi fruit in each treatment turned brown at ３ and １ DIS,
respectively.

4.0
2.0
（B）
0.0
0

1

2

3

4

5

6

7

8

9

Ascorbic acid content（mg 100mℓ−1）

2.0

1.0
（C）
0.0
0

1

2

3 4 5 6 7
Days in storage

8

9

Fig．
３ Effects of ethanol vapor treatment on total soluble
solids（TSS）（A）,titrable acidity（TA）（B）, and TSS ／TA ratio （ C ） in the pulp juice of sudachi fruit duringstorage at２
０℃
The sudachi fruit treated without（control）（●）an ethanol pador with a０．
３!（□）,０．
６!（∆）, or １!（○）ethanol pad werestored at２０℃. Measurements were conducted in triplicate. The
results presented are the means ± S. D. Some error bars andsymbols are hidden by symbols. No data on fruit treated with a ３! or ６! ethanol pad are shown in this figure because the peel ofat least one sudachi fruit in each treatment turned brown at ３
and １ DIS, respectively.

40
30
control
0.3g
0.6g
1g

20
10
0
0

Dehydroascorbic acid content（mg 100mℓ−1）

TSS / TA ratio

50 （A）

1

2

3

4

5

6

7

8

9

2

3 4 5 6 7
Days in storage

8

9

10
（B）
8
6
4
2
0
0

1

Fig．
５ Effects of ethanol vapor treatment on L-ascorbic acidcontent（A）and L-dehydroascorbic acid（B）in the pulp
juice of sudachi fruit during storage at２
０℃
The sudachi fruit treated without（control）（●）an ethanol pador with a０．
３!（□）
,０．
６!（∆）
, or１!（○）ethanol pad were stored
at２０℃. Measurements were conducted in triplicate. The resultspresented are the means±S.D. Some error bars and symbols are
hidden by symbols. No data on fruit treated with a ３! or６!
ethanol pad are shown in this figure because the peel of at least
one sudachi fruit in each treatment turned brown at ３ and １ DIS,
respectively.

（ 29 ）

〔Article〕 Postharvest Ethanol Treatment of Sudachi

might be effective to suppress the weight loss

Discussion

during storage because the weight loss of fruit

In the present study, it was found that theethanolvapor

treatmentof191

sudachiretard degreening because thefruit

decreaseintreated with a １! ethanol pad during storage was

could

less than that of the control fruit（Fig．
３）. １-MCP

thetreatment, even at an appropriate concentration,

chlorophyll contents of fruit treated with a １!

could induce some negative impact, including chilling

ethanol pad was slower than that of the control

injury （ CI ）, decay, and off-flavor５）. In contrast,

fruit. While sudachi fruit is marketed and stored,

ethanol treatment can decrease CI symptoms indegreening is the primary factor with the potential

cucumber１９） and prevent the development of decay

to reduce the commercial value of sudachi. Thus,

inthis finding indicates that ethanol vapor treatment

ethanol

could be beneficial to prolong sudachi fruit quality.

degreening

Degreening in citrus fruit is involved in ethylene

Furthermore, ethanol vapor treatment of sudachi

through chlorophyll degradation by it１５）. Exogenous

fruit

３）

grapes２０） during
vapor

at

storage.

treatment

but
an

may

also

is

possible

inhibitthat

notpostharvest

appropriate

only

disorders.

concentration

has

the

internal

quality

there

were

no

no

ethylene induces the degreening of the peel . Also,

negative

impactit is suggested that endogenous ethylene is related

storagebecausewith natural degreening of citrus because ethylene

differences of changes in TSS, TA, pH, and AsA

response inhibitor inhibits degreening, though ethylene

during storage between the control fruit and fruit

４）

on

It

duringsignificant

production is extremely low . Ethanol treatment oftreated with a １! ethanol pad（Figs．
４, ５, andsome horticultural crops suppresses ethylene-induced

６）and the flavor of the juice from ethanol-treatedripening and senescence, accompanying chlorophyll

fruit was not different from one from control fruit

degradation. Ethanol vapor treatment retards theby organoleptic test（data not shown）. Ethanol vapor

ripening of tomato fruit from mature green to red-treatment of citrus fruit for storage could have

８）

１
１）

ripe and keeps florets of harvested broccoli green .

severalFurthermore, it has been suggested that this is due

compared to even １-MCP treatment.

to the inhibition of ethylene synthesis and ethylene

Peeladvantages
browning

inofretainingsudachiquality

when

is

most

fruit

the

response by ethanol vapor treatment１６）. Thus, wecommon storage disorder. At the circulation level,

assume that the retardation of the degreening of

its occurrence tends to increase when it is stored

the sudachi fruit by ethanol vapor treatment, as

without

curingorunderextremely

high

CO２

２）

demonstrated in this study, is due to the same

conditions . On the other hand, an appropriate １-

mechanism.

MCP treatment may prolong postharvest life, but

Therechlorophyllase,
substance,are

Mg-dechelatase

pheophorbide

chlorophyll catabolite
pathway

of

four
a

known
or

chlorophyll

Mg-dechelating

oxygenase,reductase,induringenzymes,thetreatmentathigherconcentration

causespeel

redbrowning２１）. As indicated by our results, ethanol

catabolictreatment also induced peel browning at higherandsenescenceandconcentrations than the optimum（Fig．
２）. Ethanol

ripening. These enzymes are indispensable in thetreatment

degradation

increase in the permeability of cellular membranes２２）

ofchlorophylltoacolorless

blue-

１
７）

at

a

high

concentration
cells２３），２４） .

causesan

fluorescing intermediate . In the present study, theand,retardation of chlorophyll degradation of sudachi

browning observed in this study may be due to thefruit by ethanol treatment might be due to themembraneinhibition of the activities and gene expression ofconcentrations of ethanol and not by the high CO２

theethanolconditions, because polyethylene bags for package

treatment or by other unknown factors, related towere perforated. Further research on application of

ethanol that affect the metabolism of sudachi fruit.

ethanol will need to be done in order not to cause

Further studies are needed to clarify these issues.

peel browning.

chlorophyll-degrading

enzymebyfurthermore,injuresdamagescaused

by

The
the

peel
high

Loss of fresh weight after harvest is an important

Ethanol vapor treatment of horticultural products

factor in degradation in the marketable quality ofwith an ethanol pad has advantages for storage inhorticultural products. Ethylene and １-MCP do notthat it is simple and cost-effective. In this study, we

have any effect on loss of fresh weight of citrus

demonstrated

５）
，
１
８）

fruit in storage.

However,ethanol

treatmentthepossibility

thatethanolvapor

treatment of sudachi fruit could prolong the storage

192

Food Preservation Science

VOL．３
５ NO．
４ ２
０
０
９

（ 30 ）

period without a negative impact on the internal１０）CORCUFF, R., ARUL, J., HAMZA, F., CASTAIGNE, F.

quality at ambient temperature. This finding isand MAKHLOUF, J. : Storage of broccoli florets inimportant and has widespread implications because

ethanol vapor enriched atmospheres, Postharvest

it could also be available to other citrus, including
lime, kabosu, and Nagato-yuzukichi, in which the
green

color

is

desirable

toconsumers.

Biol. Technol ., ７, ２
１
９〜２
２
９（１
９
９
６）
１
１）SUZUKI, Y., UJI, T. and TERAI, H. : Inhibition of

Furthersenescence in broccoli florets with ethanol vapor

studies are needed for the commercial utilization of

from alcohol powder, Postharvest Biol. Technol ., ３
１,this treatment.

１
７
７〜１
８
２（２
０
０
４）
１
２）WU, M. J. , ZACARIAS, L. , SALTVEIT, M. E. andReferences

Reid, M. S. : Alcohols and carnation senescence,

１）TAMURA, H., PADRAYUTTAWAT, A. and TOKUNAGA,
T. :Seasonal change of volatile compounds of

HortScience, ２
７, １
３
６〜１
３
８（１
９
９
２）
１
３）MORAN,R. :Formulaefordetermination

of

N,

N-

citrus sudachi during maturation, Food Sci. Technol.

chlorophyllous

Res., ５, １
５
６〜１
６
０（１
９
９
９）

dimethylformamide, Plant Physiol ., ６
９, １
３
７
６〜１
３
８
１

２）KITAGAWA, H., KAWADA, K. and TARUTANI, T. :pigments

extracted

with

（１
９
８
２）

Effects of temperature, packaging and curing on

１４）ROE, J. H., MARY, B. M., OESTERLING, M. J. andthe storage of sudachi （ Citrus sudachi Hort. ex.

CHARLOTLE, M. D. : The determination of diketo-

Shirai ）, J. Japan. Soc. Hort. Sci. , ５
１, ３
５
０〜３
５
４

gulonic（１
９８
２）

ascorbic acid in the same tissues extract by ２,

３）SHIMOKAWA, K. , SHIMADA, S. and YAEO, K. :４-dinitorophenyl hydrazine method, J. Biol. Chem.,

Ethylene-enhanced chlorophyllase activity during
degreening of Citrus unshiu Marc, Sci. Hort. , ８ ,
１
２
９〜１
３
５（１
９
７
８）

acid,

dehydro-L-ascorbic

acid,

and

L-

１
７
４, ２
０
１〜２
０
８（１
９
４
８）
１
５）PURVIS, A. C. and BARMORE, C. R. : Involvementof ethylene in chlorophyll degradation in peel of４）GOLDSCHMIDT, E. E., HUBERMAN, M. and GOREN,

citrus fruits, Plant Physiol ., ６
８, ８
５
４〜８
５
６（１
９
８
１）

R. : Probing the role of endogenous ethylene in１６）ASODA, T., TERAI, H., KATO, M. and SUZUKI, Y. :theethylene

Effects of postharvest ethanol vapor treatment on

antagonists, Plant Growth Regul . , １
２, ３
２
５〜３
２
９

degreeningof

citrus

fruit

with

ethylene responsiveness in broccoli, Postharvest Biol.
Technol ．
（in press）

（１
９
９
３）
５）PORAT, R., WEISS, B., COHEN, L., DAUS, A., GOREN,
R. and DROBY, S. :Effects of ethylene and １ -methylcyclopropene on the postharvest qualities of‘ Shamouti’ oranges, Postharvest Biol. Technol . , １
５,
１
５
５〜１
６
３（１
９
９
９）
KANLAYANARAT,

S. :Effectsofdifferent

concentrations of １-MCP on the yellowing of West
Indianlime （ Citrus

senescence, Annu. Rev. Plant Biol . , ５
７, ５
５〜７
７
（２
０
０
６）
１
８） COHEN, E. :Ethylene concentration and theduration of the degreening process in Shamouti６）WIN, T. O., SRILAONG, V., HEYES, J., KYU, K. L.
and１７）HÖRTENSTEINER, S. : Chlorophyll degradation during

aurantifolia,

Swingle ） fruit,

Postharvest Biol. Technol ., ４
２, ２
３〜３
０（２
０
０
６）
７）MCCOLLUM, G. and MAUL, P.：１-Methylcyclopropene
inhibits degreening but stimulates respiration andorange fruit, J. Hort. Sci., ５
３, １
３
９〜１
４
２（１
９
７
８）
１
９）SALTVEIT, M. E. : Exposure to alcohol vapours
reduceschilling-induced

injuryexcised hypocotyl segments, J. Exp. Bot., ４
５, ８
１
３〜
８
２
１（１
９
９
４）
２
０）LURIE, S., PESIS, E., GADIYEVA, O., FEYGENBERG, O.,

ethylene biosynthesis in grapefruit, HortScience, ４２,

BEN-ARIE,R. ,KAPLUNOV,

T. ,LICHTER,A. :Modifiedethanolof

ethylenesynthesis

andactioninripening

tomato fruit by ethanol vapors, J. Am. Soc. Hort.
Sci., １
１
３, ５
７
２〜５
７
６（１
９
８
８）
９）RITENOUR, M. A., MANGRICH, M. E., BEAULIEU, J.
C., RAB, A. and SALTVEIT, M. E. : Ethanol effects
on the ripening of climacteric fruit, Postharvest Biol.
Technol ., １
２, ３
５〜４
２（１
９
９
７）

excisedcucumber cotyledons, but not of seedlings or

１
２
０〜１
２
４（２
０
０
７）
８）SALTVEIT, M. E. and MENCARELLI, F. : Inhibitionof

ZUTAHY,Y.

atmosphereandtocontrol decay of table grapes during storage,
Postharvest Biol. Technol ., ４
２, ２
２
２〜２
２
７（２
０
０
６）
２
１）ISSHIKI, M., TERAI, H. and SUZUKI, Y. : Effect of
１-methylcyclopropene on quality of sudachi（Citrus
sudachihort. ex Shirai）during storage, Food Preser.
Sci., ３
１, ６
１〜６
５（２
０
０
５）
２
２）SALTVEIT, M. E. : Effect of alcohols and theirinteraction

withethyleneontheripeningof（ 31 ）

〔Article〕 Postharvest Ethanol Treatment of Sudachi

epidermal pericarp discs of tomato fruit, Plant
Physiol ., ９
０, １
６
７〜１
７
４（１
９
８
９）
２
３）PERATA, P. and ALPI, A. : Ethanol metabolism insuspension cultured carrot cells, Physiol. Plant., ８
２,
１
０
３〜１
０
８（１
９
９
１）
H. : Ultrastructural evidence for the inhibition ofchloroplast-to-chromoplast

収穫後のエタノール蒸気処理がスダチ果実の品質に及ぼす影響
野間雄太＊１・鈴木康生＊１・寺井弘文＊１・山内直樹＊２＊１２４）SUZUKI, Y., KIMURA, T., TAKAHASHI, D. and TERAI,
conversioninbroccoli

193

神戸大学大学院農学研究科

（〒６
５
７―８
５
０
１ 神戸市灘区六甲台町１―１）＊２

山口大学農学部

（〒７
５
３―８
５
１
５ 山口市吉田１
６
７
７―１）

floret sepals by ethanol vapor, Postharvest Biol.
Technol ., ３
５, ２
３
７〜２
４
３（２
０
０
５）

スダチは香酸カンキツの一種であり，常温では脱緑しやすい。エタノール処理は収穫された園芸作物の追熟や老化を遅延する。貯蔵期間を延長することを目的に，エタノールパッドを用いた収穫後のエタノール蒸気処理がスダチ果実の品質に及ぼす影響が調べられた。スダチの緑熟果は，０．
３，０．
６，１，３，６!のエタノールパッド
とともに有孔ポリエチレン袋に同封され，２０℃暗所下で貯蔵された。また，エタノールパッドを同封しない処理をコントロールとした。１!のエタノールパッドで処理された果実のクロロフィル含量の減少はコントロールに比べて遅く，また新鮮重の減少も抑制された。果汁中の全可溶性固形物含量，滴定酸，糖酸比，pH，アスコル
ビン酸含量，酸化型アスコルビン酸含量などの内部品質に，コントロールとエタノール蒸気処理した果実間で差異は認められなかった。しかし，３!と６!のエタノー
ルパッドで処理された果実では果皮が褐変した。これらの結果から，スダチにエタノール蒸気処理を行うと脱緑が遅延し，内部品質に悪影響なく貯蔵期間を延長できる可能性が示された。
（平成２
１年１月１
９日受付，平成２
１年３月２
６日受理）

（ 33 ）

日本食品保蔵科学会誌 VOL．３
５ NO．
４ ２
０
０
９ 〔技術報告〕

195

ナッツ類を用いたチーズ様食品の製造
本 間 裕 人＊§・徳 田 宏 晴＊・中 西 載 慶＊
＊ 東京農業大学短期大学部醸造学科

Cheese-like Food Production from Various Nuts
HOMMA Hiroto＊§，TOKUDA Hiroharu＊ and NAKANISHI Kotoyoshi＊＊

Department of Brewing and Fermentation, Junior College of Tokyo University of Agriculture,1−1−1, Sakuragaoka, Setagaya-ku, Tokyo 156−8502

Nuts are rich in vegetable proteins and lipids, and can be easily made into a milk-like emulsion suchas coconut milk or hemp milk. In this study we attempted to produce a cheese-like food from various
nuts instead of from cows’ milk. First, we investigated the curdling activity of various proteases on
emulsions made individually from １
３ species of nuts. The proteases bromelain, papain, subtilisin,
thermolysin and a protease from E. faecalis could curdle the milk-like emulsion made from １
２ speciesof nuts. The emulsions made from hemp nuts, coconuts, almonds, pine nuts and macadamia nuts
curdled especially well. We then successfully produced cheese-like foods from ６ species of nuts（hempnuts, coconuts, almonds, pine nuts, cashew nuts and macadamia nuts）. Compared to cheese made fromcows’ milk these nut-cheeses were softer, the curd yield from the emulsion was almost the same, theprotein content was lower and the lipid content was higher. Hemp nut cheese was the most similar tocows’ milk-cheese in its composition and gave the highest yield of cheese among all of the tested nuts.
（Received Nov. １３, ２
００８；Accepted Mar. ４, ２
０
０
９）
Key words：cheese production, nuts, curd formation, protease
チーズ生産，ナッツ類，カード形成，プロテアーゼ

近年，中国やインドなどの人口大国でも経済的に豊か

その酵素学的諸性質などを明らかにしてきた７）。現在もな中流階級層の人口が増加し，食生活の欧米化が進んだ

当該酵素を利用した豆乳チーズの品質改善について検討

ためチーズやバターなど乳製品の消費が急速に拡大してを進めているが，それと並行して豆乳以外の新規なチー

１）

いる 。それに伴い原料である牛乳の増産が求められてズ原料の検索も試みてきた。

いるが，放牧地をこれ以上拡大するのは熱帯雨林の保全

今回筆者らは，大豆と同様に植物性のタンパク質と脂など環境問題との兼ね合いで難しく，さらに近年急激に質を豊富に含むナッツ類に注目した。ナッツ類の多くは飼料価格が高騰していることなどもあり，これ以上の増

ココナツやアーモンドなど独特の官能的に好ましい香り

産は難しいと考えられている２），３）。そのため牛乳以外のを有しており，加水して圧搾することによりココナツミ

原料を用いたチーズ様食品の開発などに興味がもたれてルクやヘンプミルク（麻の実より製造）などといった豆いる。

乳に類似したミルク様の乳液が簡便に得られることが知このような背景から，牛乳に非常によく似た性質を有られている。さらにナッツ類は牛乳と異なり保存性も高している豆乳が，牛乳の代替原料として注目を集めておく，輸送も容易である。しかしながらこれまでのところ，り，豆乳を利用したチーズ様食品の開発がこれまで盛ん

ナッツ類を原料とした酸・プロテアーゼ法によるチーズに試みられてきた４）〜６）。しかしながら牛乳由来チーズに製造についての報告はない。そこで本報告ではナッツ類匹敵しうるような品質を有する豆乳チーズの製造は難しのチーズ原料としての可能性を探ったところ，利用でき

く，多くの研究すべき課題が残されている。筆者らも豆る可能性が示されたので報告する。

乳チーズの品質を改善すべく研究を行っており，これま
でに新規な豆乳凝固活性を有する乳酸菌（Enterococcus
faecalis TUA ２４９５ L）由来プロテアーゼを見い出し，
＊ 〒１
５
６―８
５
０
２ 東京都世田谷区桜丘１―１―１§ Corresponding author, E-mail : h3homma@nodai.ac.jp

196

日本食品保蔵科学会誌 VOL．３
５ NO．
４ ２
０
０
９

実験方法
１．使 用 原 料
麻の実ナッツ（カナダ産），アーモンド（アメリカ産），（ 34 ）

まりを崩しながら３
０分かけてゆっくりと品温を５０℃に昇温させた（カッティングおよびクッキング処理）。ここにオートクレーブ滅菌（１２１℃１０分）した２０％（w／v）
食塩水１
５０!を添加し，３０分ほど静置した。

カシューナッツ（インド産），クルミ（アメリカ産），ピ

十分にホエーとカードが分離したらこれをモールドーナッツ（中国産）は，生ナッツを使用した。白ゴマ（直径１１#，高さ１
１．
５#，底部にまき簾を設置し，内側（国産），黒ゴマ（国産），マカダミアナッツ（アメリカ

をさらしで覆った）に徐々に流し込み，ホエーを排出さ産），松の実（中国産）は炒られたものを使用した。ませた。約１時間後，カードの上面にさらしをしいて重石

た，カボチャの種（中国産），ピスタチオ（アメリカ産），

を乗せ，その重量を徐々に増加（１〜２時間の間隔でヘーゼルナッツ（アメリカ産）は炒ったうえで加塩され

０．
５&ずつ，計１．
５&）させた。カードがある程度しっかたものを使用した。これらのナッツ類は，殻付きのもの

りしてきたら天地返し（モールドの上下を入れ替えてさについてはすべて殻を剥いてから使用した。ココナツはらしを取り替える操作）を行い，重石を１．
２５&加重してマレーシア産のココナツミルク（缶詰）を使用した。

（計２．
７５&）室温で一晩静置した。その後再び天地返し

２．使 用 酵 素をし，さらに重石を２．
５&加重して（計５．
２５&）ホエー

プロテアーゼは，レンネット（SIGMA社製，子牛胃を排出させた。ホエーの排出が停止したら，モールドか由来），微生物レンネット（SIGMA社製，Mucor miehei

ら出し得られた固形物をナッツチーズとした。

由来），ブロメライン（MERCK社製），パパイン（和光

６．一 般 分 析

純薬社製）
，サーモリシン（SIGMA社製），サブチリシ水分含量は，８０℃で試料を乾燥させて恒量を求め，乾ン（SIGMA社製），を使用した。これら市販酵素剤につ燥前後の重量差から水分含量を算出した。脂質量はソッ

いては，１０$／!になるよう５００ mMのリン酸緩衝液（pH

クスレー法により求めた。全タンパク質量はケルダール６．
０）に溶解して用いた。またEnterococcus faecalis TUA法により定量した。このとき窒素タンパク変換係数は，

２４９５ L株およびIAM １００６５株の生産する凝乳活性を有

アーモンドでは５．
１８，その他のナッツでは５．
３０とした８）。

するプロテアーゼを佐藤らの報告に基づき調製し７），５００

灰分は灰化法により求めた。すなわち，試料を電気炉を

mMのリン酸緩衝液（pH ６．
０）で２倍希釈して使用し用いて５５０℃で２時間灰化させ恒量を求め，灰化前後のた。

重量から算出した。炭水化物は差し引き法により，水分，

３．乳液の調製および凝乳試験

脂質，タンパク質，灰分の含有量を全量から差し引いてナッツ類５０%に 蒸 留 水１０
０! を 加 え，フ ー ド ミ ル

求 め た。pHはpHメ ー タ ー（HORIBA社 製pH METER（Tescom社製）で破砕した。ただし白ゴマおよび黒ゴ

M-１２）により，測定した。酸度は滴定法により求めた。

マについては蒸留水１００!では流動性が低く破砕が困難

すなわち，精秤した試料１０%に煮沸した蒸留水４０!を加だったため，蒸留水１
５０!を加えて破砕した。その後４０℃

え撹拌し試料溶液を調製し，これを０．
１ N水酸化ナトリ

で１０分撹拌しながらインキュベートし，晒布で包んでジウム溶液で滴定することにより求めた。このとき，pHュースプレス（Master Life社製）で圧搾して乳液を得

７．
０までの滴定量を滴定酸度!とし，pH ７．
０〜８．
３まで

た。この乳液５!を試験管に入れ４０℃で１０分予熱し，酵の滴定量を滴定酸度"とした９）。

素液１!を加え，十分に撹拌した。これを４０℃にて静置

実験結果および考察

し，乳液からのカード（固形物）の分離の有無と生じた
カードの凝固の度合いを経時的に観察した。
４．スターターの調製
スターターは，Lactococcus lactis subsp. lactis １
５４株，

１．凝 乳 試 験
１
３種類のナッツから乳液を調製して，そこに凝乳活性を有するプロテアーゼを添加し，カード（固形物）の形

Lactococcus lactis subsp. cremoris ４３９株，Lactobacillus

成の有無と生じたカードの凝固の度合いを観察した。そparacasei １
５３
２株の３株の乳酸菌をそれぞれ個別にスキの結果，ブロメライン，パパイン，サーモリシン，サブムミルク培地を用いて３０℃で２日間培養し調製した。

チリシン，TUA ２４９５ LあるいはIAM １００６
５株 由 来 プ

５．チーズ製造

ロテアーゼを添加した場合において，乳液の多くがカー

ナッツ類７
０
０〜７
５
０%に適量の蒸留水を加え，上記と同

ドとホエーに分離し，カードが明確に凝固することが観様の方法で１，
０
０
０〜１，
５
０
０!の乳液を調製した。得られた察された（Table １）。特に麻の実，ココナツ，アーモ

乳液を４
０℃で３
０分予熱し，ここに酵素液を乳液に対して

ンド，松の実あるいはマカダミアナッツを原料とした乳

０．
５％（v／v）添加した。酵素はココナツについてはサ液において良好な凝固が認められ，反応液を水平に傾け

ブチリシンを，ほかのナッツについてはブロメラインをても分離したカードの形状が全く崩れないほどしっかり使用した。そこに乳酸菌スターター３種類を，使用したと凝固しているものも観察された。一方，レンネットお乳液に対して１％（v／v）ずつ計３％（v／v）添加した。

よび微生物レンネットを用いた場合には乳液の状態はコ

その後，４
０℃で１
２
０分間保持し凝固させた後，適度に固ントロールとあまり差はなく，カードの形成は認められ

（ 35 ）

〔技術報告〕 ナッツ類からのチーズ様食品製造

197

Table １ Curd formation test of various curdling enzymes on the emulsions made from various nuts
Nut emulsion
Protease

Control（no enzyme
added）

Time White Black
HazelPeanut Walnut（h） sesame sesame
nut
１
２
３

Rennet（From １
calf stomach） ２
３Rennet（From Mucor
miehei）

１
２
３

Papain１
２
３Bromelain

Thermolysin

１
２
３
１
２
３

Subtilisin A

１
２
３

TUA ２４９５ L

IAM １００６５

１
２
３
１
２
３

Hemp
Pine Pump- Cashew Macada- PistaCoconut Almondnut
nut kin seed nut mia nut chio nut

−

−

−

−

−

−

−

−

−

−

−

−

−

−

−

±

−
−

−
−

−
−

−
−

−
−

−
−

−
−

++
−

−

−

−

−

−

−

−

＋

−

−

−

−

−

−

−

++

−

−

−

−

−

−

−

±

−
−

−
−

−
−

−
−

−
−

−
−

−
−

++
+++

−

−

−

−

−

++

−

++

−

−

−

＋

−

++

−

++

−

−

−

＋

±

++

−

+++

−

−

−

−

−

＋

−

+++

−
−

−
−

−
−

−
−

＋
＋

＋
＋

−
−

+++
+++

−

−

−

±

−

++

++

+++

−

−

−

++

＋

++

++

+++

＋
−

−
−

＋
−

＋
＋

＋
±

++
++

++
+++

+++
+++

−

−

−

＋

＋

++

+++

+++

±

−

±

＋

＋

++

+++

+++

−

−

−

＋

−

＋

−

+++

−
−

−
−

−
−

＋
＋

＋
＋

＋
＋

−
−

+++
+++

−

−

−

−

−

＋

−

+++

−

−

−

−

＋

++

−

+++

−

−

−

−

＋

++

−

+++

−
−
−
−
−
−
−
−
−
++
+++

−

−

−

−

−

−

−

−

−
−

−
−

−
−

−
−

−

−

−

−

−

−

−

−

−

−

−

−

−
−

−
−

−
−

−
−

−

−

＋

±

−

−

＋

＋

−

−

++

＋

++
++
+++

−

−

++

±

−
±

−
±

++
++

±
±

+++
+++

＋

−

＋

−

+++

+++
+++
+++
+++
++
+++
+++
++
++
+++

＋

−

＋

−

＋
−

−
−

++
++

±
−

−

−

+++

＋

−

±

+++

＋

−

−

−

−

−
−

−
−

＋
＋

−
−

−

−

±

−

−

−

±

−

−

−

±

−

Activity was evaluated based on curd conditions as follows : −, curd was not formed；±, the emulsion separated into whey
and curd and the curd was of low viscosity；＋, the emulsion separated into whey and curd, and the curd was of highviscosity；＋＋, the emulsion separated into whey and curd and the curd kept its shape for more than ３ seconds when thetest tube was canted horizontally；and +++, the emulsion separated into whey and curd and the curd kept its shape for morethan １０ seconds when the test tube was canted horizontally.

なかった。レンネットと微生物レンネットは極めて基質が観察された。アーモンド自体に凝乳性プロテアーゼが特異性の高いプロテアーゼであり，ナッツ類から得られ含まれている可能性が推察された。

る乳液中のタンパク質の構造はミルクカゼインとは異な以上より，ナッツ類を用いて調製した乳液においてもるため，あまり特異性の高くないブロメライン等の酵素

酵素との組み合わせしだいでは，カードの形成が可能でのほうが高い凝乳活性を示したものと推察された１０）。

あることが示された。

また松の実，カボチャの種，カシューナッツ，マカダ

２．チーズ製造試験

ミアナッツ，ピスタチオではプロテアーゼを添加しない

プロテアーゼと反応させることによりカードを得られコントロールの条件下において，１〜３時間経過するとることが示されたナッツ類の中から，麻の実，ココナツ，

沈殿が生じることが観察された。しかしこの場合生じた

アーモンド，松の実，カシューナッツ，マカダミアナッ

沈殿は主にコロイド粒子が沈殿したものと思われ，プロツの６種類のナッツを選択し，実際にチーズ製造を試み

テアーゼ存在下において形成されるカードとは異なり全た。その結果，試験したすべてのナッツにおいて実際にく凝固していなかった。アーモンドはコントロールでもチーズ様食品（以下「ナッツチーズ」とする）を製造す

乳液がホエーとカードに分離し，カードが凝固することることができた。また，炒ったアーモンドを原料として

198

日本食品保蔵科学会誌 VOL．３
５ NO．
４ ２
０
０
９

（ 36 ）

Table ２ Materials used for cheese production. The weight of the nuts used, the volume of water added, theemulsion volume and the wet weight of the cheese-like food that was produced are indicatedHemp nuts

Coconuts１）

Almonds

Pine nuts

Cashew nuts

Macadamia nuts

Nuts（g）
Added water（!）

７
０
０
１，
４
０
０

１，
２
０
０
０

７
５
０
１，
５
０
０

７
５
０
１，
５
０
０

７
５
０
１，
５
０
０

７
０
０
１，
４
０
０

Emulsion volume（!）
Produced cheese（g）

１，
５
０
０
５
６
４

１，
２
０
０
２
２
０

１，
０
５
０
９
２．
４

１，
５
０
０
２
２
５

１，
５
０
０
２
４
９

１，
５
０
０
２
６
５

Material nuts

１）

Canned coconut milk was used.

同様に製造を試みたが，ほとんどカードを得ることができず，ナッツチーズは製造できなかった。製造に使用しAたナッツの量および得ることのできたナッツチーズの量をTable ２に示す。得られたナッツチーズの量は麻の実の乳液を用いた場合に最も多く，使用した乳液の１／３程度の重量のチーズが得られた。得られたナッツチーズの量が最も少なかったのはアーモンドを用いた場合であり，得られたチーズの重量は使用した乳液の１／１０程度であった。他のナッツではおおむね使用した乳液のおよ
そ１／６程度のチーズが得られた。牛乳などからつくられる一般的なチーズではカード収量は原料乳の１２〜２０％程度であるのでナッツチーズの収量は牛乳チーズとあまり変わらないと思われる１０）。
製造したナッツチーズは一般的な牛乳チーズよりも軟B質の物が多く，最も軟質だったアーモンドチーズはクリーム状であった（Fig．
１-A）
。製造したチーズの中ではココナツチーズが最も硬く形状がしっかりしていたが，バター程度の硬さであり一般的なチーズ分類に照らし合わせると軟質チーズに分類されると思われた（Fig．
１-B）
。
そのため食感はほとんどのナッツチーズにおいてクリー
ムチーズ様の極めて柔らかいものであった。また，油分が多いと感じられる物が多かった。香りについては原料
ナッツの香りと乳酸発酵によるヨーグルト様の香気を有している物が多く，特にココナツチーズ，アーモンドチ
ーズ，麻の実チーズは原料ナッツの香りが強く官能的に好ましかった。色については，ココナツチーズ，麻の実

Fig．
１ Appearance of nut-cheeses

チーズおよびカシューナッツチーズが白色で，松の実チ

A : Almond-cheese, B : Coconut-cheeseーズとマカダミアナッツチーズは淡黄色，アーモンドチ
乳チーズの２倍近い脂質を含んでいることが示された１１）。

ーズは淡褐色であった。
製造したナッツチーズの一般成分，pH，および滴定

ナッツチーズの中では麻の実チーズが水分４
０．
８％，タン

酸度をTable ３に示した。ナッツチーズのタンパク質含パク質１７．
２％，脂質３
５．
８％と比較的牛乳を原料とする軟量は，麻の実チーズのみ１
７．
２％であったがそれ以外はい

質チーズに近い値を示した。pHについてはナッツによずれも２〜３％ほどであった。一般の牛乳から製造され

り４．
４
１から５．
４
７までの値を示した。牛乳から製造されるるチーズのタンパク質含量は，クリームチーズで８．
２％，

チーズではpHは５〜６の間にあることが多く，ナッツ

カマンベールチーズで１
９．
１％，チェダーチーズで２
５．
７％

チーズのpHはそれと同等からやや低い範囲にあるといという分析値が報告されており，ナッツチーズのタンパ

うことが示された１０）。滴定酸度の結果とあわせて考える

１
１）

ク質含量はかなり少ないといえる 。逆に脂質はナッツと，滴定酸度IIが低い物が多いことから，緩衝能が低くチーズでは麻の実，カシューナッツを除いて６０％前後で乳酸発酵によるpHの変動を受けやすいものと推察されあったが，牛乳チーズではクリームチーズで３
３．
０％，カ

る。

マンベールで２
４．
７％，チェダーチーズで３
３．
８％とおよそ今回製造したナッツチーズの中では，ココナツチーズ

３０％前後であることが知られており，ナッツチーズは牛が硬度や香りの点で優れていた。また，原料あたりのチ（ 37 ）

〔技術報告〕 ナッツ類からのチーズ様食品製造

199

Table ３ Composition, pH and acid degree of nut-cheeses
Content

Nut-cheese
Hemp nut

Coconut

AlmondPine nut

Cashew nut

Macadamia nut

Moisture（％）
Protein（％）

４
０．
８
１
７．
２

２
６．
１
３．
２

２
９．
２
３．
７

３
５．
３
２．
０

６
８．
９
３．
３

２
５．
３
２．
２

Lipid（％）
Carbohydrate（％）

３
５．
８
３．
８

６
９．
９
０．
５

６
３．
７
１．
３

５
８．
１
３．
３

６．
０
１
９．
５

６
６．
７
５．
５

Ash（％）

２．
５

０．
３

２．
１

１．
３

２．
３

０．
３

pH
Acid degree I１）

５．
３
０

４．
５
０

５．
４
７

４．
４
１

４．
５
２

４．
７
１

２．
３
６

１．
１
０

０．
８
０
２

１．
４
０

１．
４
０

０．
６
０
２

Acid degree II２）

２．
９
１

０．
５
０
２

０．
９
５
３

０．
７
０
２

０．
７
０
２

０．
５
０
２

Acid degree!indicates the volume of a ０．
１ N NaOH solution required to neutralize １０" of nut-cheese to pH ７．
０.
Acid degree II indicates the volume of a ０．
１ N NaOH solution required to alkalize １０" of nut-cheese from pH ７．
０ to ８．
３.

１）
２）

文

ーズ収量は麻の実チーズが最も多く，一般成分やpHなども牛乳チーズに近かった。いずれにしろ本研究で，ナ

献１）EVANS, L. T.・日向康吉：家畜の飼養と飼料，１００

ッツ類を原料としたチーズ様食品の製造が可能であるこ

億人への食糧（学会出版センター，東京）
，pp．
２
０
６〜

とが示された。本研究は牛乳や豆乳以外のチーズ原料の

２
０
８（２
０
０
６）

検索に主眼を置いていたため製造法の検討は行わず，牛２）金田憲和・雁和邦昭：世界の食糧問題と地球温暖化，

乳チーズと同じ製造法を用いているが，今後ナッツチー

食と環境（東京農業大学出版会，東京）
，pp．
８
７〜９
０

ズに適した製造法について詳細な検討を加えることによ

（２
０
０
５）

りさらなる品質の向上が図れるものと推察される。

３）EVANS, L. T.・日 向 康 吉：食 糧 生 産 を 高 め る 道

要

約

チーズ製造における牛乳の代替原料として，植物性の筋，１００億人への食糧（学会出版センター，東京）
，
pp．
２
１
５〜２
２
２（２
０
０
６）
４）松岡博厚・笹子謙治・関口正勝：大豆乳を利用したタンパク質と脂質を豊富に含むナッツ類に注目し，各種

チーズよう食品の製造に関する研究（第１報），日本

ナッツ類を絞って得た乳液に対してプロテアーゼを添加

食品工業学会誌，１
５，１
０
３〜１
０
８（１
９
６
８）

し凝乳試験を行い，チーズ様食品の製造を試みた。１
３種５）松岡博厚・笹子謙治：大豆乳を利用したチーズよう

類のナッツを用いて凝乳試験を行った結果，ブロメライ

食品の製造に関する研究（第２報），日本食品工業学

ン，パパイン，サーモリシン，サブチリシン，E. faecalis

会誌，１
９，２
６
２〜２
６
７（１
９
７
２）

TUA ２
４
９
５ L株由来プロテアーゼ，あるいはE. faecalis

６）松岡博厚・福家洋子：大豆乳を利用したチーズよう

IAM １
０
０
６
５株由来プロテアーゼを添加した場合におい食品の製造に関する研究（第３報），日本食品工業学て，多くのナッツ類で乳液がカードとホエーに分離し，

会誌，２
２，４
３
６〜４
４
２（１
９
７
５）

カード形成能が認められた。特に麻の実，ココナツ，ア７）SATO, S., TOKUDA, H., KOIZUMI, T. and NAKANISHI,

ーモンド，松の実あるいはマカダミアナッツを原料としK. :た乳液で良好な凝固が認められた。

extracellular proteinase having milk-clotting activity

そこで麻の実，ココナツ，アーモンド，松の実，カシューナッツ，マカダミアナッツの６種のナッツ類を用いて実際にチーズ様食品の製造を試みたところ，試験したすべてのナッツにおいてチーズ様食品を製造することが可能であった。製造したチーズはいずれも牛乳チーズよりも軟質で，収量については多くのもので牛乳チーズと同程度であった。香りについては原料ナッツ由来の香りと乳酸発酵によるヨーグルト様の香りを有している物が多かった。一般成分については，多くのナッツチーズで牛乳チーズよりも低タンパク高脂質であった。製造した

Purificationofanfrom Enterococcus faecalis TUA ２
４
９
５ L, Food Sci.
８）文部科学省科学技術・学術審議会資源調査分科会：
五訂増補

日本食品標準成分表

分析マニュアル（国立印刷局，東京）p．
１
３（２
０
０
５）
９）安藤達彦・吉田宗広：身のまわりの食品化学実験（三共出版，東京）p．
８
９（２
０
０
１）
１
０）伊藤肇躬：第８章

チーズ，乳製品製造学，光琳

（２
０
０
４）
１
１）文部科学省科学技術・学術審議会資源調査分科会：
五訂増補

点で優れていた。また，原料あたりのチーズ収量は麻の

p．
２
０
９（２
０
０
５）

に近かった。

characterization

Technol. Res., １
０, ４
４〜５
０（２
０
０
４）

ナッツチーズの中では，ココナツチーズが硬度や香りの実チーズが最も多く，一般成分やpHなども牛乳チーズ

and日本食品標準成分表（国立印刷局，東京）
，

（平成２
０年１
１月１
３日受付，平成２
１年３月４日受理）