（ 3 ）153

日本食品保蔵科学会誌
３
７

VOL．

会
長
編集委員長
編集委員

４

NO．

高井 陸雄
上田 悦範
石田
裕
津久井亜紀夫＜報文＞（英文）
飲酒前後のカキ 西条

タンゴール

清見

副

会

長

稲熊 隆博
東尾 久雄

上田

悦範

小宮山美弘

早坂

薫

井上 茂孝
古庄
律

今堀 義洋
松田 茂樹

竹永

章生

果実摂取がヒト血中のエタノール濃度に及ぼす影響 …………………………（１５５）／板村裕之・孫 寧静・二村正之・下崎俊介
中務 明・安藤はるか・永松ひろ子・勝部拓矢
鶴永陽子・浅野祐二郎・荒川長巳・亀井 勉
村田幸治・杉中克昭・舟木賢治・松本敏一
児島靖典・上村栄介・村上直之・松崎 一

果実のこはん症発生と品質に及ぼす収穫後処理の効果 ……………………………（１６７）／テチャヴィセス ナッタコーン・疋田慶夫・河野俊夫

コムギ由来プロテインジスルフィドイソメラーゼの発現，精製と性状 …………………………………（１７３）／新井智美・野口智弘・内野昌孝・"野克己＜講座＞
身近な野菜・果物〜その起源から生産・消費まで（１３） …………………………………………………（１８５）
リンゴ（!）／工藤亞義＜文献抄録＞ ……………………………………………………………………………………………………（１９１）＜本会記事＞ ……………………………………………………………………………………………………（１９２）＜会
告＞ ……………………………………………………………………………………………………（２０１）

154

（ 4 ）

Food Preservation Science

CONTENTS OF VOL．
３
７ NO．
４（２
０
１
１）
＜Article＞（English）
Effect of Ingestion of the Japanese Persimmon ‘Saijo’ Fruit on Ethanol Levelsin the Blood of Humans and RatsITAMURA Hiroyuki, SUN Ningjing, NIMURA Masayuki, SHIMOSAKI Shunsuke,
NAKATSUKA Akira, ANDO Haruka, NAGAMATSU Hiroko, KATSUBE Takuya,
TSURUNAGA Yoko, ASANO Yujiro, ARAKAWA Osami, KAMEI Tsutomu,
MURATA Koji, SUGINAKA Katsuaki, FUNAKI Kenji, MATSUMOTO Toshikazu,
KOJIMA Yasunori, UEMURA Eisuke, MURAKAMI Naoyuki and MATSUZAKI Hajime ……………（１５５）
A Comparative Study of the Effects of Postharvest Treatments on Occurrenceof Kohansho and Quality of ‘Kiyomi’ Tangor
TECHAVISES Nutakorn, HIKIDA Yoshio and KAWANO Toshio ………………………………………（１６７）
Purification and Characterization of Wheat Protein Disulfide
Isomerase Expressed in Escherichia coli
ARAI Satomi, NOGUCHI Tomohiro, UCHINO Masataka and TAKANO Katsumi ……………………（１７
３）＜Serialization Lecture＞（Japanese）
Apple（Part!）
KUDOU Tuguyoshi …………………………………………………………………………………………（１８５）（ 5 ）

Food Preservation Science VOL．
３
７ NO．
４ ２
０
１
１ 〔Article〕

155

Effect of Ingestion of the Japanese Persimmon ‘Saijo’ Fruiton Ethanol Levels in the Blood of Humans and RatsITAMURA Hiroyuki＊１§，SUN Ningjing＊２，NIMURA Masayuki＊３，＊４，＊５，SHIMOSAKI Shunsuke＊４，
NAKATSUKA Akira＊１，ANDO Haruka＊１，NAGAMATSU Hiroko＊１，KATSUBE Takuya＊６，
TSURUNAGA Yoko＊７，ASANO Yujiro＊１，ARAKAWA Osami＊８，KAMEI Tsutomu＊９，＊１０，＊１１，
MURATA Koji＊９，SUGINAKA Katsuaki＊６，FUNAKI Kenji＊７，MATSUMOTO Toshikazu＊１，
KOJIMA Yasunori＊１，UEMURA Eisuke＊１，MURAKAMI Naoyuki＊１ and MATSUZAKI Hajime＊１２＊１ Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690−8504＊２ The United Graduate School of Agricultural Sciences, Tottori University, 4−101 Koyama−Minami, Tottori 680−8553＊３ Organization for the Promotion of Project Research, Shimane University, 1060 Nishikawatsu, Matsue 690−8504＊４ Department of Experimental Animals, Center for Integrated Research in Science, Shimane University,89−1 Enya, Izumo 693−8501＊５ Kokoro Medical and Welfare Professional College, Nagasaki 850−0048＊６ Shimane Institute for Industrial Technology, 1 Hokuryo, Matsue 690−0816＊７ Faculty of Education, Shimane University, 1060 Nishikawatsu, Matsue 690−8504＊８ Health Administration Center, Shimane University, 1060 Nishikawatsu, Matsue 690−8504＊９ Shimane Institute of Health Science, 223−7 Enya, Izumo 693−0021＊１
０ Institute for Transcultural Health Studies, European University Viadrina.
August−Bebel Str. 12, Frankfurt (Oder) 15207, Germany＊１
１ Internal Medicine Unit, Meiji University of Integrative Medicine,
Honoda, Hiyoshi−cho, Nantan 629−0392＊１
２ Shimane Agricultural Technology Center, 388−3 Shimoko, Hamada 697−0006

The Japanese astringent-type persimmon ‘Saijo’ fruit is considered to contain bioactive compounds
that help to alleviate the deconditioning seen after excessive intoxication with alcohol in humans. To
evaluate the contribution of ingestion of persimmons in lowering blood ethanol levels in intoxicatedhumans, we investigated the blood ethanol concentration with the ingestion of persimmons（fresh or
semi-dried persimmon fruit and persimmon extract）before or after alcohol consumption（including acomparison with that of apples before alcohol consumption）. For rats, we investigated their blood
ethanol concentration with the ingestion of persimmon extract before alcohol administration, and also
detected the antioxidant activity （ measured as ferric reducing antioxidant potential （ FRAP ）） andflavonoid content in their blood after administration of persimmon extract. Ingestion of persimmons
before alcohol consumption significantly reduced human blood ethanol levels compared with control
values １ h after alcohol consumption, and this was even more effective than that observed withapple ingestion. Rat data also suggested the same tendency of persimmon extract, but the difference
was not significant. There was no significant difference in the human blood level of ethanol between
ingestion of persimmons after alcohol consumption and that of control. In addition, neither an increasein the level of flavonoids nor an increase in antioxidant activity were detected in rat plasma ; there
was even a slight decrease in FRAP after administration of persimmon extract. Taken together, thesedata showed that the functional compound kaki-tannin alone or together with other components inpersimmon fruit ingested by humans before alcohol consumption was effective in lowering blood
ethanol levels. These components are not entirely absorbed to blood capillaries, so they might adsorb
the ethanol in the human digestive system to depress the absorption coefficient of ethanol on thesurface of the gastrointestinal epithelium.
（Received Mar. ３, ２０
１０；Accepted May ２
３, ２
０
１
１）
Key words：‘Saijo’ persimmon, drunken sickness, blood ethanol levels, kaki−tannin, gastrointestinal epithelium
西条柿，悪酔い，血中エタノール濃度，カキタンニン，胃腸上皮組織§ Corresponding author, E-mail : itamura@life.shimane-u.ac.jp

156

Persimmon （ Diospyros

kaki

Food Preservation Science

VOL．
３
７ NO．
４ ２
０
１
１

Thunb. ） has

Committee for Animal Experimentation at Shimane

been

（ 6 ）

cultivated in Japan for several centuries, and is

University （ Matsue,

Japan ）. The

animalswerethought to have originated in Southern China１）. It

handled according toinstitutionalguidelines.

Allcontains

subjects

manybioactive

carotenoids,tannins,aromatics,

flavonoids,

compounds,

sugars,includinghydrocarbons,terpenoids,

lipids,steroids,

provided

writtencertification

of

the

Shimanenaphthoquinones, amino acids２） and minerals３）. It is

Administration Center.

also

２．Reagents and solutions

a

good

sourceofnutritional

antioxidant

informed

consent

toparticipate in all experiments in compliance with the
University

Healthpolyphenols４），５）, β-carotene６） and dietary fiber３）. The

Ethanol（９９．
５％）was obtained from Wako Pure

fruit and leaves of the persimmon are used to treat

Chemical Industries（Osaka, Japan）. Distilled water

２）

７）
，
８）

. The fruit and leaves

was used to prepare the solutions. The standardof the persimmon as well as extract from the peel

curve for calculation of ethanol concentration was

also have several health - promoting effects. These

prepared in the range of ０．
０〜４
０
０ ppm（３２６!／!）

include : free radical-scavenging activity９） ; prevention

using samples of whole blood obtained from eachcoughsand hypertension

１
０）of a rise in the level of lipids in plasma
oxidative

effects （ fromthe

; anti -fruit３）） ; anwholehuman subject before administration of fruit or
alcohol.

Allstandards

wereprepared

at ０℃ .

improvement in lipid levels in plasma and the liver

Artificial stomach liquid was prepared with ０．
２％（from compounds in the leaves１１））; anti-oxidative andNaCl（Wako）, ０．
３
２％ pepsin（Sigma-Aldrich, St Louis,

anti-genotoxic effects（from compounds in the peel

MO, USA）and adjusted to pH ２ by HCl（０．
１％ final

extracts１２） ; and an anti - mutagenic effectagainst

１
６）
，
１
７）
concentration）. Purified kaki-tannin was obtained

ultraviolet irradiation（found in kaki-shibu（crude kakifrom Maruzen Pharmaceuticals Company Limited

１
３）-tannin extract）and its tannin-degraded products ）.

（Onomichi, Japan）.

Using gas chromatography（GC）, OGATA１４） showed

３．Plant materials

that Japanese persimmon ‘Fuyu’（sweet-type）fruit（１）
Fresh persimmon fruit

juice（including the peel）significantly decreased thepersimmon（Diospyros kaki Thunb. ‘Saijo’）fruit after

ethanol

excessive

removal of astringency. The fruits used were of

drunkenness in rabbits after alcohol administration.

uniform weight（〜２００"／fruit, containing〜１．
５％ kaki

level

in

blood

and

alleviatedWe used the mature

OGATA suggested that the pectin and／or kaki-tannin-tannin）, without visual defects, and were harvested

contained in persimmon fruit juice may “coat” thein Shimane prefecture（Japan）. For the measurement

gastrointestinal epithelium and exert an uptake -of polyphenol content, ‘Saijo’ persimmon harvested

inhibitor effect. KOIKEDA et al .１５） reported that taking

from farm fields in the Shimane prefecture were

‘ Saijo’ persimmon extract before drinking alcohol

used. Astringency was removed from fruit by dry

could significantly help in lowering

ice（１．
５％ of total fruit weight）.

theethanolconcentration in blood for people who have a low（２）Semi - dried persimmon fruit

tolerance for alcohol.

persimmon‘ Saijo’

fruit

is

available

October

December.

It

is

In the present study, we examined the activity of

to

not

The fresh
only

from

availablefor‘Saijo’ persimmon in lowering the concentration ofexperimentation during other seasons. JUNG et al .６）

ethanol in blood after administration of the whole

found that the contents of dietary fibers and trace

fruit or fruit extract to humans or rats. We also

elements in fresh and equivalent quantities of driedestablished a time - course for absorption of thepersimmon fruit were comparable, and proposed

persimmon

gastrointestinalthat the latter could substitute for the fresh fruit.

epithelium to the blood vessels of rats. Using a

extractfromtheWe therefore used the semi-dried persimmon fruit

ferric-reducing antioxidant power（FRAP）assay, we（containing ３
２％ of the water content of the fresh

also examined changes in antioxidant activity in the

fruit）made from the fresh, mature persimmon. This

plasma of rats after administration of persimmon

semi-dried fruit was used in the experiments carried

extract.

out in winter in place of the fresh persimmon

Materials and Methods
１．Ethical approval of the study protocol
The animal studies were approved by the Ethics‘Saijo’ fruit.
（３）Persimmon extract

The fruit extract wasused in the experiments carried out in winter as a
substitute for the persimmon fruit. Fruit without

（ 7 ）〔Article〕 Effects of Persimmon on Drunken Sickness

calyces

were

processor

or157

washedandcrushed

in

a

food

apple fruit ３
０ min before consuming sake, the same

blender.

The

crushed

fruit

was

way as in the control subjects.
The second, third, and fourth samples of blood in

extracted in hot water for ３
０ min and filteredthrough a clean cloth. The filtered extract was

the control and treatment subject were taken at １,

subsequently centrifuged at ３，０００ rpm for １
０ min

２, and ３ h after alcohol administration, respectively.

supernatant２）Rats：Male rats（age, ２４ weeks ;３
８
０〜４
４
０ g）

（designated ‘persimmon extract’）stored at −２
０℃

were randomly divided into two groups of five after

at

room

temperature,

and

the

until use. A ３５-! persimmon extract was equivalent

overnight

to 〜２
０
０! of fresh fruit based on the amount ofvehicle（distilled water）only, whereas each rat in thetannin content.

test group received a dose of １
０! ／ " of １
０％

４．Animals

fasting.

The

controlgroup

received

persimmonextract.

Incorporated,administeredorallythealcohol（Magensonde）before ３
０ min of alcohol loading. Bothadministration experiment. Male Wistar rats（Charles

groups were given alcohol（２．
０!／"）. Baseline blood

WKY ／ Izm

Male

Hamamatsu,

rats （ SLC,

Japan ） were

used

for

Persimmonextract

was

using

gastric

tube

aRiver Laboratories, Tokyo, Japan）were used for the

samples were collected before alcohol administration

FRAP（０ h）and test samples were collected at １, ２，
４ and

assay

and

extractionofflavonoidsfromplasma. All animals were housed at the Institute of

６ h after alcohol loading.

Animal Experiment of Shimane University with a（２）Experiment ２：ingestion of persimmon fruit
after alcohol administration

controlled lighting period（lights on from ０
７：０
０ to
１
９：０
０）. Rats were fed a commercial diet（CLEA

１）Human subjects： A study population of １
６

Japan, Incorporated, Tokyo, Japan）with tap water

individuals（１１ males and ５ females）was chosen fromad libitum for １ week.

３
３ healthy adults using the subject test selection

５．Experimental procedures

methods described above. The ratio of the sex of（１）Experiment １ ： ingestion of the persimmon

subjects（male : female＝２：１）was designed to be

fruit before alcohol administrationconsistent with that in experiment １（i.e., ingestion１ ） Human subjects ： The age range of therecruitedsubjects

population

was ２
０〜５
０ years.

Aof １
０ individuals （ ７ males

females ） who

showedexpression

the

of

homo -

or

of

the

persimmon

fruit

was

beforealcoholstudyadministration ）. This experiment was undertaken

and ３

using persimmon extract of the same persimmon

heterologous

cultivar

from

the

fruit

used

in

the

earlier

acetaldehyde - dehydrogenase ２

experiments. Each subject fasted for １
０ h before（ALDH２）gene were selected from ２
０ healthy adults

the experiment. At the end of fasting, baseline

１８）by the results of the alcohol patch test and theblood samples（５!／subject）were taken from eachscore

subject（０ h）. After drawing the blood sample, each

of

Tokyo

University

ALDH２ Phenotype

１
９）

Screening Test（TAST） .

subject immediately drank a ７
０-! serving of alcohol
fresh（‘shochu’, containing ２
５％ or １４．
３ g ethanol）over amature（in October）, semi-dried persimmon and fresh

１
５- min period. Persimmon extract was not given

apple fruit（in February）. Each subject was fasted

after

for １
０ h before the experiment. At the end ofexperiments.

This experimentfasting,

the

first

was

undertakenbaseline

blood

using

sample （５! ／alcoholforthecontrol

After a ２-day interval, each subject repeated theexperimentsubject）was taken from each subject（０ h）.

consumption

exceptthat, ２
０ min

after

alcohol

In control subjects, each volunteer drank a ９０-!

consumption, they drank a ３５-! persimmon extract

serving of the Japanese alcoholic beverage sakecontaining approximately the same concentration of（containing １
５〜１
６％ or １
１．
０ g ethanol）over a １
５-

soluble polyphenols found in the whole fruit. The

min period. There was an interval of ５〜７ days

second, third, and fourth blood samples were taken

between conduction of the control experiment andat １, ２,treatment experiment because the same subject

respectively.

could not repeat alcohol consumption in a short（３）Ethanol

and ３ hafter

concentration

alcoholinadministration,

blood

Whole

subject

blood（０．
５!）was added to a ５．
０-! screw-cap vial

ingested either a single ripe fresh persimmon fruit（SY-２ ; Nichiden-Rika Glass, Kobe, Japan）. The vialor two semi - dried persimmon fruits or one fresh

was immediately sealed with a silicone-septum cap,

period.

Inthetreatmentgroup,each158

Food Preservation Science

VOL．
３
７ NO．
４ ２
０
１
１

（ 8 ）

placed in an incubator at ２
７℃ for ２
５ min, andvaried from ０ min to １
０ min with acetonitrile／０．
１％heated at ４
０℃ for ３
０ min in a water-bath. The ２５-G

formic acid（１０：９０）followed by １
０〜２
０ min with aneedle of a glass syringe（volume, １!）was thenlinear gradient ofinserted into the septum. A ０．
５-! volume of the（１０：９０）to acetonitrile／０．
１％ formic acid（３０：７０）

headspace vapor was drawn into the syringe and

and ２
０〜５
０ min with acetonitrile／０．
１％ formic acidrapidly injected into the GC port at ２
１
０℃.

（３
０：７
０） ; flow rate, １!／min ; and electron capture（４）GC condition

detector（ECD）detection was set at ８００ mV.

The GC analysis was carriedacetonitrile ／０．
１％ formic

acid

out on a GC １
４ A series gas chromatograph with（６）Experiment ４：changes in soluble polyphenol

flame ionization detector （ FID ）（ Shimadzu, Kyoto,content in persimmon fruit after incubation in

Japan）. The GC conditions were as follows : column

artificial stomach liquid

Persimmon flesh wastemperature, ９０℃（the retention time of ethanol was

cut into fine pieces, and ground into paste in a

〜１
０．
２ min）or ９
０℃ to １
５
０℃（the retention time ofmortar and pestle. Four grams of the fruit paste

ethanol was〜９．
４ min ; ７ min hold at ９
０℃, ３
０℃／

with ２０! of artificial stomach liquid or distilled

min from ９
０℃ to １５
０℃, and ８ min hold at １
５
０℃）;

water was added to a ５
０-!centrifuge tube, and theinjection temperature，２
１
０℃ ; detection temperature,

tube

２
１
０℃ ;

and

heliumflow

rate, ６０! ／ min.

wasshaken.

The

centrifuge

tube

was

A

incubated in an ultrasonic automatic washer at ３
７℃

chromosorb １
０
１（８
０／１
０
０ mesh）pre-conditioned glassfor ５ min. After further incubation （ without ancolumn（１．
６ m×３．
２ mm I.D. ; Shimadzu）was used.

ultrasonic automatic washer）at ３
７℃ for ０．
５, １, ２,Forand ３ h, the solution was centrifuged at １０，
０
０
０ rpm

GC

quantification,

the

peak

area

of

each

compound was used. Ethanol was quantified with afor ３
０ min at ４℃.

calibration curve fit using ０．
０〜４
００ ppm （３
２
６" ／

The

!）ethanol solutions prepared in whole-blood samples

supernatantfrom each subject.

method as described by SWAIN and EILLIS２２）. Briefly,（５）Experiment ３：flavonoids levels in the plasma

１! of sample（２００ µ% of supernatant diluted with

content
was

of

total

polyphenols

measured

by

the

in

the

Folin-Denis

８００ µ% of distilled water）was made up to ７! withof rats administered persimmon extract１）Sampling method of the blood for detectionof the absorption of kaki - tannin from thegastrointestinal epithelium to the blood vessels：
Each rat（age, ５ weeks ; １２０〜１４０$）was fasted for

distilled water, and mixed with ０．
５! of Folin-Denis
reagent. After ３ min, １!

ofsaturated

Na２CO３solution was added. The mixture was made up to
１
０!

with

distilled

water

at

room

absorbancewastemperature

end of fasting, a baseline blood sample（３!／rat）was

measured with a spectrophotometer （ Hitachi U -taken from each rat （０ h ）, and each rat was１１００）at ７２
５ nm. The determination was carried out

immediately given persimmon extract（１!／rat）using

thrice. The amount of phenolics（expressed as mg

a gastric tube（Magensonde）. The second and thirdcatechin／１
０
０ g fresh weight）was calculated from a

blood samples were taken at ２ h and ４ h after

standard curve（０．
０１〜０．
１# catechin／!）preparedadministration, respectively.

at the same time.

２）FRAP assay：We carried out the FRAP assay

The

left

１
６ h before the experiments were started. At theaccording to the methods of LOTITO and FREI２０）. In

for ６
０ min.

and（７）Experiment ５：determination of the adsorption
effect of kaki-tannin on ethanol gas/liquid

this assay, the antioxidant capacity of plasma wasFirst, purified kaki-tannin（powder ; １００ mg）wasmeasured as ferric - reducing antioxidant potential

sealed in a ６- ! glass vial with an open - topscrewcap and Teflon ／ silicon disk （ Nippon Electricusing sampled blood.
３ ） Extraction and determination of flavonoids

Glass, Shiga, Japan）. In the control experiment, kaki-from plasma：We extracted the flavonoids fromtannin was not added. Half a milliliter of saturated

the plasma of rats administered persimmon extract

ethanol gas or ２! of ethanol（９９．
５％）liquid was２
１）

according to the methods of SESINK et al . . The

added to the sample or control ６-! vial. The vial

extract product （２０ µ% ） was analyzed by high -wasperformance liquid chromatography （ HPLC ）. The

ethanol gas was prepared as follows. Four milliliters

shakenwelltoensuremixing.

Saturated

HPLC system（LaChrom, Hitachi, Tokyo, Japan）wasof ethanol（９９．
５％）solution was sealed in a １
９-! vial

fitted with an ODS-８
０ Ts column（４．
６×２
５０!）. Theand incubated at ３
５℃ for ２
０ min : the headspacesystem conditions were as follows :gas was assumed to be saturated ethanol gas.

solvent was（ 9 ）〔Article〕 Effects of Persimmon on Drunken Sickness

The ethanol gas concentration and amount of
ethanolinethanolsolution

in

the

vials

were

159

the １％ level. Fig.１-B shows that the blood ethanol
levels were lowest in the semi - dried persimmon

６．
３
７±１
５．
７
４! ／ " ）, and also that levels in thedetermined ２ h after incubation. Headspace gas（０．
２． （９!）or １ µ" of the solution was injected into the gas

fresh apple（１
３
３．
１
１±１
４．
３
３!／"）were lower thanchromatograph. The GC condition was identical tothose in the control group （１７
４．
７
５±１
３．
９
５! ／ " ）.

that described in Experiment ２ except that the

Therecolumn, injection and detector temperatures weretreatment and control at the ５％ level.

weresignificant

differences

among

each

３）Two hours after alcohol consumption：In the

１
６
０℃，２
０
０℃, and ２
００℃, respectively.
６．Statistical analyses

fresh persimmon group, the ethanol concentrations

The mean ± SE value from experiments

waswere lower（８．
５７±８．
５７!／"）than in the control

thegroup （６
１．
１
１±２
８．
９
９! ／ " ）, but there was noconcentration of ethanol in human blood between

significant difference at the ５％ level（Fig．
１ A）. In

control and fresh persimmon or persimmon extract

the semi-dried persimmon（１
６．
３
８±１
２．
３
８!／"）andgroups was evaluated by two - factor analysis of

freshvariance（ANOVA）with only one observation in eachconcentrations were almost identical with those incell （ P ＜０．
０５）. Among the controls, semi - dried

the control group（２
９．
４
４±１
９．
１
５!／"）
（Fig.１ B）.

determined.

Time - dependentchangesinpersimmon and apple, pairwise comparisons weremade using the Ryan-Einot-Gabriel-Welsch Q test（Papple （１
７．
５
８±１
４．
６
９! ／ " ）,４）Three

hours

after

alcoholtheethanolconsumption ：The mean whole - blood ethanol concentration of＜０．
０５）. Time - dependent changes in the ethanol
250

concentration in rat blood between control andA

Control 1

persimmon extract groups were evaluated by one -Fresh persimmon

200

factor ANOVA（P ＜０．
０５）. A multiple comparison**

method for mean values of changes in the FRAP
150

was undertaken using the Tukey-Kramer test（P ＜
０．
０
５）
.

Results
１．Ingestion of the persimmon fruit before alcohol
consumption（１）Human subjects

Among the data of １０

subjects, we excluded outlier values from ３ subjects.
The mean values of the remaining ７ subjects who
had a similar tendency with respect to changes in

Ethanol concentration (mg・ℓ-1)

assay in rat plasma among different time points

100

50

0
250
Control 2
Semi-dried persimmon
Fresh apple

B
200

a
150

b

blood ethanol level were calculated and statistical
100

analyses undertaken.
In the control group, the ethanol concentration inwholeblood （ n ＝７） peaked １ h

after

alcoholadministration and returned to baseline level at ３ h.
Blood ethanol levels in the fresh and semi - driedpersimmon and fresh apple almost returned to thebaseline level by ２ h（Fig.１）.
１）Baseline results before alcohol consumption：
The subjects in each group had whole-blood ethanol
concentrations of〜０!／".
２）One hour after alcohol consumption：Fig.１-A
shows that blood ethanol levels were lower insubjects treated with fresh persimmon （１
０
１．
８
５±
２
９．
８
９!／"）than those in the control（１
８
４．
１
６±２
９．
２
７
!／"）, and that this difference was significant at

c

50

0

a
a a
a
0

a
1

2

a

a
a a
3

Time after ethanol administration (hour)
Fig．
１ Time-course of the whole-blood ethanol level in ７
human subjects who ingested fresh persimmon（A），orsemi-dried persimmon fruit and fresh apple（B）before
alcohol consumption
Each value is expressed as the mean of ７ replications±SE.
A :＊＊ significant at P ＜０．
０１（two-factor ANOVA）; B : pairwise comparisons were made using the Ryan-Einot-GabrielWelsch Q test, different letters annotating values within eachhour indicate significance at P ＜０．
０
５ among treatments andcontrol.

160

Food Preservation Science（ 10 ）

was a slight decrease in antioxidant activity over ４
Controlh（Fig.４）.

Persimmon extract

1,600

1,200

800
400
400

Control

0
1

2

4

6

Time after ethanol administration (hour)
Fig．
２ Time-course of the whole-blood ethanol level inmale rats who ingested persimmon extract before
alcohol consumption
Each value is expressed as the mean of five replications±SE.
Significant differences between treatment and control were
tested by one - factor ANOVA at the P ＜０．
０
５ level. Eachvalue was not significant.

Ethanol concentration (mg・ℓ-1)

Ethanol concentration (mg・ℓ-1)

2,000

VOL．
３
７ NO．
４ ２
０
１
１

Persimmon extract

300

200

100

0
0

1

2

3

Time after ethanol administration (hour)

subjects in each group returned to baseline levels（Fig.１）.
（２）Rats

Blood ethanol concentrations measured

at appropriate intervals after administered alcohol
are shown in Fig.２. The peak blood level wasreachedbetween １ hadministration.

Theand ２ hpersimmon

after

alcohol

extract

group

Fig ．
３ Time - course of whole - blood ethanol levels inhuman subjects who ingested persimmon extract after
alcohol consumption
Each value is expressed as the mean of １
６ replications±SE.
Significant differences between treatment and control were
tested by two - factor ANOVA at the P ＜０．
０５ level. Eachvalue was not significant.

400

showed lower blood ethanol concentrations １, ２ anddifference was not statistically significant.
２． Ingestion of persimmon extract after alcohol
consumption in human subjects
There was no statistical significance at the ５％
level

in

changesinwhole - bloodconcentrations （ n ＝ １６ ） between

ethanolpersimmon（ persimmon extract ）- treated groups and control

FRAP (μmol TE / g)

４ h compared with the control group, though the
300

200

100

groups １〜３ h after alcohol consumption. However,
the results suggested that blood ethanol levels in
the treatment group were even higher than thosein the control groups １ h and ２ h after alcohol
consumption（Fig.３）.
３． Flavonoid levels in the plasma of rats afteradministration of persimmon extract
In the plasma of rats given persimmon extract,kaki-tannin consisting of four types of catechins was
not detectable by HPLC（data not shown）, and anincrease in FRAP was not seen. However, there

0
0

2

4

Time after kaki-drink administration (hour)
Fig ．
４ Time - course of plasma antioxidant capacity inmale rats after consumption of persimmon extract
Each value is expressed as the mean of ２〜５ replications±SE. Statistical analyses were undertaken by one - factor
ANOVA at the P ＜０．
０
５ level. Significant differences among
respective time periods were tested by the Tukey-Kramer
post−hoc analysis. Each value was not significant.

〔Article〕 Effects of Persimmon on Drunken Sickness

４． Changes

insolublepolyphenol

content

persimmon

fruit

after

incubation

inin

Soluble polyohenol content (mg / 100 gfw)

（ 11 ）

artificial

stomach liquid
There was a larger content of soluble polyphenols
in ‘Saijo’ persimmon extract when it was incubatedwith artificial stomach liquid than when it was
incubated in distilled water. The polyphenol content
being incubated with artificial stomach liquid wassignificantly larger than that when it was incubatedwith water at ３ h（Fig.５）.
５．Adsorption effect of kaki-tannin on ethanol gas/liquid
The level of ethanol gas（０．
０３!／!）after ２-h

ʻSaijoʼ persimmon (astringency removed)

100

Water

liquid after ２-h incubation with kaki-tannin showedno significant difference in ethanol amount comparedwith that in the control（Fig.６ B）
.

60

*

40

20

0
0

after alcohol consumption in humans. The time ofingestion of persimmon fruit, either before or afteranimportantfactorindecreasing the ethanol concentration in blood. The
effects of different times of fruit ingestion were also
distinguished in this work.
Subjects
beforewho

theingested

consumption

fresh
of

persimmon

alcohol

fruit

showedadecrease in blood ethanol levels when comparedwith their corresponding control levels in which
they did not receive fruit before drinking sakealcohol. One hour after alcohol consumption, thedecrease in blood ethanol levels was significant（Fig.
１ A ）. The results for the semi - dried persimmon
werenearlyidentical

to

those

for

the

fresh

persimmon（Fig.１ B）. We also observed a similar（but

1.6

1

A
Ethanol amount (g/mℓ)‘Saijo’ persimmon in lowering blood ethanol levels

is

3

Fig．
５ Time-course of soluble polyphenol content in Saijo
persimmon fruit after incubation in distilled water andartificial stomach liquid（pH ２）at ３
７℃

Ethanol gas concentration (mg/mℓ)

In the present study, we studied the effects ofconsumption,

0.5
1
2
Incubate time (h)Each value is expressed as the mean of three replications±SE. ＊significant at P ＜０．
０５（one-factor ANOVA）.

Discussion

alcoholArtiﬁcial stomach liquid

80

incubation with kaki-tannin was significantly lower
than that in the control（１．
５!／!）（Fig.６ A）. Ethanol

161

1.2

0.8

0.4

**

0
Control

B

0.8
0.6
0.4
0.2
0
ControlKaki-tannin

Kaki-tannin

Fig．
６ Ethanol gas concentration（A）or ethanol amountin liquid（B）２ h after incubation with kaki-tannin atroom temperature compared with that in the control
Each value is expressed as the mean of three replications±SE.
＊＊significant at P ＜０．
０１（one-factor ANOVA）between kakitannin treatment and control.

not significant trend）in adult rats（Fig.２）. Although
there was a tendency of lowering blood ethanol

used ; blood ethanol levels were reduced markedly

levels by administration of persimmon extract inonly if fruit juice or peel had been administered ３０rats, this effect was not significant at the ５％ level.

minThis might have been due to the excessively highsubjects

administration of alcohol, which was almost tenfold

consuming alcohol had blood ethanol levels nearly

that used in humans.

identical to the controls at each time point after

beforewho

alcoholadministration１４） .

ingested

persimmon

Conversely,

extract

after

These results that ingestion of persimmon before

alcohol consumption（Fig.３）. These results show that

alcohol consumption can reduce blood ethanol levels

the whole - blood ethanol concentrations of subjects

are in accordance with those obtained from adult

were affected only by the ingestion of persimmon

rabbits in the study of OGATA in which the fruit

fruit before alcohol consumption.

juice and peel of the ‘Fuyu’（sweet type）fruit was

These

resultssuggestthat

theingestion

of

162

Food Preservation Science

intoxicationinhumans.

et

KOIKEDA,‘ Saijo’

our artificial stomach liquid experiment（Fig.５）. Kaki

excessiveal １５） .

（ 12 ）

tannin under acid conditions, which was proved bypersimmon fruit before alcohol consumption may
alleviate the deconditioning seen after

VOL．
３
７ NO．
４ ２
０
１
１

also

-tannin was not absorbed from the surface of theextract

gastrointestinal epithelium to the blood capillaries in

before drinking alcohol could lower the ethanol

our rat experiments（Fig.４）, so we do not think thatconcentration in human blood. This is an importantitfinding, particularly forreported

thattakingpersimmon

subjects

plays

a

part

in

the

ethanolconcentration -

have

low

decreasing effect in the blood and other organs（e.g.,

reduction

whoinliver）. Thus, we hypothesize that inside the stomachethanol concentration was significant compared withthese de-polymerized soluble kaki-tannins（１
５!／１
０
０

control values.

gfw）with the initial low-molecular-weight polyphenols

toleranceforalcohol,becausethe

However, it remains unclear which components of

（３
０!／１
０
０ gfw ; total，４
５!／１
０
０ gfw）, as well as

the persimmon fruit are involved in decreasingsome dietary fibers（or probably in addition with theblood ethanol levels. According to our in vitro study,

insoluble-type tannin）form a type of “film” to adsorb

we showed that kaki - tannin （ water - soluble ） was

the ethanol gas which is released from the ingested

extremely efficient in adsorbing ethanol gas（Fig.６

ethanol liquid. This results in the disturbance ofA）, but it could not adsorb ethanol liquid（Fig.６ B）.

total ethanol absorption by the human digestive

It is therefore likely that the highly functional

system （ Fig.７）. OGATA１４） also proposed that thecompound kaki-tannin is related to the reduction of

intoxication-alleviating effect of the persimmon fruit

ethanol gas but not ethanol liquid in the humanwas dependent upon depression of the absorption

stomach. Usually, astringent - type persimmon fruit

coefficient

contains kaki-tannin in an exclusively soluble form

gastrointestinalofethanolonepitheliumtheinsurface
the

of

the

presence

of

（１．
５"／１
０
０ gfw［fresh weight］）. After the removal

persimmon fruit juice. OGATA’s hypothesis supports

of astringents in order to be edible, nearly all of the

our hypothesis to a certain extent.

soluble tannin（high-molecular-weight）condenses to

Based on the above hypothesis, we might explaina higher-molecular-weight, insoluble tannin. Insoluble

the different effects of persimmon and apple inkaki - tannin is then ingested in the stomach, anddecreasing blood ethanol levels （ Fig.１ B ）. These

part of it（１５!／１０
０ gfw）is de-polymerized to soluble

might be due to the complete deficiency of soluble

Astringent ʻSaijoʼ
persimmon fruit

ʻSaijoʼfruit,astringency removed

ʻSaijoʼfruit,
incubated in stomach

Soluble polyphenol (catechins)

Soluble polyphenol (catechins)

Soluble polyphenol (catechins)
Soluble kaki-tannin

partially solubilised
by stomach liquid(pH = 2; 37ºC)

Soluble kaki-tannin

Insoluble kaki-tanninRemova(CO2,E lofastringentOH,dcyriedfruit)

Insoluble kaki-tannin

Soluble polyphenol

Ingestionintostom

achInsoluble tannin

Soluble kaki-tannin
Alcoholconsumption(stomach andupper part of the small intestine)
Ethanol gas

Ethanol liquid

Soluble kaki-tannin adsorbs the ethanol gas
which is released from the ingested ethanol liquid.
This results in the decrease of total ethanol
absorption by the human digestive system.

Fig．
７ Interactions of soluble kaki-tannin and ethanol in the human digestive system with respect to reductionin blood ethanol levels after alcohol consumption（schematic）

（ 13 ）

〔Article〕 Effects of Persimmon on Drunken Sickness

163

Table １ Dietary fibers and tannin（polyphenol）contents in persimmons and apples（!／１００ gw）
Dietary fibersz

Japanese
PersimmonsTanniny

Total polyphenolsx

SolubleInsoluble

SolubleInsoluble

Astringent, raw

０．
５

２．
３

１．
５〜２．
０

Trw

Astringency removed, raw
Dried

０．
５
１．
３

２．
３
１
２．
７

Tr
Tr

１．
５
２．
２〜４．
２

１．
５
１v
１．
５
１
７．
４
８

Raw

０．
３

１．
２

０

０

１．
２〜２．
４u

Applesz

Data for persimmons and apples are cited from Tables of Food Composition ２００
９（Fifth Revised and Enlarged Edition）２００９２３）.
y Data for persimmons are cited from study of T
９９２２４）, １９９８２５）, and ２００
８２６）.
AIRA et al ., １
x In persimmon, total polyphenols contain kaki-tannin and other catechins ;In apple, total polyphenol usually includes variety of soluble flavonoids and other phytochemicals.
w Tr means the content is trace level.
v Data for persimmons are cited from study of J
００
５６）.
UNG et al ., ２u Data for apples are cited from study of B
００
４２７）.
OYER and LIU, ２

kaki-tannin, or both soluble and insoluble tannin inapple. Also, the contents of dietary fibers in apple
fruit are lower than that of persimmons（Table １）.

２
０
２
７（１
９
９
８）
４）MATSUO, T. and ITO, S. : The chemical structureofkaki - tannin

fromimmature

fruit

of

the

Both of these aspects might contribute to thepersimmon（Diospyros kaki L.）, Agric. Biol. Chem.,

higher efficiency in ethanol alleviation in persimmon.

４
２, １
６
３
７〜１
６
４
３（１
９
７
８）

Futureexperiments （ including

animals ） willbeessential

mechanisms

by

which

decreases

blood

ethanol

for

the

those

with

５）GORINSTEIN, S., ZEMSER, M., WEISZ, M., HALEVY, S.,

resolvingthe

DEUTSCH, J. , TILIS, K. , FEINTUCH, D. , GUERRA, N. ,persimmon

fruit

FISHMAN, M. and BARTNIKOWSKA, E. : Fluorometricalcohol

analysis of phenolics in persimmons, Biosci. Biotech.

levelsafter

Biochem., ５
８, １
０
８
７〜１
０
９
２（１
９
９
４）

consumption in humans.

６）JUNG, S. T., PARK, Y. S., ZACHWIEJA, Z., FOLTA, M.,
Acknowledgments
HirokiOHTANI,The authors deeply thank Dr.
Dr.

Jun

UDAGAWA （ Shimane

BARTON,H. ,PIOTROWICZ,

TRAKHTENBERG, S.

and

J. ,

KATRICH,

E. ,

S.

Some

GORINSTEIN,

essential

SAITO （ Shimane University Organization

potential in fresh and dried persimmon, Int. J.

for

the

Promotion of Project Research ） for many helpful
suggestions during this work. We also thank Ms.

phytochemicals

and

the

:

University Faculty of Medicine ） and Ms. Masami

antioxidant

Food. Sci. Nutr., ５
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０
５〜１
１
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０
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KASHIMAeffect of shibu against hypertension, J. Jpn. Soc.

（Shimane University Health Administration Center）

Inter. Med ., ４
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５
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６（１
９
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５）（In Japanese with

ChitoseTSUBOKURA

and

Ms.

Norikofor their cooperation with sampling of human blood.
This work was supported by a grant （ project

English abstract）
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number １
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spontaneously

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:

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treatmentontheactivity of persimmon fruit purees, J. Agric. Food
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Dietary

the rise in plasma lipids in a diet-induced murine

persimmon improves lipid metabolism in rats fed

obesity model, Biol Pharm. Bull . , ２
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（２
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２
１）SESINK, A. L. A., O’LEARY, K. A. and HOLLMAN, P.

diet, Food Chem. Toxicol ., ４
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C. H. : Quercetin glucuronides but not glucosides

１２）KIM, S. Y., JEONG, S. M., KIM, S. J., JEON, K. I.,

are present in human plasma after consumption

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１３）MATSUO, T., KINOSHITA, M., ITOO, S., SHIMOI, K.,
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:

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１
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alcohol

excessive

drunkenness,

Effects

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metabolism
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andNew

pollensimulatedallergensandinsoluble

tannins

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３）
１
８）HIGUCHI, S., MURAMATSU, T., SAITO, M., SASAO, M.,
MARUYAMA, K., KONO, H., and NIIMI, Y. : Ethanol
patch test for low Km aldehyde dehyrogenase
deficiency, Lancet, ３
２
９, ６
２
９（１
９
８
７）
NORIOKA,

T.

〜２
９
７（２
０
０
８）
（In Japanese）
２
７）BOYER J. and LIU R. H. : Apple phytochemicals

food proteins and effect of preheating on theA. ,solubleduring fruit development in nonastringent and５ ）： On the several utilizations of kaki - tannin

１７）TAKAGI, K., TESHIMA, R., OKUNUKI, H. and SAWADA,

ASAKA,

Accumulation of:andtest, J. Serb. Chem. Soc., ６
９, ５
３
３〜５
４
０（２
０
０
４）

K. ,

carbongastrointestinal

conditions measured by a self - developed ELISA

１９） YAMADA,

andastringent persimmon, J. Japan Soc. Hort. Sci., ６
７,

PETROVIC, D., VUCKOVIC, O. and JANKOV, R. M. :mugwortethanol（１９９２）（In Japanese with English abstract）

GAVROVIC-JANKULOVIC, M. D., BURAZER, L., DERGOVIC bindingof２５） TAIRA, S. , MATSUMOTO, N. and ONO, M.

Remedies, ４
４, １
０
８
１〜１
０
８
９（２
０
０
７）
（In Japanese）

allergoids exposed to

mixture:１６） POLOVIC, N. D. , CIRKOVIC - VELICKOVIC, T. D. ,

IgG

a

on

alleviating

Consultation

with

and

TAKESHITA, T.. : Questionnaire for detecting thephenotype of low Km ALDH（ALDH２）（KURIYAMA,
K., TAKADA, A. and ISHII, H., eds. : Biomedical andsocial aspects of alcohol and alcoholism ）（ Elsevier
Science Publishers B. V., Amsterdam）, pp.４
８
１〜４
８
４
（１
９
８
８）
２
０）LOTITO, S. B. and FREI, B. : The increase in
human plasma antioxidant capacity after apple

and their health benefits, Nutr. J ., ３, ５（２００４）

（ 15 ）

〔Article〕 Effects of Persimmon on Drunken Sickness

飲酒前後のカキ 西条 果実摂取がヒト血中のエタノール濃度に及ぼす影響＊１

かった。カキ抽出液を投与したラットの血漿中にはフラボノイドは検出されず，FRAP値の増加も認められず，
むしろFRAP値は減少する傾向さえみられた。これらの

＊７

＊１

・下崎俊介

＊８

鶴永陽子 ・浅野祐二郎 ・荒川長巳 ・亀井

勉

＊９，
＊１
０，
＊１
１

村田幸治＊９・杉中克昭＊６・舟木賢治＊７・松本敏一＊１
＊１

＊１

＊１

児島靖典 ・上村英介 ・村上直之 ・松崎
＊１

一

＊１
２

島根大学生物資源科学部

（〒６
９
０―８
５
０
４ 島根県松江市西川津町１
０
６
０）
＊２

鳥取大学大学院連合農学研究科

（〒６
８
０―８
５
５
３ 鳥取県鳥取市湖山町南４−１
０１）＊３

島根大学プロジェクト研究推進機構

（〒６
９
０―８
５
０
４ 島根県松江市西川津町１
０
６
０）
＊４

中エタノール濃度は，対照と比べて有意差が認められな

明＊１・安藤はるか＊１・永松ひろ子＊１・勝部拓矢＊６

寧静 ・二村正之

＊３，
＊４，
＊５

認められた。一方，飲酒後にカキを摂取した被験者の血＊４

板村裕之 ・孫
中務

＊２

165

島根大学総合科学研究支援センター実験動物分野
（〒６
９
３―８
５
０
１ 島根県出雲市塩冶町８９−１）＊５こころ医療福祉専門学校

（〒８
５
０―０
０
４
８ 長崎県長崎市上銭座町１１−８）＊６島根県産業技術センター

（〒６
９
０―０
８
１
６ 島根県松江市北陵町１番地）＊７

島根大学教育学部

（〒６
９
０―８
５
０
４ 島根県松江市西川津町１
０
６
０）
＊８

島根大学保健管理センター

（〒６
９
０―８
５
０
４ 島根県松江市西川津町１
０
６
０）
＊９

島根難病研究所

（〒６
９
３―０
０
２
１ 島根県出雲市塩冶町２
２
３−７）
＊１
０ Institute for Transcultural Health Studies,
European University Viadrina（August-Bebel Str. 12, Frankfurt（Oder）15207,
Germany）＊１
１ 明治国際医療大学附属病院内科学ユニット
（〒６
２
９―０
３
９
２ 京都府南丹市日吉町保野田）＊１２ 島根県農業技術センター
（〒６
９
７―０
０
０
６ 島根県浜田市下府町３
８
８−３）
渋ガキ

西条

果実は，酩酊したヒトの体調の悪化を緩和できることが知られている。本研究では，カキを摂取することで飲酒後血中エタノール濃度の低下に寄与できるかどうかを評価するために，ヒトを対象に飲酒前後にカキ（カキ果実，あんぽガキまたはカキ抽出液）を摂取したときの血中エタノール濃度を調査するとともに，飲酒前のリンゴ摂取との比較も行った。また，ラットに対して，アルコール摂取前にカキ抽出液を投与して血中濃度を測定した。さらに，カキ抽出液を投与したラットの血漿中の抗酸化力（鉄還元能FRAP値）の変化とフラボノイドの有無を調べた。その結果，ヒトへの飲酒前のカキ摂取において，血中エタノール濃度はアルコール摂
取後１時間で対照およびリンゴ摂取よりも有意に低くなった。さらに，アルコール摂取前にカキ抽出液を投与したラットについても，有意差はないものの同様の傾向がことから，本研究は，飲酒前のヒトに，カキ果実中に含まれる機能性成分であるカキタンニンを，単独もしくは他の成分とともに摂取させることによって，血中エタノール濃度を低下させる効果があることを示した。カキの機能性成分は血中に移行しないため，これらはヒトの胃の中でエタノールを吸収し，胃腸の上皮表面からのエタ
ノールの吸収を抑制するものと考えられる。
（平成２２年３月３日受付，平成２
３年５月２
３日受理）

（ 17 ）

Food Preservation Science VOL．
３
７ NO．
４ ２
０
１
１ 〔Article〕

167

A Comparative Study of the Effects ofPostharvest Treatments on Occurrence of Kohansho
and Quality of ‘Kiyomi’ Tangor
TECHAVISES Nutakorn＊１，HIKIDA Yoshio＊２§ and KAWANO Toshio＊３＊１ The United Graduate School of Agricultural Sciences, Ehime University, 3−5−7 Tarumi, Matsuyama, Ehime† Present address : C.P. ALL Public Co., Ltd., Thailand ;313 C.P. Tower, 23 Fl., Silom Rd., Bangrak, Bangkok 10500, THAILAND＊２ Faculty of Agriculture, Ehime University, 3−5−7 Tarumi, Matsuyama, Ehime 790−8566＊３ Faculty of Agriculture, Kochi University, 200 Monobe, Nankoku, Kochi 783−8502

790−8566

The effects of postharvest treatments, namely, perforated film packaging （ PFP ）, hot water dip（HWD）, and hot calcium dip（HCD）, on the occurrence of “Kohansho（rind disorder）” and the quality
attributes of ‘Kiyomi’ Tangor（Citrus unshiu Marc. ×C. sinensis Osb.）fruit were compared. Samples ofthe fruit were treated, packed in corrugated fiberboard boxes, and stored at １
０℃ for up to ４ weeks.
Weight loss was significantly lower and firmness was significantly higher in the fruit treated by PFP
than in those treated by other postharvest treatments or in the control fruit. All postharvest
treatments reduced the occurrence of Kohansho compared with control, and PFP and HWD
treatments were more effective than HCD in reducing the occurrence of Kohansho. None of thepostharvest treatments impaired quality attributes tested（total soluble solids［TSS］, titratable acidity［TA］, TSS／TA ratio, ascorbic acid content, and ethanol content）, when compared with the attributesof the control. We conclude that PFP treatment, which can maintain a high relative humidity and amodified atmosphere except in the presence of high CO２ levels（＞８％）and/or low O２ levels（＜５％）was the most effective and commercially practical method to control Kohansho and preserve
quality.
（Received Nov. １５, ２０
１０；Accepted May ３
０, ２
０
１
１）
Key words：postharvest treatments, perforated film packaging, hot water dip, kohansho, ‘Kiyomi’ Tangor
収穫後処理，有孔フィルム包装，温湯浸漬，こはん症，タンゴール清見‘Kiyomi’ Tangor（Citrus unshiu Marc. ×C. sinensis‘ Kiyomi’ fruit should be stored under modified

Osb. ） is recognized as a high - quality late - season

atmosphere conditions, with high relative humidity,

citrus fruit. Unfortunately, this variety is vulnerable

and should not be exposed to high CO２ levels（＞to Kohansho, a serious, non-chilling physiological rind

４）８％）and/or low O２ levels（＜５％）.

disorderthatduringstorage,

Hot water dip（HWD）treatment（５０℃ for ３〜５

periods.

Kohanshomin）reduced the occurrence of Kohansho in Navelinitially appears as small pitting, later developing

orange５）, Natsudaidai５） and Hassaku５），６）. Similar HWD

into a brown sunken area. The disordered fruit istreatment（５０〜５３℃ for ２ 〜 ３ min ） reduced theusually rejected by the market.

incidencetransportation,

lowersandqualitymarketingofchilling

injuryin‘ StarRuby’

Individual seal -packaging reduces the occurrence

grapefruit７） and various cultivars of blood oranges８）of Kohansho１）, and storage under low temperature

without impairing quality. Calcium dip treatment

andwithout

was reported to lessen the damage due to many

prestorage conditioning reduces the occurrence ofphysiological disorders and preserve fruit quality byrind injury in ‘ Kiyomi’ fruit２）. In our previous

maintaining firmness and reducing weight loss andstudies,bedecay in a variety of fresh fruits and vegetables９）.

suppressed by reducing fruit weight loss during

A combined calcium and hot water（hot calcium dip,

high

we

relative

humidity

observedthat

conditions

Kohanshocan３）storage , and it was recommended that in order toHCD）treatment１０） is therefore expected to be moreprevent

effective than either HWD or calcium dip treatment

Kohanshoandpreserve

highquality,§ Corresponding author, E-mail : hikida.yoshio.mc@ehime-u.ac.jp

168

Food Preservation Science

VOL．
３
７ NO．
４ ２
０
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１

（ 18 ）

vapor pressure of the surrounding air from ９
８％ ofalone.
However, there is no information about the effectsaturated vapor pressure at the surrounding airof HWD and HCD treatment on ‘Kiyomi’ Tangor.

temperature.

The aim of the present study was to compare the

３．Evaluation of Kohansho

effects of perforated film packaging（ PFP ）, HWD,

Three replicates of １
６ fruits in each treatment

and HCD treatments on the occurrence of Kohansho

were checked for Kohansho by visual inspection at

and the quality of ‘Kiyomi’ Tangor fruit.

１−week intervals. The number of fruit and thediameter of affected areas were measured and rated

Materials and Methods

using the following index : ０ ＝ no occurrence of

１．Plant material

Kohansho ; １＝one spot of less than ５㎜ ; ２＝one‘ Kiyomi’ Tangor fruit （ diameter, ７．
３〜８．
０㎝ ）
harvested on March １
９，２
０
０
８, from Misaki, Ehime,
were transported to the Laboratory of Agricultural
Process Engineering, Ehime University. The fruit
without Kohansho were used for the experiment on
the following day（March ２６，
２
０
０
８）
.

more than ４ spots of ２０㎜.
The results were expressed as the percentage
and degree of Kohansho :Percentage of Kohansho（％）＝
total number of fruit incurring Kohansho
×１
００
total number of fruit

２．Postharvest treatments
The fruit were treated with different postharvest
treatments as follows.
（１）PFP treatment

spot of ６〜２
０ ㎜ ; ３＝２〜３ spots of ２
０ ㎜ ; ４＝

The fruit were sealed in a

perforated low-density polyethylene film package（aDegree of Kohansho＝
Σ（Kohansho index×number of fruit incurring Kohansho in each index）
×１００
４×total number of fruit４．Determination of quality attributes

０．
０
１
９- ㎜ film of dimensions ６
０×５
０! , with １
５

Three replicates of １
６ fruits in each treatment

perforations, each ５㎜ in diameter）and packed in a

（ the same samples that were used to evaluate

corrugated fiberboard box （ dimensions ３
６×２
５×１
５

Kohansho ） were weighed at １- week intervals to! ）. This perforated package was designed

inassess percentage weight loss. The firmness, andaccordance with the modified atmospheric conditions

the content of total soluble solids（TSS）, titratable

recommended by TECHAVISES and HIKIDA４）.

acidity（TA）, ascorbic acid（AA）, and ethanol were

（２）HWD treatment

The fruit were dipped indetermined in ３ replicates of ４ fruits each, on day

a hot water bath at ５
３℃ for ２ min, dried at room

０（fresh）and day ２
８（end of storage）. Fruit firmnessawas determined with a fruit hardness tester using acorrugated box. The temperature in the hot water１-" weight. Juice TSS content was measured withtemperature
bath

was

for

about ２ h,maintained

andpackedat ５
３ ± ０．
５℃

in

using

an

a digital refractometer, juice TA was measured bytitration with ０．
１ M NaOH, and the results were

electronic thermostat.
（３）HCD treatment

The fruit were dipped in

expressed as citric acidpercentages.

Juice

AA

hot １％（w／v）calcium chloride solution at ５
３℃ forcontent was determined by the indophenol titration

２ min, dried at room temperature for about ２ h,method１１）.

and packed in a corrugated box.
（４）Control

The

fruit

Ethanol, the dominant fermentative metabolite, canwerepackedinamandarin fruit１２）. The acetaldehyde and ethanol that

corrugated box without treatment.
Three replicates of ５" of ‘Kiyomi’ fruit（about
２
８ fruits ） were

used

in

each

treatment.

All

corrugated boxes were stored at １
０℃ for up to ４
weeks.

The

temperaturedevelopmentofoff - flavors,

thus

impairing

fruit

quality１３）. Juice ethanol content was determined byhumidity

incubation of １
０-! aliquots of juice in ５
０-! glassvials sealed with an aluminum cap at ３
０℃ for ３
０

PFP treatment, and inside the storage chamber

min following SCHIRRA et al .８）. Aliquots of １
０-! eachwere monitored throughout the storage period byof ethanol standards of １，
０
０
０, ２，
０
０
０, ３，
０
０
０, ４，
０
０
０,

using temperature and humidity sensors. The vaporand ５，０００（ µ# ／ # ） werepressure deficit（VPD）between the fruit surface andcalibration curve. After incubation, a １-!gas samplethetranspiration,

was

air,

a

major

calculatedbyrelative

accumulate during fermentation can lead to the（RH）inside the boxes, inside the packages in thesurroundingandbe used to indicate fermentative metabolism for

drivingusedtogenerate

a

force

of

was withdrawn from the headspace and analyzed

subtracting

thewith a gas chromatograph （ GC ;model GC -８ A,

（ 19 ）

〔Article〕 Postharvest Treatments on Kohansho and Quality

Shimadzu, Japan ） coupled with an FID anda

Results and Discussion

stainless steel column（length and internal diameter,
２０
０ × ０．
２ ㎝ ） containing １
５％

Carbowax

169

The ‘Kiyomi’ Tangor fruit in all treatments lostandChromosorb W ８
０／１
００ mesh. The temperatures ofweightthe injector, column, and detector were １００, ９０, andsignificantly lower in the PFP treatment than in

１
００℃, respectively, and nitrogen was used as theother treatments and control（Table １）. This was

carrier gas.

because the notably higher RH led to lower vapor

５．Atmosphere analysis

pressure deficit（VPD）in the PFP treatment than induringstorage ;theweightloss

was

A gas sample of ０．
５! was taken from inside

the other postharvest treatments or control（Table

each PFP package and from inside the storage２）. VPD is a major driving force for weight loss,

chambersince higher VPD results in higher transpiration.

at ３ - dayintervals.

O２

and

CO２

concentrations were determined using a GC（model

The fruit that underwent all the postharvest

GC-８ A ; Shimadzu, Japan）coupled with a TCD andtreatments showed lower percentage and degree ofainternal

Kohansho than the control fruit, but the PFP andstainlesssteel

column （ lengthanddiameter, １
８０ cm×０．
６ cm）containing WG-１
０
０. The

HWD treatments were more effective than the HCD

temperatures of the injector, column, and detector

treatment on reducing the occurrence of Kohansho

were ８０, ５０, and ８
０℃ , respectively. Helium was（Table ３）. In the PFP treatment, the average O２

used as the carrier gas.

and CO２ concentrations inside the package during

６．Statistical analysis

the storage period were ２０．
４０％ and ０．
９７％ , aAll data were analyzed by one - way analysis ofslight change from normal atmospheric level （ P ＜variance（ANOVA）by the SPSS software（Windows

０．
０５）. According to our previous study４）, such aversion １
１．
５; SPSS Inc., Chicago, IL, USA ）. The

slight change does not affect the occurrence ofsignificance

Kohansho.

ofdifferences

between

means

was

determined by the Tukey HSD test（P＜０．
０５）.

Therefore,theeffectiveness% Weight loss
Treatment
Week １

Week ２

Week ３

Week ４

０．
１
７±０．
０
１c
１．
７
４±０．
０
４ ab
１．
８
２±０．
０
４a
１．
６
５±０．
０
４b

０．
３
０±０．
０
１b
２．
９
５±０．
０
６a
３．
０
９±０．
０
７a
２．
９
３±０．
０
８a

０．
４
８±０．
０
１b
４．
４
０±０．
０
８a
４．
６
１±０．
１
０a
４．
５
４±０．
１
２a

０．
６
６±０．
０
２b
５．
８
９±０．
１
１a
６．
１
３±０．
１
３a
６．
１
４±０．
１
５a

Data are represented as mean±S.E. of ３ replicates of １
６ fruits each.
Column means followed by different letters are significantly different at P〈０．
０５．

Table ２ Average temperature and RH inside the corrugated fiberboard box（HWD,
HCD, control ）, inside the PFP package, and inside the storage chamber
throughout the storage period, and the vapor pressure deficit（VPD ）, in eachtreatment
Treatment
PFP
HWD
HCD
Control
Chamber＊

PFP

treatment results from the generation of high RH

Table １ Weight loss of ‘ Kiyomi’ Tangor fruit during storage at １
０℃ under different
postharvest treatments

PFP
HWD
HCD
Controlof

VPD＊

Temperature
（℃）

RH
（％）

（kPa）

１
０．
６
７
１
０．
０
６
１
０．
２
５
９．
８
２
９．
７
３

９
９．
２
１
７
９．
１
６
７
９．
８
５
８
２．
３
５
４
９．
１
７

０．
０
１
０．
２
７
０．
２
６
０．
２
２
−

VPD was calculated by subtracting the vapor pressure of the surrounding air from ９
８％of saturated vapor pressure at the surrounding air temperature.

170

Food Preservation Science

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０
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（ 20 ）

Table ３ Effect of postharvest treatments on occurrence of Kohansho in ‘Kiyomi’ Tangor fruit during storage at１０℃
Percentage of Kohansho（％）

Degree of Kohansho

Treatment
PFP
HWD
HCD
Control

Week １

Week ２

Week ３

Week ４

Week １

Week ２

Week ３

Week ４

０．
０
０
０．
０
０
０．
０
０
２．
０
８

０．
０
０
０．
０
０
０．
０
０
２．
０
８

２．
０
８
０．
０
０
２．
０
８
４．
１
７

２．
０
８
２．
０
８
６．
２
５
１
４．
５
８

０．
０
０
０．
０
０
０．
０
０
０．
５
２

０．
０
０
０．
０
０
０．
０
０
０．
５
２

０．
５
２
０．
０
０
１．
０
４
１．
５
６

０．
５
２
１．
０
４
２．
６
０
５．
７
３

Data are means of ３ replicates of １
６ fruits.

surrounding the fruit ; this result is consistent with

the PFP treatment group at the end of storage.

our previous findings３），４）. The HWD treatment wasThis result is consistent with the observation thatas effective as the PFP treatment（Table ３）. Similar

the weight loss was lower in the PFP treatment

effects of the HWD treatment have alsobeengroup than in the other treatment or control groups

and（Table ４）. Our previous study４） found that use ofreported

in

Navel

５）

５）

orange ,Natsudaidai ,

５）
，
６）
，
１
４）

Hassaku

modified. There was no significant difference inatmosphericconditions

maintainstheweight loss between the HWD and control groups ;firmness of ‘Kiyomi’ Tangor via the maintenance of

therefore,limit

a high RH inside the package. BEN-YEHOSHUA et al １５）.

Kohansho. For example, MAKITA５） reported that thealso reported that the high humidity of the micro-

HWD treatment of Navel oranges enhanced theatmospherereducing

ispackaging maintains the turgidity of the tissue andassociated with Kohansho tolerance. The fruit thatmarkedly inhibits water loss and softening in citrus

underwent the HCD treatment also showed reduced

fruits.

other

sugar

mechanisms

content

in

may

the

act

peel,

to

which

createdinsidemodified

atmosphereoccurrence of Kohansho compared with the control

The TSS content did not change, but the TA

fruit but was not as effective as the PFP and HWD

content tended to decrease, resulting in an increase

treatments（Table ３）. This implies that calcium wasin the TSS／TA ratio after ４ weeks of storage in all

not as effective as expected ;in contrast, thetreatment and control groups, compared with thecalcium

effectivenessofratio in the fresh fruit（Table ４）. The AA content

HWD. MANAGO６） also reported increased occurrence

treatment

decreased slightly and the ethanol content tended toof Kohansho in Hassaku after dipping in a ２％

increase in all the treatment and control groups

CaCO３solution. These results suggest that the bestcompared with the contents in the fresh fruit（Table

results can be achieved by a combination of the４）. Ethanol was seen to accumulate during the

PFP and HWD treatments.

storage period even in the control group, consistent

Firmnesswasreducedthethehighest

in

fresh

fruit

with our previous observation４）. However, there（determined on day ０）and decreased during storage

were no significant differences in TSS, TA, TSS／TA

in all treatments, but it was significantly higher inratio, AA content, and ethanol content between any

Table ４ Effect of postharvest treatments on the quality attributes of ‘Kiyomi’ Tangor fruit
Treatment
Fresh
PFP
HWD
HCD
ControlFirmness
（kg）

TSS
（％）

TA
（％）

TSS/TA
ratio

AA
０
０!）
（!／１

Ethanol＊（µ"／"）

０．
９
２
２±０．
０
０
５a
０．
８
８
７±０．
０
０
９b
０．
８
４
７±０．
０
０
５c
０．
８
５
７±０．
０
０
５c
０．
８
３
３±０．
０
０
６c

１
２．
４±０．
４a
１
２．
５±０．
２a
１
２．
２±０．
３a
１
２．
３±０．
３a
１
１．
９±０．
５a

１．
０
９±０．
０
５a
０．
９
３±０．
０
４ ab
０．
９
８±０．
１
１ ab
０．
８
１±０．
０
６b
０．
７
９±０．
０
５b

１
１．
６±０．
６a
１
３．
６±０．
６a
１
５．
６±３．
２a
１
６．
２±１．
４a
１
５．
４±０．
９a

５
８．
３±１．
８a
４
８．
７±１．
６b
５
１．
１±１．
４b
５
２．
６±１．
６ ab
５
０．
９±１．
７b

１
５
６
８±２
１
５a
１
８
１
７±３
４
０a
２
５
３
３±２
２
７a
２
５
９
６±３
０
４a
２
０
０
０±２
５
０a

Data are represented as mean±S.E. of ３ replicates of ４ fruits each.
In the case of the ‘fresh’ condition, all parameters were determined on day ０（initial day of experiment）, while in the case of
all other treatments, the parameters were determined on day ２
８（end of storage）.
Column means followed by different letters are significantly different at P＜０．
０５.
＊Ethanol is an indicator of fermentative metabolism.

（ 21 ）

〔Article〕 Postharvest Treatments on Kohansho and Quality

171of the postharvest treatment groups and the control

（２
０
０
８）

group after ４ weeks of storage.

５）MAKITA, Y. : Histochemical observations on the

On

the

basis

of

the

results

obtainedandpitting rind of some citrus varieties and effects ofconsidering the recommended modified atmosphereprestorage hot - water treatments, Bull. Shizuoka

conditions４）, the PFP treatment, which maintains

Citrus Exp. Sta., ２
１, ４
５〜４
７（１
９
８
５）

high RH and a modified atmosphere except in the６ ） MANAGO,

M. :Studies‘ Kohansho’ :onapresence of high CO２ levels（＞８％）and／or low O２

physiological disorder of fruit rind in Hassaku

levels（＜５％）, appears to be the most effective（Citrus hassaku Hort. ex Tanaka ）, J. Japan. Soc.

means to control Kohansho and preserve quality.

Hort. Sci., ５
７, ２
９
５〜３
０
３（１
９
８
８）
７ ） PORAT, R. , PAVONCELLO, D. , PERETZ, J. , BEN -Conclusions

YEHOSHUA, S. and LURIE, S. :The results clearly showed that PFP is better

Effects of various

heat treatments on the induction of cold tolerance

reducingand on the postharvest qualities of ‘ Star Ruby’

weight loss and incidence of Kohansho, as well as ingrapefruit, Postharvest Biol. Technol ., １
８, １
５
９〜１
６
５

than

other

postharvest

treatments

inretaining firmness and other quality attributes. Since

（２
０
０
０）

Kohansho is caused mainly by fruit weight loss３）, we

８）SCHIRRA, M., MULAS, M., FADDA, A. and CAULI,

conclude that PFP treatment reduces Kohansho by

E. : Cold quarantine responses of blood oranges torestricting

postharvest hot water and hot air treatments,

water

loss.

The

HWD

and

HCD

treatments reduce the occurrence of Kohansho evenwhen the fruit lose weight, and these treatmentsareeffective

duringmarketingperiods

expected.

Since

storage,
where

the

PFP

transportation,

some

water

loss

treatmentis

Postharvest Biol. Technol ., ３
１, １
９
１〜２
０
０（２
０
０
４）
９ ） MARTIN - DIANA, A. B. , RICO, D. , FRIAS, J. M. ,and

BARAT, J. M., HENEHAN, G. T. M. and BARRY -RYAN,

is

C. : Calcium for extending the shelf life of fresh

more

whole

and

minimally

processed

fruitsanda review, Trends in Food Sci. andconvenient and economical than the HWD or HCD

vegetables :treatment, the PFP which can maintain a high RH

Technol ., １
８, ２
１
０〜２
１
８（２
０
０
７）

and a modified atmosphere except in the presence

１０）LUNA-GUZMÁN, I., CANTWELL, M., and BARRETT, D.

of high CO２ levels（＞８％）and／or low O２ levels

M. : Fresh-cut cantaloupe : effects of CaCl２ dips

（ ＜ ５％ ）， may

practical

and heat treatments on firmness and metaboliccommercial method of Kohansho prevention andactivity, Postharvest Biol. Technol . , １
７, ２
０
１〜２
１
３

be

preferable

asaquality maintenance in ‘Kiyomi’ Tangor.

（１
９
９
９）
１
１）HELRICH, K. : AOAC Official Methods of Analysis

References（ AOAC, Arlington, VA, USA ）, pp.１
０
５
８〜１
０
５
９

１）ABE, K., BENG, H., YANO, M., HASEGAWA, Y. and
IWATA, T. : Effects of polyethylene packaging ontheoccurrenceofrind - oilspotphysiological and chemical changes

１２）LUENGWILAI, K., SUKJAMSAI, K. and KADER, A. A. :the

Responses of ‘ Clemenules Clementine’ and ‘ W.

TangorMurcott’ mandarins to low oxygen atmospheres,

and
in

（１
９
９
０）

‘Kiyomi’ fruits, J. Japan. Soc. Cold Preser. Food , １
８,
１
４
８〜１
５
４（１
９
９
２）

Postharvest Biol. Technol ., ４
４, ４
８〜５
４（２
０
０
７）
１
３）PESIS, E. : The role of the anaerobic metabolites,２ ） FUJISAWA, H. , TAKAHARA, T. and OGATA, T. :acetaldehyde

andethanol,in

Influence of prestorage conditioning treatment andenhancement

of

fruit

quality

fruit

ripening,

and

fruit

optimal temperature and humidity for prolonged

deterioration, Postharvest Biol. Technol ., ３
７, １〜１９storage of ‘ Kiyomi’ Tangor, J. Japan. Soc. Hort.

（２
０
０
５）

Sci., ７
０, ７
１
９〜７
２
１（２
０
０
１）

１
４）HIDEMI, I. : Ascorbic acid treatments reduce the３ ） HIKIDA, Y. , TECHAVISES, N. and KAWANO, T. :occurrence and development of Kohansho in citrus

Occurrence of rind - oil spot in and transpiration

Hassaku and Tangor ‘Kiyomi’ fruits（Studies on

considerations for packaging design for ‘ Kiyomi’vitamin C of fruits and vegetables - Part X）, Food

Tangor, Food Preser. Sci., ３
２, ２
０
１〜２
０
７（２
０
０
６）

Preser. Sci., ２
４, ８
１〜８
６（１
９
９
８）

４）TECHAVISES, N. and HIKIDA, Y. : Effect of MA

１５）BEN-YEHOSHUA, S., PERETZ, J., MORAN, R., LAVIE, B.

conditions on occurrence of Kohansho and quality

and

KIM,

of ‘Kiyomi’ Tangor, Food Preser. Sci., ３
４, １
９
７〜２
０
２

superficial flavedo necrosis（noxan）of ‘Shamouti’

J.

J. :

Reducingtheincidence

of

172

Food Preservation Scienceoranges（Citrus sinensis, Osbeck）, Postharvest Biol.
Technol ., ２
２, １
９〜２
７（２
０
０
１）

タンゴール 清見 果実のこはん症発生と品質に及ぼす収穫後処理の効果
テチャヴィセス

ナッタコーン＊１

疋田慶夫＊２・河野俊夫＊３＊１

愛媛大学大学院連合農学研究科

（〒７
９
０―８
５
６
６ 愛媛県松山市樽味３丁目５―７）†

現在 C. P. ALL Public Co., Ltd., Thailand（３１
３ C.P. Tower, ２
３ Fl., Silom Rd., Bangrak,
Bangkok １０５
００, THAILAND）＊２

愛媛大学農学部

（〒７
９
０―８
５
６
６ 愛媛県松山市樽味３丁目５―７）＊３高知大学農学部

（〒７
８
３―８
５
０
２ 高知県南国市物部乙２００）
有孔フィルム包装（PFP），温湯浸漬（HWD），温湯
カルシウム浸漬（HCD）の収穫後処理が清見

果実のこはん症発生と品質に及ぼす効果を比較した。３種類の処理を施した果実を段ボール箱に収納し，１０℃で４週間貯蔵した。PFP処理区では，ほかの処理および対照区（無処理）と比較して，重量減少が明らかに小さく，果
実硬度が明らかに高い結果が得られた。すべての処理は
こはん症の発生を減少させ た が，PFPとHWD処 理 はHCD処理より高い減少効果を示した。また，これらの処 理 は 対 照 区 と 比 較 し て，糖 度（TSS），滴 定 酸 度（TA），TSS／TA比，アスコルビン酸含量，エタノール
含量を損なうことはなかった。これらの結果より，高湿度条件を保ち，かつ８％以上のCO２濃度環境と５％以下のO２濃度環境を避けたPFP処理はこはん症の抑制と品質保持において最も効果的で，商業的に実用的な方法であると考えた。

VOL．
３
７ NO．
４ ２
０
１
１

（ 22 ）

（ 23 ）

Food Preservation Science VOL．
３
７ NO．
４ ２
０
１
１ 〔Article〕

173

Purification and Characterization of Wheat Protein Disulfide
Isomerase Expressed in Escherichia coli
ARAI Satomi＊１，NOGUCHI Tomohiro＊２§，
UCHINO Masataka＊１ and TAKANO Katsumi＊１＊１ Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture,1−1−1 Sakuragaoka, Setagaya−ku, Tokyo 156−8502＊２ Food Processing Center, Faculty of Applied Bioscience, Tokyo University of Agriculture,1−1−1 Sakuragaoka, Setagaya−ku, Tokyo 156−8502

A protein disulfide isomerase（PDI）coding sequence was cloned from wheat（Triticum aestivum cv‘Haruyutaka’ ）. The cDNA contained ３ distinct sequences ; ２ of them, wPDI １ and wPDI ３, were １５４８
bp in length, and the other, wPDI ２, was １５
３
９ bp in length. Sequences wPDI １ and wPDI ２ shared
９８．
６％ identity, wPDI １ and wPDI ３ shared ９９．
２％ identity, and wPDI ２ and wPDI ３ shared ９９．
０％identity. The total PDI activity of recombinant（r）PDI was １
１
４ U, and the specific activity was ３
３
６
U ／ ! . The rPDI catalyzed the formation of disulfide bonds in disulfide reduced and denatured
ovalbumin. The optimal temperature for the ２ PDIs（rPDI and native PDI）was ３
５℃, and PDI activity
was highest at pH ８．
５. Furthermore, the rPDI was superior to the native PDI with respect to heatand pH stability ; however, the rPDI and native PDI had similar optimum conditions. This study isthe first to demonstrate recombinant expression of wheat PDI.
（Received Jan. ２３, ２０１
１；Accepted May ３
０, ２
０
１
１）
Key words：protein disulfide isomerase, disulfide bonds, PDI
プロテインジスルフィドイソメラーゼ，ジスルフィド結合，PDI

Protein disulfide isomerase （ PDI ５．
３．
４．
１） is analfalfa６），７）, barley８）, common wheat９）, maize５）, andessential protein for the formation, reduction, or

castor bean１０）. Wheat PDI cDNA has been isolated

isomerization of protein disulfide bonds, dependingfromon the conditions in the lumen of the endoplasmic

recombinantly expressed or characterized. In thisreticulum（ER）. PDI is a member of the thioredoxin

paper, we report the isolation and characterization

superfamily and it contains ２ thioredoxin-like activeof a cDNA encoding PDI from wheat and thesites（CGHC）, which act as the catalytic sites forexpression of wheat PDI. Subsequently, we analyze

１）

isomerase activity . This family contains thioredoxin,
２）３）thioredoxin-２ , DsbA , etc. Thioredoxin, thioredoxin-

root

regulation

of

not

been

Materials and methods
１．Primers for PCR amplification
Primersfortheamplification

of

PDI

were

development, and its physiological role are not well

Spring１１）and cv Wyuna cDNA sequences１２）．Primers

known.

plant

PDI

duringhas

synthesized based on the common wheat cv Chineseonexpression

proteinplant

Studies

its

this

PDI.

Thioredoxin-２ is a mitochondria -type thioredoxin２）,prokaryotes. The characteristics of plant PDI, thebutthe characteristics of the expressed recombinant

２,and DsbA contain only thioredoxin active sites.
whereas DsbA is localized to the periplasm oftip,

expression

andwPDI-F（５′-TGCTCCCCAGTYCCTTCCGCC-３′; bases

４）
，
５）

have

５−２
５ of AF ２６２９７９） and wPDI - R （５′- CTGGC -indicated that the enzyme may play an important

GTTGGAAGCTGCCTTGCCA-３′; bases １
６
２
３−１
６
４
６

intracellular localization in wheat and maize

proteins,of AF ２６
２９７９）were used for the amplification ofparticularly in the formation of endosperm protein

PDI cDNA including the full - length open reading

bodies. Plant PDI or PDI-like cDNA sequences haveframe（ORF）. To express precursor PDI（prePDI）

been cloned and sequenced from species such as

cDNA using pET-２
１ a（Novagen, USA）in Escherichia

role in the folding of plant secretory§ Corresponding author, E-mail : tomo@nodai.ac.jp

174

coli,

Food Preservation Science

theforwardprimerwPDI - Nde

VOL．
３
７ NO．
４ ２
０
１
１

The ３- dimensional

I - F （５′-（ 24 ）

proteinstructure

of

each

ATGCCATATGGCGATCTSCAAGG - YCTGGATC -recombinant PDI was predicted based on the amino

TCGCT-３′; bases ２
６―５
４ of AF ２
６２
９７９）, containing anacid sequence using ３ D-JIGSAW（http : ／／www.bmm.

NdeI site and the reverse primer wPDI-Xho I-R（５′-icnet.uk／servers／３djigsaw／）and Chimera software１７），１８）.

GCATCTCGAGGCAGAGCTCGTCCTTCAGAGGCTC -４．Overexpression and purification of recombinant

３′; bases １
５
４
９―１
５
７
３ of AF ２
６２
９７９）, containing an

PDI from E.coli.

XhoI site were used.

MEADOWS and ROBINSON１９） previously reported that

２．Preparation of T. aestivum cv Hruyutaka cDNA

wheat protein precursors are expressed in E. coli,

Dormant seeds were soaked in water for １
５ min

and that mature protein is produced in these cells.

at room temperature. Young roots were harvested

Therefore, for effective expression of wheat PDI inafter cultivation for ２ days at ２
８℃. Harvested roots

E. coli, the presence and location of signal peptide

were immediately frozen in liquid nitrogen and usedcleavage sites in prePDI were predicted using thefor the preparation of total RNA using the RNeasy

gram - negative prokaryote database in the SignalPplant mini kit （ Qiagen, USA ） according to the３．
０ server（http : ／／www.cbs.dtu.dk／services／SignalP

manufacturer’s

concentrations

２０）／）
before expressing recombinant PDI. The result

were determined by measuring the absorbance atshowed that the location of the signal peptide

２６０ nm. The isolated RNA was transcribed intocleavage site corresponds to that of wheat, andcDNA using the PrimeSTAR RT-PCR Kit（TaKaRa,

computational analysis showed that this site wasJapan）, with thermal modification.

probably cleaved（９９．
９％ probability）in E. coli（data

３． Cloning and Sequence analysis of PDI （ T.

notaestivumcv Hruyutaka ） and phylogenetic
analysis of Poaceae
Amplification of PDI from cDNA was performedby PCR using PrimeSTAR HS DNA Polymerase（ TaKaRa, Japan ）. The PCR was performed asfollows : ３
５ cycles of １
０ s at ９
８℃, ５ s at ６
０℃, and１
２
０ s at ７
２℃. The amplified fragment was inserted
into pUC １
１８（TaKaRa, Japan）, and was sequenced
by the dideoxy chain - termination method１３）using a
BigDyeTerminator v ３．
１ Cycle Sequencing Kit（Applied Biosystems, USA）. The PDI sequences ofT. aestivum and neighbor related taxa were obtained
from DDBJ, and the distances were calculated
according to the method of JUKES and CANTOR１４）. A
phylogenic tree was constructed by the neighbor joining method１５）using the CLUSTAL X program（version １．
８;１６））.

recombinant PDI expressed from DNA encoding

instructions.

RNAshow ）.

PDI,

Therefore,

including

the

we

wheat

predicted
PDI

signalthatthepeptide（prePDI）would be nearly identical to native PDI.
For

intracellular

recombinant

expression,

the

prePDI cDNA was inserted into the expression
vector, pET ２
１ a（Fig．
１）. The resulting expression
plasmid, pET ２
１ a／prePDI, was used to transform E.
coli BL ２１（DE ３）（Stratagene, USA）
. E. coli BL ２
１
（DE ３）＋pET ２
１ a／prePDI was cultured in １００!
of LB broth containing ampicillin（１００!／!）at ３
７℃,
until

the

opticaldensity

at ６０
０ nm

was ０．
６.

Expression of rPDI was induced by incubation with
IPTG（０．
２ mM）for １
６ hr at ２
０℃.
The cells were harvested by centrifugation at
５，０
００×" for ２
０ min at ４℃. The cell pellets were
resuspended in １
０! of ２０ mM Tris-HCl（pH ８．
０）
containing １
００ mM NaCl, and were disrupted by an

f1 origin
prePDIAmp
pET‑21a（+）/pr-wPDI/NheI/XhoI
ATATACATATGGCGATCTGCAAGGTCTGGATC
M A I
C
K V W
I

pET‑21 a（+）/prePDIGAGCCTCTGAAGGACGAGCTCTGCCTCGAGCACCACE
P L
K D E L
C L E
H H

lacI
ori

Fig．
１ Expression plasmid for wheat PDI
pET-２
１／prePDI : a plasmid containing the wheat prePDI cDNA including signal
peptide inserted between the NdeI and XhoI sites

〔Article〕 Wheat PDI Expressed in E. coli

（ 25 ）

175

ultrasonic oscillator（US １５０; Nihon Seiki Seisakusho,

２
５℃ with a spectropolarimeter（J-７
２
０ WI ; JASCO,Japan ）.

byJapan ）. The CD data were expressed as meancentrifugation（１４０，
０００×#, ６０ min, ４℃）. Then, theresidue ellipticity（degrees・!・dmol−１）using １
１
１ asclear supernatant was used for protein purification.

the mean residue weight of ovalbumin.

A TALON metal affinity resin column （ TaKaRa,

８． Two - dimensional

The

cell

debriswasremovedgelelectrophoresis

andJapan）was first equilibrated with １
０ column volumes

Western blot native PDI comparison with rPDI

of start buffer（２０ mM Tris-HCl, ３００ mM NaCl, pH

Separation of proteins by first - dimensional IEF

８．
０）. Then the clarified sample was applied to theelectrophoresis was performed using agar-GEL（pHcolumn. The column was washed with １
０ column

３―１
０, ATTO, Japan）according to the manufacturer’s

volumes

instructions２７）.

of

start

buffer,andtheproteinwasPartially

purifiednativePDI

andsubsequently eluted with elution buffer（start buffer

purified rPDI samples dissolved in sample solvent

containing ４
０ mM imidazole ）. The protein content

buffer２
１）

was determined by the Bradford methodusing

５．Assay for PDI activity

incubation

Lundstrom
mixtureusingasamplecup.

IEF

electrophoresis was performed at ３００ V for ２
１
０ min.
PAGE,

The activity of PDI was measured using theofloadedBefore carrying out the second-dimensional SDS-

BSA as a standard.

methodwere

andHolmgren２２），２３） .

The

contained ０．
５ " ／ ! insulin（TaKaRa, Japan）, ０．
１ M potassium phosphate（pHtheequilibrated.

agar - GEL
The

was

entrenchedandequilibrated

agar - GEL

wastransferred onto the SDS - PAGE, and then, SDS PAGE

wasperformed

using

the

methodof

２
４）

LAEMMLI .

８．
０）, ２ mM EDTA, and PDI protein extract （２０The rPDI was electrophoresed and the gel wasµ$）. The reaction was initiated by the addition ofstained with CBB R-２
５
０ and G-２
５
０. Meanwhile, the１
３
２ µ$of ５ mM DTT in a final volume of ２ ! ,electrophoresed gel of the partially purified native

after incubation for ４
０ min at ２
５℃. The increasing

PDI was electroblotted onto a PVDF membrane andturbidity of insulin peptide chains was measured atincubated

６
５
０ nm.

detected with horseradish peroxidase-conjugated anti

６．Amino acid sequence analysis and research on- mousewithanti - wPDI

IgG （ GE

antibody,

which

２
８）
Japan ）.

Healthcare,

was

Bound

the cubic structure of proteins

antibodies

The eluted fractions, cleared lysate, and total cellchemiluminescence system （ GE Healthcare ）. Gel

extract were analyzed using SDS - PAGE by theimages were obtained using a gel image analyzer

２４）method of LAEMMLI

on a １０％（v／v）acrylamide gel.

werevisualized

using

an

ECL - Plus

（Gel-Doc ; Bio-Rad, USA）. Antiserum was obtained

The gels were stained with Coomassie Brilliant Blueby immunizing a mouse with rPDI antigen.

R -２
５０ and G -２
５
０. The purified recombinant PDI,

９．Influence and stability of pH and temperature

which was designated rPDI, was stored at ４℃ until

on native PDI and rPDI

analysis.

The relative activity was determined at various

The N - terminal amino acid sequence of the ２
automatedEdman

degradation

using

a

pH and temperatures. Experiments were carried outbywith the purified rPDI and the partially purified

proteinnative PDI from wheat seed２９）. The influence of pH

kinds of recombinant PDI were determined２
５）

sequencer（PPSQ-２
１; SHIMADZU, Japan） .

on PDI activity was determined at ２
５℃ by using

７．Assay for PDI folding activity（rPDI）

the assay protocol described above. To test pH

The PDI folding activity was measured by the２６）stability, rPDI was diluted to １ µM（about ３４ U）,

mixturewhile the native PDI was diluted ２- fold using

contained ０．
５"／! disulfide reduced and denatured

respective buffers with pHs ranging from ４ to １
０,

ovalbumin, ５０ mM Tris-HCl（pH ８．
２）, １ mM EDTA,

and then the rPDI and native PDI were incubatedand either ０．
５ µM rPDI orat ４℃ for ８ hr and ４ hr, respectively. The residual

methodof

KAWAMURA.

The

reactionno

PDI （ negative

control）. The prepared ovalbumin was refolded by
PDI at ２
５℃ and samples were taken from thereaction after ０, ５, １
０, and １
５ hr of incubation.

activity was determined using these enzymes.
Todeterminetheeffectoftemperatureon

enzymatic activity, standard assay reaction mixtures

These reaction samples were diluted １
０-fold with ５０were incubated at temperatures ranging from ２
０ to

mM Tris-HCl（pH ８．
２）and １ mM EDTA, then the

４
５℃. In order to assess stability, rPDI was diluted

far-UV CD spectrum of ovalbumin was recorded at

to １ µM while undiluted native PDI was used. The

176

rPDI

Food Preservation ScienceandnativePDI

wereincubated

VOL．
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４ ２
０
１
１

（ 26 ）

at-F-S）（Fig．
２）. The wPDIs contained ５ domains : a,temperatures from １
０ to ７０℃ for ３ hr and １ hrb, b ′, a ′, and c. The a and a ′ domains had arespectively. The residual activity was measured

thioredoxin - like catalytic site. The c domain was

following the procedure described above.

alanine rich, and alanine was ９ of ３
２, ７ of ２
９, and ９of ３
２ amino acids in wPDI １, wPDI ２, and wPDI ３,

Results and discussion

respectively. This feature appears to be conserved

１．Cloning of full-length PDI cDNAin wheat and barley. These results were nearly

The cDNA fragments were amplified by RT-PCR

identical to those of M. CIAFFI２）. The shared identity

using RNA isolated from wheat roots. To isolate abetween wPDI １ and wPDI ２ was ９８．
６％ and thatfull - lengthof wPDI １ and wPDI ３ was ９９．
２％ , while the

cDNA,

we

screened ３ wheatcDNA

libraries using １．
５ kb cDNA fragments. The isolatedshared identity between wPDI ２ and wPDI ３ was

cDNAs containing the PDI genes were sequenced.

９９．
０％ . Ciaffi reported the sequences of ３ wheat

Three distinct sequences were identified ; ２ of themprotein disulfide isomerases（wheat PDI １, ２, and ３）,（wPDI 1 and wPDI 3 ）were １
５
４
８ bp in length, andand wPDI １ and wPDI ３ shared ９９．
６％ identity

the other（wPDI 2 ）was １
５３
９ bp in length. The ３

with wheat PDI ３２）, while wPDI ２ shared ９９．
８％sequences, wPDI 1, wPDI 2, and wPDI 3 encode ５１
５,identity with wheat PDI ２２）. However, we did not

５
１２, and ５１
５ amino acid polypeptides, respectively.

obtain any wPDI that was highly homologous toEachencodedwheat PDI １. The reason for this was that the １

polypeptide with a putative signal sequence at thecultivar had numerous isotypes, at least ３. In the

N-terminus and the ER retention signal KDEL at

future we will try again to obtain the cDNA of

the C-terminus, as well as ２ thioredoxin-like catalytic

wheat PDI １.

of

these

sequenceshadansites（CGHC）and a putative N-glycosylation site（NUsing phylogenic analysis based on deduced PDI

Signal sequence

Active sit (thioredoxin motif)

N glycosylation site (NFS)

ER retention signal

Fig．
２ Primary structure of wPDI
Domain organization of wPDI２（A）and alignment of the ３ wPDI sequences（B）.（A）The CGHC motifs（thioredoxin motifs）indicate the location of the active sites.（B）Active-site residues in the a and a’domains are shown as solid boxes. The dashed line box represents a putative N-glycosylation site（NFS）.
The double line box indicates the endoplasmic retention signal（KDEL）.

〔Article〕 Wheat PDI Expressed in E. coli

（ 27 ）

177
0.02

Sweet potato＜Q6IV17＞
Ethiopian mustard＜Q38HW3＞
Cotton＜A4L9H0＞
Cotton＜A4L9I3＞
Cotton＜A4L9G4＞
Balm of Gilead＜A9PJ12＞
Castor bean＜Q43116＞
Durango root＜Q9XF61＞
Grape＜A7NVF0＞
Soy bean1＜AB300660＞
Alfalfa＜P29828＞
wPDI3
Wheat PDI3＜AF262981＞
wPDI1
wPDI2
Wheat PDI2＜AF262980＞
Wheat PDI1＜AF262979＞
Barly＜P80284＞
Rice＜Q52PJ0＞
Maize2＜Q5EUE0＞
Maize1＜Q5EUE1＞

Fig．
３ The phylogenetic relationships of the amino acid sequences of the isolated wPDIs and someplant PDIs
The branching pattern was generated by the neighbor - joining method. The bar represents ０．
０２ nucleotide
substitution per site. The sequence data were obtained from DDBJ.

amino acid sequences from wheat and related taxa,
２ potentially independent clusters were constructed

c

（Fig．
３）. One cluster contained the Poaceae family,
including maize, and was located on an outer group

Active site

in the cluster. All wheat PDIs were in a single
subcluster in the family Poaceae, neighboring barley.

a´Wheat PDI １ reported by Ciaffi is somewhat far
from the other wheat PDIs. However, the distancesamong PDI isotypes from maize and cotton werealong compared with the distances among wheat
PDIs. Therefore, this result may be attributed to
species variety. The homology of PDI sequences ofb´

neighboring organisms in this group to wPDI wasin the range from ９６．
３〜９６．
７％（barley）to ７
９．
８〜
８
１．
０％（maize ２）. In addition, the phylogenic tree
based on PDI sequences had a shape similar to thatbof the taxonomic tree based on the DUF ６
４２-domain
３０）gene family . The ３-dimensional protein structure isshown in Fig．
４. The ２ thioredoxin-like catalytic sites
face each other. Furthermore, analysis of the aminoacid sequence of Poaceae PDI was carried out toinvestigate their ３-dimensional protein structure, andFig．
４ Surface representation of PDI with the a, b, b′,
and a′
domains in hot pink, blue, aquamarine, and pink,respectively, and the C-terminal extension in yellow
The side chains of the active site cysteines in the a and a′domains are shown in red.

the results showed that the structure of Poaceae
PDI was similar to that of the Poacese family

from wheat. On the basis of phylogenic analyses,（data not show）. Hence, we propose that the PDI

wPDI ２ was positioned at the center of the wheatgene

PDI branch. In addition, this isotype did not diverge

evolvednaturally,

similar

to

the

above

３
１）

phylogenetic data .

in

２．Expression and purification of Recombinant PDI

homologous to that of wheat PDI ２ reported by

In this study, we obtained ３ PDI gene cDNAs

CIAFFI２） ; therefore, we chose to express wPDI ２.

cultivars,

becausethesequence

washighly178

Food Preservation Science

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０
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１

（ 28 ）

Then, the presence and the location of the signal

activity of ３
３２．
７
４ U ／ mg. In addition, we tried to

peptide cleavage sites of prePDI were determined

express PDI without a signal peptide under theusing the Gram-negative prokaryote database in thesame conditions. However, the resultant activity was

２０）

SignalP ３．
０ server . The signal peptide of wheat

２０％ of the activity of PDI with the signal peptide

PDI had a peptidase recognition site with high（data not show）. This may be due to the transporthomologysystem of E. coli.

to（ maximumtheGram - negative

cleavagesite

signalpeptidePurifiedprobability ９
９．
９％ ）.

rPDI

had

an

apparent

MWofTherefore, we expressed rPDI with the wheat PDI

approximately ６
３ kDa as observed by SDS - PAGE,

signal peptide. The target protein was expressed inand a pI of ５．
３ on a ２- dimensional gel, and theE. coli BL ２１（DE ３）and induced by IPTG. Next,

partiallyrPDI was extracted from E. coli. SDS-PAGE showedapproximately ６
３ kDa and a pI of ５．
３ as observedthat rPDI purified from the cell extracts using aby ２- dimensional gel electrophoresis and western

purifiednative

PDI

had

a

MW

of

TALON metal affinity resin column had a MW ofblotting（data not show）; thus, the molecular weightapproximately ６
３ kDa （ Fig ．
５）（ Table １）. We（ MW ） and pI values of rPDI and native PDI

confirmed removal of the rPDI signal peptide using

correspondN - terminal amino acid sequencing, which showedcarbohydrate moiety bound to N-F-S was found to

to

each

other.

Therefore,

thethat rPDI did not have any signal peptide（data not

be small, and it did not affect the MW or pI.

show ）. The amount of purified recombinant PDI

Soybean, alfalfa, cattle, and yeast PDIs had MWs ofprotein was ０．
３
４ mg, and its total activity was

６
３ kDa,

６
０ kDa,

５
７ kDa,

１
１
４．
１
３ U. Further, １ µM rPDI exhibited a specificrespectively３２）〜３５） as

determined

and ７
０ kDa,bySDS - PAGE.

Therefore, the molecular weight of wheat PDI wasM12

nearly3

identical

to

that

of

other

plant

PDIs.

（kDa）

Meanwhile, the isoelectric point of wheat PDI was

250
160

similar to those of cattle, yeast, and alfalfa, whichwere ４．
２ , ４．
０２,

respectively３６），３３），３４） .

and ４．
６
５,

However, it differed from that of soybean PDI at

105

７．
６
５３４）（Table ２）. The pI of wheat PDI was acidic,

75

which is the case for most PDIs.
３．Analysis of folding activity of PDI（rPDI）

50

Protein disulfide isomerase （PDI）is an essential
protein for the formation, reduction, or isomerization

37

ofproteindisulfidebonds,

therebyfacilitating

protein folding. This mechanism has been analyzed
using several techniques. We employed a technique25

using disulfide reduced and denatured ovalbumin as
a substrate, then CD spectrum was measured to【T-10% Me（+）】

Fig ．５ Overexpression
derivative in E. coli

of

rPDI

from

detect conformational change.
a

pET２
１ a

We examined the capability of rPDI to catalyze

SDS-PAGE analysis of the expressed and purified rPDI from
E. coli harboring pET２
１ a／prePDI. Lane M, molecular weightmarkers（Precision Plus Protein Standards ; Bio-Rad, USA）;
lane １, cleared lysate ; lane ２, inclusion bodies ; lane ３, after
TALON column chromatography.

folding for different times in the presence of rPDI

the formation of disulfide bonds. In Fig．
６, protein
is shown. At ５ hr, the refolded proteins from therPDI

reaction

spectrum

exhibited

at ２
１
２ nm,

an

with

intermediateCDabout ５０％ oftheTable １ Expression and purification of rPDI in E.coli
Purification step
Crude extracta
TALON metal affinity
a The

Total protein（!）

Total Activity（U）

Specific activity（U／!）

Yield（％）

２
６
５．
８
６
０．
３
４

６
３
９．
５
８
１
１
４．
１
３

２．
４
１
３
３
５．
６
７

１
０
０
１
７．
８
５

starting material was crude extract from １
０
０! of induced E. coli culture.

〔Article〕 Wheat PDI Expressed in E. coli

（ 29 ）

179

Table ２ Characteristics of various PDIs
molecular weight
（kDa）

pI

Optimum pH

Optimum Temp.
（℃）

Stability pH

Stability Temp.
（℃）

６
３ kDa
６
３ kDa
６
３ kDa
７
０ kDa
５
７ kDa
６
０ kDa
６
０ kDa

５．
３
０
５．
３
０
７．
６
５
４．
０
２
４．
２
０
４．
６
５
−

８．
５
８．
５
８．
０
８．
５
７．
５
−
７．
６

３
５
３
５
−
−
−
−
３
０

６〜１
０
７〜９
９〜１
０
−
−
−
−

４
０
３
０
５
０
＜５
５＊
−
−
−

Wheat（pr-PDI-His）
Wheat（native）
Soy bean
Yeast
CattleAlfalfa
Chlamydomonas reinhardtii＊

Yeast PDI was significantly inactivated when incubatedat ５
５℃ for １
５ min.

B
6

6

4

4

2

2
[θ](deg・cm2・dmol‑1)[θ](deg・cm2・dmol‑1)

A

0
2
4
6
8
10

2
4
6
8
10
12

12
14
200

0

14
210

220

230

240

250

200

210

220

230

240

250

Wavelength (nm)Wavelength (nm)native0hr

Scan speed:200nm/min

5hr

15hr

Accumulation:25

Fig．
６ The far UV CD spectra of ovalbumin in various states were recorded at
２
５℃ after ０, ５, and １
５ hr（A）in the absence of rPDI.（B）with the addition of rPDI（０．
５ µM）.（A）and（B）heavyline, native ovalbumin ; dotted line, after ０ hr ; double line, after ５ hr ; solid line, after
１
５ hr

absolute ellipticity of the native form. The absolute

However,

ellipticity increased with increased folding time ; andsoybean PDI were not clear.

thereactionconditions

reportedforthe CD spectra of proteins that were refolded for

４．Enzyme characteristics of Native PDI and rPDI

１
５ hr were nearly identical to that of the native

The activities of PDIs from various organisms

protein （ Fig ．
６ B ）. These results were similar to（mammal, plant, fungi, yeast, and thermophiles）have

those

of

soybean

PDI３２）.

reactions

been reported. However, few characteristics of PDIs

without prePDI did not show an intermediate CDhave been reported （ for example, soybean, andspectrum, and did not change over time（Fig．
６ A）.

cattle ）, because the assay for PDI activity

This shows that the rPDI catalyzed disulfide bondcomplicated and the activity is generally very low.

formation in the disulfide reduced and denatured

Therefore, data on PDI characteristics are very

ovalbumin. Ovalbumin was almost completely folded

important for PDI research. In thisstudy,

weobserved

pHs

and

In

contrast,

２
６）

by rPDI in １
５ hr, while soybean PDI took ２
０ hr .

enzymeactivityatvariousis180

Food Preservation Science

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７ NO．
４ ２
０
１
１

（ 30 ）

maintained up to ４
０℃, while the partially purified

temperatures.
The effect of temperature on PDI activity isnative PDI was maintained up to ３
０℃（Fig．
７―c）. At

shown in Fig．
７ a, and the results indicate that the５
０℃ , the activity of rPDI and partially purified

optimal temperature for the ２ PDIs （ rPDI andnative PDI was ５０％ and less than １
０％ of that at

native ） is ３５℃ . The ２ PDIs had similar activity

theversus temperature curves. The activity of the ２stability of rPDI was maintained between pH ６ andPDIs was highest at pH ８．
５. However, at alkaline

pH ９; while, the stability of partially purified native

optimum

temperature,

respectively.

EnzymepHs, the partially purified native PDI had higher

PDI

activity than rPDI. In fact, at pH １
０ the partially（Fig．
７―d）. Therefore, rPDI was superior to native

purified native PDI maintained ３
０％ of the activity

PDI with respect to heat and pH stability. KISHI andat optimal pH ;WATAZU３８） reported

detectedathowever, no rPDI activity was

pH １０（ Fig ．７― b ）.

The

was

maintained

betweenthat

pH ７ andrecombinant

pH ９cholesterol

optimaldehydrogenase（CDH）was superior to native CDH,

temperature of the ２ wheat PDIs（３５℃）was very

and they supposed that the reason for this was a３
７）similar to that of Chlamydomonas reinhardtii （３０℃）.

difference in primary protein structure ; hence, r -The optimal pH of the ２ wheat PDIs at pH ８．
５,

CDH

was essentially the same as that of cattle PDI（pH

Therefore, rPDI is thought to be superior to native

３
４）
３
２）
７．
５）
, Soybean PDI（pH ８．
０）
and yeast PDI（pHPDI because of a difference in structure or the

３
２）
８．
５）
（Table．
２）. Regarding the enzyme stability atpresence of the histidine tag. The pH-stability profile

various temperatures and pHs, the enzyme retained

of

more

soybean PDI. In addition, wheat PDI was stable

than ８０％ activity
The

thermostability

theoffollowing
rPDI

was

higher

PDI

was

order

broad

proteincomparedb

100

Relative activity（%）

Relative activity
（%）

wheat

a

80
60
40
20
0

0

10

20
30
40
50
Temperature
（℃）

100
80
60
40
20
0

60

5

6

7

8

9
pH

10

11

12

9

10

11

d
Relative activity
（%）

c
100
80
60
40
20
0

0

10

20

30

40

50

60

Temperature
（℃）
rPDI

70

80

structure.

with

the

between neutral and alkaline pHs ; while, soybeanaRelative activity（%）

conditions.

over

had

100
80
60
40
20
0

3

4

5

6

7

8

pH
Partlypuriﬁed native PDI

Fig．
７ Enzyme characterization of native PDI and rPDI
（A）Temperature dependency of rPDI and partially purified native PDI.
（B）pH dependency of rPDI and partially purified native PDI.
（C）Effect of temperature on the stability of rPDI and partially purified native PDI.
（D）Effect of pH on the stability of rPDI and partially purified native PDI.

〔Article〕 Wheat PDI Expressed in E. coli

（ 31 ）PDI was stable only at alkaline pHs（９〜１０）（Table３
４）

181

Residue That Cause Accumulation of Enzymatic２） . Further, the native PDI was stable at ３
０℃

Reaction Intermediates of DsbA, a Member of theduring a １ hr incubation, while rPDI was stable atThioredoxin Superfamily, J. bacteriol , １
８
７, １
５
１
９〜

４
０℃ during a ３ hr incubation. Heat treatment at
５５℃ for １
５ min significantly inactivated yeast PDI ;

１
５
２
２（２
０
０
５）
４）SHIMONI, Y., ZHU, X., LEVANOY, H., SEGAL, G., ZHU,

however, the PDIs of cattle３４）, thermophilic fungus３９）,

X. and GALILI, G. : Purification, characterization,soybean３２）, and wheat retained activity. In particular,

andrPDI was shown to be more stable than yeast PDI

Protein Disulfide Isomerase from wheat grains,

at high temperature, although it was less stable

Plant Physiol ., １
０
８, ３
２
７〜３
３
５（１
９
９
５）

３
２）

intracellular

localizationofglycosylated５）LI, C. P. and LARKINS, B. A. : Expression ofthan soybean PDI at ５
０℃ .

protein disulfide isomerase is elevatedConclusions

in

the

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（〒１
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２ 東京都世田谷区桜丘１―１―１）＊２

東京農業大学食品加工技術センター

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２ 東京都世田谷区桜丘１―１―１）

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６％，wPDI１とwPDI

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９．
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を用いて発現用ベクター構築した後，大腸菌を形質転換

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させリコンビナントPDI（rPDI）を調製した。インスリンを基質としたrPDIの総活性は１
１
４ U，比活性は３３６U／!であった。また，rPDIの酸化活性は，還元変性オボ
アルブミンのリフォールディングにて測定した。rPDIとコムギ粉中のPDI（native PDI）の性状を比較したところ，両PDIともに温度３
０℃，pH ８．
５で最大活性を示したことから，rPDIはwPDIと類似の挙動を示すことを確認した。さらに，rPDIはnative PDIよりも温度，pHともに高い安定性を示した。本研究は，小麦由来PDIの発現に成功した初めての報告である。
（平成２
３年１月２
３日受付，平成２３年５月３０日受理）