J. Biol. Macromol., 12(3), 72-80 (2012)

Review

ACE inhibitory substances derived from soy foods
Atsushi Shimakage*, Mamoru Shinbo and Seihan Yamada

Yamadafoods. Co. Ltd, 279 Aza-kaidounoue, Noaramachi, Misato-cho, Senboku-gun,
Akita 019-1301

Because about half of Japanese population have been suffering from hypertension orare about to suffer from it, hypertension is a typical lifestyle related disease in Japan.
Therefore, it is important to eat foods with antihypertensive effect in order to preventthe onset or aggravation of hypertension. It is known that soy foods such as soy sauce,miso, and natto show antihypertensive effect by inhibiting angiotensin convertingenzyme (ACE), the key enzyme in renin-angiotensin system. ACE inhibitory substances
contained in soy sauce and miso are nicotianamine and the peptide Ser-Trp respectively,
but there is almost no evidence about ACE inhibitors in natto. We purified andidentified 5 novel ACE inhibitory peptides containing Ile-Ile and Ile-Asp fromprotease-treated hikiwari-natto, whose ACE inhibitory activity was about 1.4 times
higher than that of protease-untreated hikiwari-natto. In addition, we identified 8 novel
ACE inhibitory peptides containing Phe-Phe-Tyr-Tyr and Trp-His-Pro derived fromprotease-treated soymilk, whose ACE inhibitory activity was about 36 times higher
than that of protease-untreated soymilk.
Received October 26, 2012; Accepted November 5, 2012
Key words：soy foods, renin-angiotensin system, angiotensin converting enzyme(ACE),
ACE inhibitory peptide

Introduction
Hypertension is a typical lifestyle relaE-mail：shimakage@yamadafoods.co.jp

ted disease in Japan and is defined as thesituation

wherethesystolicbloodpressure is above 140 mmHg, or theACE inhibitors in soy foods
73

diastolic blood pressure is above 90

In a situation like this, due to theirmmHg according to The Japanese Society

antihypertensive effect, soy foods such asof Hypertension(1). The patients suffering

soy sauce, miso, and natto (fermentedfromnosoybean)isprevention of onset or aggravation of

detrimental to life to leave hypertension

hypertension in Japan. It is deduced that

untreated for a long period becausesoy foods show antihypertensive effect byhypertension may lead to miocardinal

inhibiting angiotensin converting enzyme

infraction, brain infraction, and stroke.

(ACE),

The

renin-angiotensin

hypertension

subjective

have

symptoms,

numberof

almostbutpatients

initJapan,

have

the

been

keyinfocus

enzymeinsystem.(2-4)

fortheACE

suffering from hypertension and people

inhibitory substances in soy sauce andwho are in danger of suffering from it are

miso have been already identified as

aboutnicotianamine(2) and Ser-Trp(3) , respecti-40million

and15million

respectively(1). Therefore, we conclude

vely.

However,

there

that hypertension is a big problem toinformation

healthy living in Japan.

substance derived from natto.

aboutisACE

almostnoinhibitory

Shimakage, Shinbo and Yamada
74

ACE inhibitory activity(U/g)

400
350
300
250
200
150
100
500
1611Day

Fig.2 ACE inhibitory activities of nattoes，Hikiwari-natto ；hikiwari-natto．

，Protease-treatedEach bar represents the mean ± SEM(n=3).
This figure is created based on data from [6].
In

thisreview,

thenovelACE

into the blood circulation in response toinhibitory

peptidesidentified

fromvarious

stimuli.

Renincleaves

theprotease-treated hikiwari-natto and soy

N-terminus of angiotensinogen produced

milk by us and our collaborators arein the liver to yield the 10-amino acidintroduced. In addition, we elaborate thepeptide, angiotensin I (AI). AI is further

attempt toprocessed

realize the development ofbyACEtoproduce

theFood for Specified Health Uses (FOSHU)

octapeptide, angiotensin II (AII).

AII

related to blood pressure.

stimulates the synthesis and release ofaldosterone, the hormone responsible for

Renin-angiotensin system(5)Aschematic

representation

elevatingof

bloodpressure,

fromtheadrenal cortex in the adrenal gland.

renin-angiotensin system is shown in
Fig.1. In this system, Renin produced

ACE inhibitory peptides derived frommainly in the kidney cortex is released

protease-treated hikiwari-natto

ACE inhibitors in soy foods
75

Table1 Novel ACE inhibitory peptides
isolated from protease-treated hikiwari-natto
Amino acid sequence

S oy protein-derived

Ile-Ileβ-amylase etc.

Ile-Asp

Cyclin etc.

Ile-Phe-Tyr

US P12 etc.

Leu-Phe-Tyr

RNA-binding protein etc.

Leu-Tyr-Tyr

Lipoxygenase etc.

It was found that ACE inhibitoryofheat

hikiwari-natto is more than twice that ofresistance(4,6), and so far only a few oftsubu-natto(6). The main constituent of

the different natto have been tested.

soybean is protein and therefore, we

ACE inhibitory activity of natto is about

deduced that ACE inhibitor of natto is a1.3 times higher than that of steamed

peptide that is produced because of theSys tolic blood pressure (mmHg)

substancesnatto

havesoybean(7) and ACE inhibitory activity of220200180###160

**

**

***

140

****

120
0123456Time after administration (h)Fig.3 Change in systolic blood pressure after a single
oral administration of the extract of natto and distilledwater in SHRSPs，SHRSPs that distilled water was administered to ；，SHRSPs that the extract of natto was administered to．
Each point and bar represents the mean ± SEM (n=6). **p<0.01，*******
p<0.001， p<0.0001 ；compared with the group that distilledwater was administered. ### indicates a significant value (p<0.001)
on change in systolic blood pressure of each group. This figure iscreated based on data from [6].

Shimakage, Shinbo and Yamada
76

degradation of soy protein by proteases

inhibitory peptides from protease-treatedsecreted by Bacillus subtilis natto.

hikiwari-natto(6). It was found that 5 of 11We

preparedprotease-treatedpeptidesarenovel

ACE

inhibitory

hikiwari-natto made by the addition ofpeptides(6) (Table 1). In addition, as

spore suspension of B. subtilis natto

shownandinFig.3,

theextractof

theend-type

neutralprotease,protease-treated hikiwari-natto showed

PROTIN

SD-NY10(theproteasesignificant reduction in systolic blood

preparation used as a food additive) topressure, 1 h after oral administration tothebefore

stroke prone spontaneously hypertensive

fermentation. ACE inhibitory activity ofrats (SHRSPs) in a single dose (80mg/kg

natto prepared in this fashion is aboutof body weight)(6). It was deduced that the1.4

strong in vitro ACE inhibitory activity

steamedtimessoybeanshigherthan

protease-untreatedthat

ofhikiwari-natto(6)seensurmisedthat

antihypertensive effect on SHRSPs ofprotease-treated hikiwari-natto cotains

the natto extracts were in response topeptides with strong ACE inhibitory

the presence of ACE inhibitory peptidesactivities.

described in previously.

(Fig.2).

Hence,

weinprevious

We purified and identified 11 ACE

Table 2 Novel ACE inhibitory peptides
isolated from protease-treated soymilk
Amino acid sequence

IC50(μM)

Phe-Phe-Tyr-Tyr

1.9

Trp-His-Pro

4.8

Phe-Val-Pro
Leu-His-Pro-Gly-Asp-Ala-Glu-Arg

10.1
10.3

Ile-Ala-Val
Val-Asn-Pro

27.0
32.5

Leu-Glu-Pro-Pro
Trp-Asn-Pro-Arg

100.1
880.0

studiesandACE inhibitors in soy foods
77

ACE inhibitory peptides derived fromprotease-treated soymilk

strong ACE inhibitory activities.
We isolated and identified 11 ACE

Soymilk is one of major soy foods, andinhibitory peptides from protease-treated

due to its consistency, it is easy to add asoymilk(8). It was found that 8 of 11

protease

peptidespreparation

tosoymilk

arenovelACEinhibitory

uniformly and control the enzymatic

peptides(8) (Table 2). In these peptides,

reaction after the addition of the protease

the IC50 values of Phe-Phe-Tyr-Tyr andpreparation. Thus, our collaborators andTrp-His-Pro are 1.9 µM and 4.8 µM

we confirmed the ACE inhibitory activity

respectively. The IC50 values of 111 ACEof protease-treated soymilk (the soymilk

inhibitory peptides derived from various

treatedfoodswith0.1%[w/w]

PROTIN

reportedsoare

0.32–945.5

ACE inhibitory activity of protease-activities of these 2 peptides are strong

treated soymilk is about 36 times higher

enough compared with existing ACE

than that of untreated soymilk (Fig.4). It

inhibitory peptides.

deducedthatACE

between

SD-NY10). As a result, it was found thatwasµM(9-10),

far

protease-treatedsoymilk also contains the peptides with

ACE inhibitory activity (U/ml)

300
250
200
150
100500Soymilk

Protease-treated soymilk

Fig.4 ACE inhibitory activities of soymilks
Each bar represents the mean ± SEM(n=3).

inhibitory

Shimakage, Shinbo and Yamada
78Summaryour plant. Therefore, we want to establish

Soy foods such as soy sauce, miso, andthe development of Food for Specified

natto show antihypertensive effect byHealth Uses (FOSHU) related to blood

inhibiting ACE, the key enzyme ofpressure by developing a beverage thatrenin-angiotensin

Ourcontains protease-treated soymilk backed

ACEwith scientific evidence necessary to gain

collaborators

andsystem.
wepurified

inhibitory substances derived from natto

FOSHU approval in the future.

that have not been identified so far. Weidentified

5peptidesnovelfromACE

inhibitory

Acknowledgements

protease-treatedThis research was supported in part byhikiwari-natto. In addition, the extract ofa Grant for the City Area Program fromthis natto showed significant reduction in

the Ministry of Education, Culture,systolic blood pressure when it was orallySports, Science and Technology (MEXT)

administered to SHRSPs in a single dose.

of Japan. We would like to express ourWe also identified 8 novel ACE

deep gratitude to Dr. Saori Takahashi, Dr.

inhibitory peptides from protease-treatedMakotosoymilk. In these peptides, the IC50

Hiwatashi at Akita Research Institute ofvalues

andFood and Brewing (ARIF) for purification

Trp-His-Pro are 1.9 µM and 4.8 µM

and identification of ACE inhibitory

respectively,inhibitory

peptides. We also would like to express

activities of these 2 peptides are strong

our deep gratitude to Dr. Michio Komai,

enough when compared to existing ACEDr.

inhibitory activities from other foods.

Ardiansyah at Laboratory of Nutrition,of

Phe-Phe-Tyr-TyrandACE

Tomatsu

HitoshiandDr.

Shirakawa,KazuyukiandDr.

Our company has been manufacturing

Graduate School of Agricultural Science,soymilk products and the production ofTohoku University for experiments onprotease-treated soymilk is possible atSHRSPs.

ACE inhibitors in soy foods
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Communicated by Ishijima Sumio