Você está na página 1de 7

Bioscience, Biotechnology, and Biochemistry

ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20

Angiotensin I-Converting Enzyme Inhibitory


Peptides Isolated from Tofuyo Fermented Soybean
Food
Megumi KUBA, Kumi TANAKA, Shinkichi TAWATA, Yasuhito TAKEDA &
Masaaki YASUDA
To cite this article: Megumi KUBA, Kumi TANAKA, Shinkichi TAWATA, Yasuhito TAKEDA &
Masaaki YASUDA (2003) Angiotensin I-Converting Enzyme Inhibitory Peptides Isolated
from Tofuyo Fermented Soybean Food, Bioscience, Biotechnology, and Biochemistry, 67:6,
1278-1283, DOI: 10.1271/bbb.67.1278
To link to this article: http://dx.doi.org/10.1271/bbb.67.1278

Published online: 22 May 2014.

Submit your article to this journal

Article views: 289

View related articles

Citing articles: 84 View citing articles

Full Terms & Conditions of access and use can be found at


http://www.tandfonline.com/action/journalInformation?journalCode=tbbb20
Download by: [82.196.1.179]

Date: 01 May 2016, At: 00:59

Biosci. Biotechnol. Biochem., 67 (6), 12781283, 2003

Angiotensin I-Converting Enzyme Inhibitory Peptides Isolated from Tofuyo


Fermented Soybean Food
Megumi KUBA,1 Kumi TANAKA,1 Shinkichi TAWATA,1 Yasuhito TAKEDA,2
and Masaaki YASUDA1,
1Department

of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru,


Nishihara-cho, Okinawa 903-0213, Japan
2Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University,
1-21-24 Korimoto, Kagoshima 890-0065, Japan

Downloaded by [82.196.1.179] at 00:59 01 May 2016

Received December 2, 2002; Accepted January 20, 2003

Angiotensin I-converting enzyme (ACE) inhibitory


activity was observed in a tofuyo (fermented soybean
ml. Two
food) extract with an IC50 value of 1.77 mg W
ACE inhibitors were isolated to homogeneity from the
extract by adsorption and gel ltration column chromatography, and by reverse-phase high-performance
liquid chromatography (HPLC). The puried substances reacted with 2,4,6-trinitrobenzensulfonic acid
sodium salt. The amino acid sequences of these inhibitors determined by Edman degradation were Ile-PheLeu (IC50, 44.8 mM) and Trp-Leu (IC50, 29.9 mM). The
Ile-Phe-Leu sequence is found in the a- and b-subunits
of b-conglycinin, while the Trp-Leu sequence is in the
B-, B1A- and BX-subunits of glycinin from soybean.
Both of the peptides are non-competitive inhibitors.
The inhibitory activity of Trp-Leu was completely
preserved after a treatment with pepsin, chymotrypsin
or trypsin. Even after successive digestion by these
gastrointestinal proteases, the activity remained at 29%
of the original value.
Key words:

angiotensin I-converting enzyme inhibitor; bioactive peptide; fermented soybean


food; tofuyo

The angiotensin I-converting enzyme (ACE) is a


dipeptidyl carboxy peptidase associated with the
regulation of blood pressure. It converts angiotensin
I to the potent pressor peptide, angiotensin II, and
also degrades depressor peptide bradykinin.1,2) ACE
inhibitors from various foods have recently been
studied in terms of their ability to prevent and alleviate hypertension. Physiologically functional foods
enriched with ACE inhibitors such as tripeptide (ValPro-Pro, Ile-Pro-Pro) from sour milk,3) dipeptide
(Val-Tyr) from a sardine muscle hydrolysate4) and
dodecapeptide (Phe-Phe-Val-Ala-Pro-Phe-Pro-GluVal-Phe-Gly-Lys) from a casein hydrolysate5) are
used as supplements to improve hypertension. In

respect of soybean, Kawamura6) has puried ACE inhibitory peptides from a soybean protein hydrolysate
and observed that the peptide fragments existed in
the primary structures of b-conglycinin and glycinin.
Wu and Ding7,8) have also conrmed the hypotensive
eect of ACE inhibitory peptides derived from a soy
protein alkaline hydrolysate on spontaneously hypertensive rats; they isolated and characterized two ACE
inhibitory peptides. ACE inhibitors have also been
isolated from fermented soybean foods, and were
identied to be nicotianamine from soy sauce,9) as
well as phytic acid10) and peptide (Ser-Tyr) from miso
paste.11) Although the ACE inhibitory activity in
natto has already been studied, the chemical structure of the inhibitor has not yet been reported.12)
More information on the ACE inhibitors in soybean
foods is necessary for the development of healthy
foods.
Tofuyo is a traditional fermented tofu from
Okinawa in Japan. It has a mild avor and ne taste
similar to cream cheese. It also has high nutritional
value because it is from soybean. We have been
studying the production of tofuyo and have characterized it.1316) However, the physiological functions
of tofuyo remain to be claried.
We report here the isolation and identication of
ACE inhibitors from tofuyo. Moreover, we investigated the mechanism underlying the inhibition at the
kinetic level and the stability of the puried ACE
inhibitors against digestion.

Materials and Methods


Materials and reagents. Tofuyo prepared from red
and yellow kojis16) was supplied by Benihama Co.,
Ltd. The enzymes used throughout this study were
the angiotensin I-converting enzyme (rabbit lung, 2.5
units), pepsin (porcine gastric mucous membrane,
mg of solid), chymotrypsin (bovine pan3,900 units W

To whom correspondence should be addressed. Tel: 81-98-895-8807; Fax: 81-98-895-8734; E-mail: yasudaagr.u-ryukyu.ac.jp

ACE Inhibitory Peptides from Tofuyo

Downloaded by [82.196.1.179] at 00:59 01 May 2016

creas, 4060 units W


mg of protein), and trypsin
(bovine pancreas, 8,600 units W
mg of solid) from
Sigma Chemical Co. Hippuryl-L-histidyl-L-leucine
(Hip-His-Leu) and isoleucyl-phenylalanyl-leucine
were obtained from Peptide Institute Inc., and tryptophanyl-leucine was obtained from Nikka Techno
Service Co. SEPABEADS SP825 and Sephadex G-25
resin were from Mitsubishi Chemical Co. and Amersham Bioscience, respectively. All other chemicals
used were of analytical grade.

Assay for ACE inhibitory activity. The ACE


inhibitory activity was assayed by using the modied
method of Lieberman.17) For each assay, 65 ml of a
sample solution and 50 ml of 12.5 mM Hip-His-Leu as
a substrate in a borate buer (pH 8.3) containing 1 M
NaCl were incubated with 10 ml of ACE (2 mU) for
1 h at 379C. The reaction was stopped by adding
125 ml of 0.5 M HCl. The liberated hippuric acid was
extracted with 750 ml of ethyl acetate, and 200 ml of
the resulting extract was then evaporated to dryness
with a centrifugal concentrator (Tomy CC-105). The
precipitate was dissolved in 1.2 ml of 1 M NaCl, and
the absorbance at 228 nm was then measured. The
sample concentration required to inhibit 50z of the
ACE activity under the assay conditions is taken as
the IC50 value.
Protein assay. The protein concentration in each
sample was determined with a micro BCA protein
assay reagent kit (Pierce Chemical), with bovine
serum albumin used as the standard.
Preparation of the tofuyo extract. Tofuyo was suspended in 3 volumes of distilled water, homogenized
with a labo-stirrer (Yamato L-35) and then shaken in
an incubator (Yamato BT-21) at room temperature
for 1 h. The mixture was then centrifuged at 12,000
g for 15 min and the resulting precipitate was
removed. The turbid supernatant was ltered, and
the ltrate was boiled for 20 min. The ltered solution was used as the tofuyo extract.
Purication of the ACE inhibitors. The extract
obtained from 600 g of tofuyo was applied to an
adsorptive column of SEPABEADS SP825 (q5 cm
21.4 cm) that had been pre-equilibrated with distilled
water. After washing with the same distilled water,
the inhibitors were eluted with a step-wise gradient of
an ethanol solution from zero to 70z. Each fraction
was evaporated in a rotary evaporator (Yamato
RE-46), and the resulting residue was dissolved in
distilled water.
The active fraction was subjected to reverse-phase
HPLC in a column of Cosmosil 5C18-AR-300, elution
being carried out with a linear gradient of acetonitrile
from zero to 40z in 0.05z triuoroacetic acid for 80
min with monitoring at
min at a ow rate of 0.5 ml W

1279

Table 1. IC50 Values for Typical Fermented Soybean Foods


Fermented soybean food*

ml)
IC50 (mg W

Soy sauce
Miso paste
Natto
Tofuyo

3.44
1.27
0.16
1.77

* Miso paste and natto were extracted by following the same method as
that for tofuyo described in the text. Soy sauce was used directly for the
assay.

220 nm. The fraction corresponding to an active peak


was rechromatographed in the same column. Purication of the other potent inhibitor was done by gel
ltration (Sephadex G-25, q1.2 cm142.5 cm)
before reverse-phase HPLC.

Amino acid sequence analysis. The amino acid


sequences of the ACE inhibitors were determined by
automated Edman degradation with a gas W
liquid
phase protein sequencer (Applied Biosystems 473A).
Digestion test. The stability of each puried ACE
inhibitory peptide against gastrointestinal proteases
in vitro was assessed. An inhibitor solution (1.5 mM,
0.2 ml) was incubated with 0.2 ml of a 0.05z pepsin,
chymotrypsin or trypsin solution (0.1 M HCl at pH
2.0 or 0.1 M potassium phosphate buer at pH 8.0)
and 0.2 ml of each buer for 6 h at 379C. The inhibitor solution was then subjected to successive digestion tests. The pepsin solution was evaporated in a
centrifugal concentrator and then redissolved in
0.6 ml of a solution containing both 0.025z (w W
v)
chymotrypsin and 0.025z (w W
v) trypsin. The reaction mixture was incubated for 6 h at 379C and then
boiled for 5 min to stop the digestion. After the
enzymatic treatment, each sample was centrifuged,
and the supernatant was adjusted to pH 8.3 for
measuring the ACE inhibition.

Results and Discussion


ACE inhibitory activity in tofuyo
The ACE inhibitory activity of the tofuyo extract
compared with that of other fermented soybean
foods is shown in Table 1. The miso paste and natto
extracts were obtained by using distilled water with
the same method as that used for the tofuyo extract,
while soy sauce was used directly. Before the assay,
each extract and the soy sauce were adjusted to pH
8.3 with a 0.1 M sodium hydroxide solution. The
natto extract showed the highest inhibitory activity,
and the ACE inhibitory activity in the tofuyo extract
was almost as strong as that in the miso paste extract.
Purication and identication of the ACE inhibitory peptides
The crude tofuyo extract was loaded into a column

1280

M. KUBA et al.

Table 2. Separation of ACE Inhibitors from the Tofuyo Extract


by Chromatography in a SEPABEADS SP825 Column
Fraction
0z
20z
40z
70z

ethanol
ethanol
ethanol
ethanol

Protein (g)

Inhibition (z)*

498.91
6.83
0.59
0.04

5.1
25.7
10.1
28.6

* The nal concentration of protein in each fraction was 0.1 mg W


ml in the
reaction mixture.

Downloaded by [82.196.1.179] at 00:59 01 May 2016

Fig. 2. Chromatography, Using a Sephadex G-25 Column, of


the 20z Ethanol Fraction Eluted from SEPABEADS SP825.

Fig. 1. Reverse-phase HPLC of the ACE Inhibitors in a Cosmosil 5C18-AR-300 Column.


The 20z ethanol fraction from SEPABEADS SP825 was
subjected to reverse-phase HPLC. The column was eluted with a
linear gradient of acetonitrile (040z for 80 min) in 0.05z
triuoroacetic acid at a ow rate of 0.5 ml W
min (A). The fraction corresponding to an active peak was rechromatographed in
the same column with a moderate acetonitrile gradient (022z
for 10 min and 2224z for 20 min) (B).

of SEPABEADS SP825 and eluted with a step-wise


gradient of ethanol (0, 20, 40 and 70z). The ACE
inhibitory activity in each of these fractions is shown
in Table 2. Potent activity and a large sample amount
were found in the 20z ethanol fraction, so this fraction was concentrated. The concentrate was subjected to reverse-phase HPLC (Cosmosil 5C18-AR-300
column), elution being performed with a linear
gradient of an acetonitrile solution as shown in
Fig. 1(A). ACE inhibitory activity was detected in
several peaks. Peak I, which had the highest activity,
was used for the next step of purication. After peak
I had been concentrated, it was rechromatographed
in the same column with a moderate acetonitrile
gradient. As shown in Fig. 1(B), a homogeneous
preparation (compound I) was obtained.
Purication was carried out on the other potent
inhibitor in the 20z ethanol fraction from

Fig. 3. Reverse-phase HPLC of Fraction No. 6 Eluted from the


Sephadex G-25 Column.
Fraction No. 6 was subjected to reverse-phase HPLC. The
column was eluted with a linear gradient of acetonitrile (040z
for 40 min) in 0.05z triuoroacetic acid at a ow rate of 0.5 ml W
min (A). The peak was rechromatographed in the same column
with a moderate acetonitrile gradient (034.8z for 10 min and
34.835z for 20 min) (B).

SEPABEADS SP825 already described. The active


solution was loaded into a column of Sephadex G-25
and then eluted with distilled water. Seven active
fractions were obtained as shown in Fig. 2. The
major active fraction, No. 6, was then obtained. The
active solution was subjected to reverse-phase HPLC
(Cosmosil 5C18-AR-300 column), elution being
performed with a linear gradient of an acetonitrile
solution as shown in Fig. 3. Strong inhibitory activity
was found in peak II that was eluted at 35 min

Downloaded by [82.196.1.179] at 00:59 01 May 2016

ACE Inhibitory Peptides from Tofuyo

(Fig. 3(A)). A homogeneous preparation of compound II was nally obtained (Fig. 3(B)).
Since both substances (compounds I and II) reacted with 2,4,6-trinitrobenzensulfonic acid sodium
salt, which binds only to primary amines and the
sulfhydryl group, they both seemed to be peptide-like
substances.
The chemical structures of compounds I and II
were identied to be Ile-Phe-Leu and Trp-Leu,
respectively, by an amino acid sequence analysis.
Their IC50 values were determined to be 44.8 and
29.9 mM, respectively. The contents of Ile-Phe-Leu
and Trp-Leu were estimated to be 23.9 and 0.3 mg in
100 g of the tofuyo extract. This is the rst report of
ACE inhibitory peptides derived from tofuyo. A
computer search of the SWISS-PROT protein
sequence database showed that the amino acid
sequence Ile-Phe-Leu exists in the primary structures
of the a- and b-subunits of b-conglycinin (Ile
(476)-Phe (477)-Leu (478) in the a-subunit, and Ile
(247)-Phe (248)-Leu (249) in the b-subunit), whereas
Trp-Leu exists in the primary structures of the B-,
B1A- and BX-subunits of glycinin (Trp (44)-Leu (45)
in the B- and BX-subunits, and Trp (43)-Leu (44) in
the B1A-subunit). Since the a?-, a-, and b-subunits in
b-conglycinin and the acidic subunit in glycinin are
degraded to low-molecular-weight elements during
tofuyo fermentation for 3 months,1416) it is likely that
Ile-Phe-Leu was liberated from b-conglycinin by proteases that were produced by Monascus purpureus
and W
or Aspergillus oryzae used in the fermentation
process. Although the basic subunit in glycinin cannot be easily degraded by these enzymes,1416) as compared with each subunit in b-conglycinin or the acidic
subunit in glycinin, Trp-Leu might have been liberated from the subunit during long-term fermentation
(3 months).

Clarication of the ACE inhibition mechanism


To clarify the mechanism underlying the inhibition
at the kinetic level, Lineweaver-Burk plots were
determined for Ile-Phe-Leu and Trp-Leu. As shown
in Fig. 4, these plots with an intersection on the 1 W
S
axis indicate that both peptides were non-competitive
inhibitors. Moreover, Dixon plots displaying the 1 W
v
versus inhibitor concentration with an intersection on
the I axis were also characteristic of non-competitive
inhibition (data not shown). Competitive ACE inhibitors have been most frequently reported, and some
non-competitive inhibitors have also been found in
such foods as natto,12) sake,18) tuna,19) sardine,20) and
chickpea.21) However, the inhibition site on ACE of
these non-competitive inhibitors has not previously
been investigated. Interestingly, captopril, enalaprilat and ramiprilat, which are competitive inhibitors
of ACE, have also shown a non-competitive mode on
the Dixon plots.22) One of the reasons for such an inhibition mode, i.e., the mixed type, has been consi-

1281

Fig. 4. Lineweaver-burk Plots of the ACE Inhibition by Tofuyoderived ACE Inhibitory Peptides.
Each point represents the mean value of three experiments.
ACE activities were measured in the absence or in the presence
of Ile-Phe-Leu (A) or Trp-Leu (B). , absence of inhibitor; #,
0.32 m M Ile-Phe-Leu; $, 1.6 m M Ile-Phe-Leu; , 0.06 mM TrpLeu; , 0.32 mM Trp-Leu.

dered to be the slow-tight binding of these inhibitors


at the ACE active site.22) Further investigation is required to determine the mechanism for non-competitive inhibition in foods containing the Ile-Phe-Leu
and Trp-Leu peptides.

Digestion test on the inhibitors


In order to understand the resistance of both the
Ile-Phe-Leu and Trp-Leu peptides in tofuyo to digestion in vivo, changes in their IC50 values before and
after treatment with gastrointestinal proteases in
vitro were examined. As shown in Table 3, the ACE
inhibitory activities of both peptides were completely
preserved after the pepsin treatment. Although the
inhibitory activity of Trp-Leu was also completely
preserved after the chymotrypsin or trypsin treatment, that of Ile-Phe-Leu had decreased to 62z and
75z of the original value following the chymotrypsin
and trypsin treatment, respectively. In spite of the
successive digestion with pepsin, chymotrypsin and
trypsin, the inhibitory activity of Ile-Phe-Leu was
found to be 38z, and that of Trp-Leu was 29z of
the original value. It has been reported that the IC50

1282

M. KUBA et al.

Table 3. Digestive Stability against ACE Inhibition of Peptides


Puried from the Tofuyo Extract
Digestion1
None
Pepsin
Chymotrypsin
Trypsin
Pepsinchymotrypsin and trypsin2

6)

IC50 ( mg W
ml)
Ile-Phe-Leu

Trp-Leu

18
18
29
24
47

10
10
11
10
35

7)

8)
1

Downloaded by [82.196.1.179] at 00:59 01 May 2016

The enzymes used in this study were porcine gastric pepsin, bovine pancreas chymotrypsin and bovine pancreas trypsin. An Ile-Phe-Leu or
Trp-Leu solution (1.5 m M, 0.2 ml) was incubated with 0.2 ml of each
protease solution (0.05z, w W
v) and 0.2 ml of the buer for 6 h at 379
C.
The reaction mixture treated with pepsin for 6 h at 379
C was evaporated
in a centrifugal concentrator, and then the residue was redissolved in
0.6 ml of the enzyme solution containing both chymotrypsin and trypsin
v for each concentration) followed by incubation for 6 h at
(0.025z, w W
C.
379

values of ``Ile-Phe''23,24) and ``Phe-Leu'',25) which are


parts of Ile-Phe-Leu, were 930 and 16 mM, respectively. Thus, Ile-Phe-Leu is likely to preserve its
activity until it is degraded to its individual amino
acids.
It is well known that di- and tripeptides are more
rapidly absorbed and reach a higher concentration in
the blood than single amino acids.26,27) Chun et al.27)
have recently demonstrated that short peptides
(average residue length of 3.2) in a soybean hydrolysate were more rapidly absorbed than the long ones
(average residue length of 5.2) when using a rat intestinal everted sac. Therefore, Ile-Phe-Leu and TrpLeu isolated from tofuyo are expected to be easily
absorbed and contribute to the antihypertensive
eect via an in vivo transport system.
Further studies are necessary and will be performed to conrm the antihypertensive eect of
tofuyo and its ACE inhibitors in vivo.

9)

10)

11)

12)

13)

14)

15)

References
1)

2)

3)
4)

5)

Yang, H. Y. T., Erdos, E. G., and Levin, Y., A


dipeptidyl carboxypeptidase that converts angiotensin
I and inactivates bradykinin. Biochim. Biophys.
Acta, 214, 374376 (1970).
Yang, H. Y. T., Erdos, E. G., and Levin, Y., Characterization of a dipeptide hydrolase. J. Pharmacol.
Exp. Ther., 177, 291300 (1971).
Takano, T., Milk derived peptides and hypertension
reduction. Int. Dairy Journal, 8, 375381 (1998).
Kawasaki, T., Seki, E., Osajima, K., Yoshida, M.,
Asada, K., Matsui, T., and Osajima, Y., Antihypertensive eect of Valyl-Tyrosine, a short chain peptide
derived from sardine muscle hydrolyzate, on mild
hypertensive subjects. J. Human Hypertension, 14,
519523 (2000).
Maruyama, S., Mitachi, H., Tanaka, H., Tomizuka,
N., and Suzuki, H., Studies on the active site and
antihypertensive activity of angiotensin I-converting
enzyme inhibitors derived from casein. Agric. Biol.

16)

17)

18)

19)

Chem., 51, 15811586 (1987).


Kawamura, Y., Peptide inhibitor for angiotensin converting enzyme of soybean protein and its antihypertensive eect. Shokuhin Kogyo (in Japanese), 40,
7382 (1997).
Wu, J., and Ding, X., Hypotensive and physiological
eect of angiotensin converting enzyme inhibitory
peptides derived from soy protein on spontaneously
hypertensive rats. J. Agric. Food Chem., 49, 501506
(2001).
Wu, J., and Ding, X., Characterization of inhibition
and stability of soy-protein-derived angiotensin Iconverting enzyme inhibitory peptides. Food Res.
Int., 35, 367375 (2002).
Kinoshita, E., Yamakoshi, J., and Kikuchi, M.,
Purication and identication of an angiotensin Iconverting enzyme inhibitor from soy sauce. Biosci.
Biotechnol. Biochem., 57, 11071110 (1993).
Teranaka, T., Ezawa, M., Matsuyama, J., Ebine, H.,
and Kiyosawa, I., Inhibitory eects of extracts from
rice-koji miso, barley-koji miso, and soybean-koji
miso on the activity of angiotensin I converting
enzyme. Nippon N ogeikagaku Kaishi (in Japanese),
69, 11631169 (1995).
Takahama, A., Iwashita, A., Matsuzawa, M.,
Takahashi, H., Nakatsuka, M., and Yahata, S.,
Anti-hypertensive peptides derived from fermented
soybean paste-miso. Int. News on Fats Oils Relat.
Mater., 4, 525 (1993).
Okamoto, A., Hanagata, H., Kawamura, Y., and
Yanagida, F., Anti-hypertensive substances in fermented soybean, natto. Plant Foods Hum. Nutr., 47,
3947 (1995).
Yasuda, M., Studies on manufacturing of tofuyo.
Nippon Shokuhin Kogyo Gakkaishi (in Japanese),
37, 403409 (1990).
Yasuda, M., Matsumoto, T., Sakaguchi, M., and
Kobamoto, N., Changes in chemical components of
tofuyo prepared by Monascus fungus during fermentation. Nippon Shokuhin Kogyo Gakkaishi (in
Japanese), 40, 331338 (1993).
Yasuda, M., Matsumoto, T., Sakaguchi, M., and
Kinjyo, S., Changes in protein and nitrogen
compounds of tofuyo prepared by Aspergillus oryzae
during fermentation. Nippon Shokuhin Kogyo
Gakkaishi (in Japanese), 41, 184190 (1994).
Yasuda, M., Matsumoto, T., Sakaguchi, M., and
Kinjyo, S., Production of tofuyo using the combination of red and yellow kojis. Nippon Shokuhin
Kagaku Kogaku Kaishi (in Japanese), 42, 3843
(1995).
Yamamoto, S., Toida, I., and Iwai, K., Re-examination of the spectrophotometric assay for serum angiotensin-converting enzyme. Nippon Kyobu Shikkan
Gakkai Zasshi (in Japanese), 18, 297303 (1980).
Saito, Y., Nakamura, K., Kawato, A., and Imayasu,
S., Angiotensin I converting enzyme inhibitors in
sake and its by-products. Nippon N ogeikagaku
Kaishi (in Japanese), 66, 10811087 (1992).
Kohama, Y., Oka, H., Yamamoto, K., Teramoto,
T., Okabe, M., Mimura, T., Nagase, Y., Chiba, Y.,
and Fujita, T., Induction of angiotensin-converting
enzyme inhibitory activity by acid-limited proteolysis

ACE Inhibitory Peptides from Tofuyo

20)

21)

22)

Downloaded by [82.196.1.179] at 00:59 01 May 2016

23)

of glyceraldehyde 3-phosphate dehydrogenase.


Biochem. Biophys. Res. Commun., 161, 456460
(1989).
Matsufuji, H., Matsui, T., Seki, E., Osajima, K.,
Nakashima, M., and Osajima, Y., Angiotensin I-converting enzyme inhibitory peptides in an alkaline protease hydrolyzate derived from sardine muscle.
Biosci. Biotechnol. Biochem., 58, 22442245 (1994).
Pedroche, J., Yust, M. M., Giron-Calle, J., Alaiz,
M., Millan, F., and Vioque, J., Utilisation of chickpea protein isolates for production of peptides with
angiotensin I-converting enzyme (ACE)-inhibitory
activity. J. Sci. Food Agric., 82, 960965 (2002).
Braudin, B., and Beneteau-Burnat, B., Mixed-type
inhibition of pulmonary angiotensin I-converting
enzyme by captopril, enalaprilat and ramiprilat. J.
Enzyme Inhibition, 14, 447456 (1999).
Cheung, H. S., Wang, F. L., Ondetti, M. A., Sabo,
E. F., and Cushman, D. W., Binding of peptide
substrates and inhibitors of angiotensin-converting

24)

25)

26)

27)

1283

enzyme. J. Biol. Chem., 255, 401407 (1980).


Seki, E., Osajima, K., Matsufuji, H., Matsui, T., and
Osajima, Y., Val-Tyr, an angiotensin I converting
enzyme inhibitor from sardines that have resistance to
gastrointestinal proteases. Nippon N ogeikagaku
Kaishi (in Japanese), 69, 10131020 (1995).
Eto, Y., Ito, T., and Nishioka, S., Angiotensin I
converting enzyme-inhibitory dipeptides in an alkaline protease hydrolysate of whey protein. Nippon
Eiyo Shokuryo Gakkaishi (in Japanese), 51, 355359
(1998).
Claft, I. L., Geddes, D., Hyde, C. W., Wise, I. J.,
and Matthews, D. M., Absorption and malabsorption of glycine and glycine peptides in man. Gut, 9,
425437 (1968).
Chun, H., Sasaki, M., Fujiyama, Y., and Bamba, T.,
Eect of peptide chain length on absorption and
intact transport of hydrolyzed soybean peptide in rat
intestinal everted sac. J. Clin. Biochem. Nutr., 21,
131140 (1996).