Isolation and Structural Analysis of Antihypertensive Peptides
That Exist Naturally in Gouda Cheese
ABSTRACT
Seven kinds of ripened cheeses (8-mo-aged and 24-
mo-aged Gouda, Emmental, Blue, Camembert, Edam,
and Havarti) were homogenized with distilled water,
and water-soluble peptides were prepared by C-18 hy-
drophobic chromatography. The inhibitory activity to
angiotensin I-converting enzyme and decrease in the
systolic blood pressure in spontaneously hypertensive
rats were measured before and after oral administra-
tion of each peptide sample. The strongest depressive
effect in the systolic blood pressure (−24.7 mm Hg) and
intensive inhibitory activity to angiotensin I-converting
enzyme (75.7%) were detected in the peptides from 8-
mo-aged Gouda cheese. Four peptides were isolated by
HPLC with reverse-phase and gel filtration modes.
Their chemical structures and origins, clarified by com-
bination analyses of protein sequencing, amino acid
composition, and mass spectrometry, were as follows:
peptide A, Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln [αs1-
casein (CN), B-8P; f 1–9]; peptide B, Arg-Pro-Lys-His-
Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln (αs1-CN, B-8P; f
1–13); peptide F, Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn
(β-CN, A2-5P; f 60–68); and peptide G, Met-Pro-Phe-
Pro-Lys-Tyr-Pro-Val-Gln-Pro-Phe (β-CN, A2-5P; f 109–
119). Peptides A and F, which were chemically synthe-
sized, showed potent angiotensin I-converting enzyme
inhibitory activity with little antihypertensive effects.
(Key words: Gouda cheese, peptides, angiotensin-con-
verting enzyme, antihypertensivity)
Abbreviation key: ACE = angiotensin I-converting
enzyme, CE = capillary electrophoresis, GPC = gel per-
meation chromatography, HHL = hippuryl-histidiyl-
leucine, IC50 = 50% inhibitory concentration, RP = re-
versed phase, SBP = systolic blood pressure, SHR =
spontaneously hypertensive rats.
Received October 3, 1999.
Accepted January 24, 2000.
Corresponding author: T. Saito; e-mail: tsaito@bios.tohoku.ac.jp.
2000 J Dairy Sci 83:1434–1440
T. Saito, T. Nakamura, H. Kitazawa,
Y. Kawai, and T. Itoh
Laboratory of Animal Products Chemistry,
Graduate School of Agricultural Science, Tohoku University,
Tsutsumidori-Amamiyamachi 1-1, Aoba-ku,
Sendai 981-8555, Japan
INTRODUCTION
Recently, bioactive peptides have been isolated and
characterized in the proteolytic products of several food
proteins. Peptides that show opiate, immunomodulat-
ing, antihypertensive, or enhancing mineral-utilization
effects have been derived mainly from milk proteins and
have been intensively studied (7, 14). Such bioactive
peptides, which are hidden in the inactive state in the
original protein sequence, may be released by later pro-
teolytic functions. These findings have led to the estab-
lishment of a new criterion for defining the nutritive
value of milk proteins.
Angiotensin I-converting enzyme (ACE; peptidyldi-
peptide hydrolase, EC 3.4.15.1) cleaves the C-terminal
dipeptide portion of angiotensin I and produces the va-
soconstrictor angiotensin II. It also inactivates the vaso-
dilator bradykinin. Many ACE inhibitory peptides have
been derived from not only milk proteins but also from
food proteins (10, 11, 19). In recent evaluations of these
studies, a demonstration of antihypertensive experi-
ments (in vivo) with spontaneously hypertensive rats
(SHR) is recommended in addition to ACE experiments
(in vitro) (2, 6, 20). Antihypertensive peptides that con-
tribute to the reduction of hypertension were also found
in protease digests of milk protein (18) and protease-
treated cheese whey protein (1, 2, 13), sour milk (6),
and yogurt-like products fermented by Lactobacillus
helveticus CPN4 (20). A new category of foods, termed
functional foods, which have beneficial effects on anti-
hypertensivity, is now commercially available in Japan.
Ripened-type cheese is an important dairy product
and contains numerous peptides that originate mainly
from casein by proteolysis during the ripening period
and contribute to the flavor, taste, and texture of the
cheese. Although there have been a few reports on ACE
inhibitory peptides in several cheeses, such as Italian
cheeses (Mozzarella, Italico, Crescenza, and Gorgon-
zola) (17) and general cheeses (Camembert, Edam,
Gouda, Cheddar, Roquefort, Emmentaler, and Parme-
san) (8, 12), there is little information on antihyperten-
sive substances that exist naturally in cheeses. In this
study, we describe the isolation and structural analysis
1434
 ANTIHYPERTENSIVE PEPTIDES IN GOUDA CHEESE
Table 1. Seven kinds of ripened-type cheese used in this study.
Sales agency
Name Country (in Japan)
Gouda (8 mo) Holland Sekai Cheese Co., Ltd.
Gouda (24 mo) Holland Sekai Cheese Co., Ltd.
Emmental Switzerland Sekai Cheese Co., Ltd.
Blue Denmark Sekai Cheese Co., Ltd.
Camembert France Nihon Maisera Ltd.
Edam Holland Sekai Cheese Co., Ltd.
Havarti Denmark Meijiya Co., Ltd.
of these antihypertensive peptides in ripened-type
cheese.
MATERIALS AND METHODS
Rats
Male SHR, aged 12 to 16 wk and weighing 265 to 330
g, were purchased from Funabashi Farm Inc. (Kyoto,
Japan). The rats were housed in cages and were under
a 14-h light and 10-h darkness cycle. The temperature
and humidity conditions were controlled at 24 ± 1°C and
60 ± 3%, respectively. The rats were fed the standard
laboratory diet Labo MR Breeder (Nihon Nosan Kogyo
Co., Ltd., Yokohama, Japan) and distilled water on tap
ad libitum.
Materials
Seven kinds of imported, ripened-type cheeses were
purchased from a retail store in Japan. The names of
cheeses, countries in which they are produced, and their
sales agencies in Japan are summarized in Table 1.
The ACE (EC 3.4.15.1, from rabbit lung), standard
amino acid H-type (each 2.5 µmol/ml in 0.1N HCl), tri-
fluoroacetic acid for HPLC, triethylamine, and phenyl-
isothiocyanate for amino acid composition analysis
were obtained from Wako Pure Chemical Co., Ltd.
(Osaka, Japan). Hippuryl-histidiyl-leucine (HHL; sub-
strate for ACE) was from Sigma Chemical Co., Ltd. (St.
Louis, MO). Constant boiling 6N HCl for acid hydrolysis
was from Pierce (Rockford, IL). Acetonitrile (CH3CN) of
HPLC grade was from Kanto Chemical Co., Ltd. (Tokyo,
Japan). Other chemicals of analytical grade were pur-
chased from Wako Pure Chemical Co., Ltd.
Chemical synthesis of peptides with N-α-9-fluore-
nylmethoxycarbonyl (Fmoc)-derived amino acids was
conducted by Sawaday Technology (Tokyo, Japan). The
peptides were passed through a Sep-Pak Plus C18 car-
tridge (Waters Co., Milford, MA) and were washed thor-
oughly by distilled water. Peptides absorbed to the car-
tridge were recovered with 90% (vol/vol) methyl alcohol
(CH3OH) and then used for the assay after removal of
solvent and lyophilization.
1435
Preparation of Free Peptides from Cheeses
Each cheese sample (50 g) was homogenized at 4°C
with three volumes of distilled water (150 ml) at 15,000
rpm for 5 min with a high-powered homogenizer (type-
II; Hirosawa Ironworks Co., Ltd., Tokyo, Japan). After
centrifugation at 13,000 rpm and 4°C for 20 min, the
water-soluble fraction located between the upper layer
(fat) and the precipitate (casein) was dialyzed by using
seamless cellulose tubing (pore size, 24Å, 12 to 14 kDa
cutoff; Viskase Sales Co., Chicago, IL) against distilled
water for 24 h at 4°C. The dialyzate (about 800 ml)
containing free peptides was mixed with Wakogel LP-
40C18 resin (100 g of dry weight; particle size, 20 to 40
µm; Wako Pure Chemical Co., Ltd.) as a batch reaction
system. After the mixture was vigorously shaken, pep-
tides absorbed to the resin were recovered with 90%
(vol/vol) CH3OH (hydrophobic chromatography). After
removal of CH3OH by rotary evaporation below 40°C,
each peptide sample was lyophilized.
Assay of ACE Inhibitory Activity (In Vitro)
The ACE inhibitory activity was measured in vitro
according to the procedure of Abubakar et al. (2), modi-
fied by scaling up by 2.5 times. A sample solution (75
µl) was mixed with an ACE solution (2.5 mU of ACE
in 5 µl of 50% glycerol) and 50 µl of 0.2 M borate buffer
(pH 8.3) containing 12.5 mM HHL and 1.0 M NaCl.
After incubation at 37°C for 60 min, the reaction was
stopped by adding 0.5 N HCl (125 µl). The absorbance of
hippuric acid, which was enzymatically liberated from
HHL by ACE, was monitored at 228 nm with a spectro-
photometer (model UV-1200; Shimadzu, Kyoto, Japan)
after ethyl acetate extraction. The 50% inhibitory con-
centration (IC50) value is the peptide concentration
(µM) that inhibits the activity of ACE by 50%. The
experiment was done in duplicates, and results are
shown as averages.
Assay of Blood Pressure Measurement in SHR
(In Vivo)
Each rat was prewarmed in a thermostatted box at
45°C for 8 min, and then systolic blood pressure (SBP)
was measured. After 6 h of oral administration of the
sample (2 mg/2 ml of distilled water, dosage of 6.1 to
7.5 mg/kg of body weight) to SHR (n = 3) by gastric
intubation, SBP was measured in each rat by the tail-
cuff method with a programmable electrosphygmoma-
nometer UR-1000 (Ueda Co., Ltd., Tokyo, Japan).
Change in SBP was expressed as the difference in SBP
before and after administration. The control value was
defined as the SBP in each SHR (n = 3) after 6 h of
administration of distilled water (2 ml). Results are
Journal of Dairy Science Vol. 83, No. 7, 2000
1436 SAITO ET AL.
shown as means and standard errors. Statistical differ-
ences between SBP in the control group (n = 3) and the
experimental group (n = 3) were evaluated by Student’s
t test.
Isolation and Purification of Peptides by HPLC
The peptide sample (8-mo-aged Gouda cheese), which
was reabsorbed to Wakogel LP-40C18 resin, was frac-
tionated by hydrophobic chromatography with stepwise
elution of CH3OH from 15 to 90% (vol/vol) at 15% inter-
vals. To isolate bioactive peptides, only the peptides
eluted with 45% (vol/vol) CH3OH were analyzed by
HPLC with reversed-phase (RP) and gel permeation
(GPC) modes under similar conditions to those de-
scribed earlier (2).
The RP-HPLC was conducted with an intelligent
pump (model L-6200; Hitachi Co., Ltd., Tokyo, Japan)
fitted with a Superiorex ODS column (5 µm, 4.6 mm ×
150 mm; Shiseido Co., Ltd., Tokyo, Japan). Solvent A
was 0.05% (vol/vol) TFA in 10% (vol/vol) acetonitrile
(CH3CN) solution, and solvent B was 0.05% (vol/vol)
TFA in 60% (vol/vol) CH3CN solution. Separations were
performed at 40°C with a flow rate of 0.5 ml/min with
a linear gradient system from 0 to 100% of solvent B
for 30 min. Peptide peaks were monitored at 214 nm
with a UV and visible light detector (L-7420; Hitachi
Co., Ltd.) that was linked to a data station (D-5500;
Hitachi Co., Ltd.).
HPLC with a GPC mode was performed using a
Superdex Peptide HR 10/30 column (1.0 cm × 30 cm;
Pharmacia Biotech AB, Uppsala, Sweden) equipped
with the same devices. Elution was isocratic with dis-
tilled water containing 0.1% (vol/vol) TFA at 40°C with
a flow rate of 0.5 ml/min, and peptides were detected
at 214 nm. After a final check of the purity of individual
peaks with capillary electrophoresis (CE), each peptide
was subjected to the following structural analyses.
CE
After filtration with an Ultrafree-MC filter (0.45 µm;
Millipore Co., Bedford, MA), samples were analyzed
by CE with a BioFocus 3000 (Bio-Rad Laboratories,
Richmond, VA) equipped with a coated column (25 µm
× 24 cm, no. 148-3031; Bio-Rad Laboratories) in the
presence of 0.1 M phosphate buffer (pH 2.5, no. 148-
5011; Bio-Rad Laboratories) at 10 kV for 15 min. The
column effluent was monitored at 200 nm, and the ca-
rousel temperature was maintained at 20°C.
Sequence Analysis
Each peptide was sequenced by the automated Ed-
man degradation method by using a protein sequencer
Journal of Dairy Science Vol. 83, No. 7, 2000
490 Procise (Perkin Elmer Co. Ltd., Applied Biosystem
Division, Foster City, CA) with a PTH-C18 column (2.1
mm × 220 mm; Perkin Elmer Co. Ltd.). Chemicals used
in the 140C Microgradient Delivery System included
two kinds of solvent (Perkin Elmer Co. Ltd.): A3 [3.5%
(vol/vol) aqueous tetrahydrofuran] and B (CH3CN
and isopropanol).
Fast Atom Bombardment-Mass Spectrometry
The molecular mass of purified peptides was analyzed
by fast atom bombardment-mass spectrometry under
a positive mode at low resolution, 8 kV, with glycerol
as the matrix.
Amino Acid Composition Analysis
Amino acid composition analysis of each peptide was
done by the method of Bidlingmeyer et al. (3) with some
modifications. The samples were hydrolyzed by the gas-
phase method with constant boiling HCl containing
10% (vol/vol) phenol in a dry block bath (MG-2; Japan
Torika Ltd., Tokyo, Japan) at 108°C for 24 h. Phenylthi-
ocarbamyl amino acids were analyzed by HPLC
through an intelligent pump (L-6200 with a Superiorex
ODS column, 5 µm, 4.6 mm × 150 mm; Shiseido Co.,
Ltd.) at 254 nm. Solvent A was 140 mM sodium acetate
buffer (pH 5.4), and solvent B was 60% (vol/vol) CH3CN
solution. Elution was performed at 40°C and at a flow
rate of 1.0 ml/min with a linear gradient system from
0 to 50% of solvent B for 20 min.
RESULTS
Preparation of Water-Soluble Peptides
from Ripened-Type Cheeses
Seven ripened-type cheeses (see Table 1) were homog-
enized with distilled water, and water-soluble peptides
were prepared by hydrophobic chromatography with
Wakogel LP-40C18 by elution with 90% (vol/vol)
CH3OH. Figure 1 shows the comparative yield (mg/
100 g of cheese) of free peptides obtained from seven
cheeses. The yield was within the range of about 70 to
240 mg/100 g of each cheese, and the highest (233 mg)
and lowest (74 mg) yields were obtained from Edam
and Blue cheeses, respectively.
Selection of Cheese with Antihypertensive Activity
The ACE inhibitory and antihypertensive effects of
the water-soluble peptides prepared from seven differ-
ent cheeses were determined (Table 2). Three peptide
samples from the two Gouda cheeses and Havarti
cheese showed strong ACE inhibitory activity (>72%).
 ANTIHYPERTENSIVE PEPTIDES IN GOUDA CHEESE 1437

Figure 1. The yield of free peptides (mg/100 g of cheese) prepared Figure 2. The inhibitory activity of angiotensin/I-converting en-
from each cheese by hydrophobic chromatography with Wakogel LP- zyme (ACE) and antihypertensive effects in spontaneously hyperten-
40C18 (Wako Pure Chemical Co., Ltd., Osaka, Japan). sive rats (SHR) of six fractionated peptides from 8-mo-aged Gouda
cheese. They were fractionated by hydrophobic chromatography with
stepwise elution of CH3OH. Fraction G8-15, 15% CH3OH eluent; G8-
30, 30%; G8-45, 45%; G8-60, 60%; G8-75, 75%; and G8-90, 90%. The
The highest inhibitory effect (average 78.2%) against values in the lower panel show the changes in systolic blood pressure
ACE was detected in Gouda cheese (at 24 mo). In the (SBP) in SHR after 6 h of administration, and each dose was 6.1 to
7.5 mg/kg of body weight. *Significantly different from the control
SHR experiments, the change in SBP was measured (P ≤ 0.05). **Significantly different from the control (P ≤ 0.01).
at 6 h after gastric intubation with each sample. The
decrease in SBP (mm Hg) was statistically significant
in four cheeses [Gouda (8 mo), Blue, Edam, and Ha- Fractionation of Water-Soluble Peptides
varti]. The strongest antihypertensive effect was ob-
from 8-Mo-Aged Gouda Cheese
served in the peptide sample from Gouda cheese (8 mo)
(−24.7 ± 0.3 mm Hg; P ≤ 0.01); therefore, only this The water-soluble peptides of Gouda cheese (8 mo;
sample was used in subsequent experiments. termed G8) were fractionated by hydrophobic chroma-
tography with stepwise elution from 15 to 90% (vol/vol)
CH3OH (at 15% intervals). The yields of peptides from
Table 2. The inhibitory activity of angiotensin-converting enzyme 100 g of the cheese that were eluted with 15, 30, 45,
(ACE) and antihypertensive activity of free peptides prepared from
seven kinds of cheese by hydrophobic chromatography. 60, 75, and 90% CH3OH were 11.9, 8.2, 9.7, 11.0, 21.4,
and 10.8 mg, respectively. Figure 2 shows the results
ACE Inhibitory Decreased
Samples activity1 SBP2
of ACE inhibitory and antihypertensive activities of the
six fractionated samples from G8. The strongest effects
(%) (mm Hg) in both assays were found in the 45% CH3OH fraction
X SE (ACE inhibitory activity, 58.4%; antihypertensive activ-
Gouda (8 mo) 75.5 −24.7** 0.3 ity, −29.3 ± 0.9 mm Hg; P ≤ 0.01). Only this peptide
Gouda (24 mo) 78.2 −17.2 4.5
Emmental 48.8 −13.0 4.1 fraction (termed G8-45) was used in subsequent exper-
Blue 49.9 −20.3* 3.8 iments.
Camembert 69.1 −7.1 3.1
Edam 56.2 −20.7* 3.3
Havarti 72.7 −20.0** 2.0 Isolation of the Antihypertensive Peptides
1
Mean of ACE inhibitory activity (%) in duplicate experiment. Figure 3 shows a typical elution profile of the peptide
2
Dose was 6.1 to 7.5 mg/kg per sample. Mean of changes in systolic fraction G8-45 by RP-HPLC. Although about 40 peaks
blood pressure (SBP) for spontaneously hypertensive rats (SHR) after
6 h of gastric intubation (n = 3). were observed in the chromatogram, only the seven
*Significantly different from the control (P ≤ 0.05). major peaks were selected and termed A to G, according
**Significantly different from the control (P ≤ 0.01). to the eluted order. The components of these seven

Journal of Dairy Science Vol. 83, No. 7, 2000

1438 SAITO ET AL.

Figure 3. A typical elution profile of the peptide fraction G8-45

obtained by reversed-phase HPLC. Column: Superiorex ODS (5 µm,
4.6 mm × 150 mm); elution A [10% acetonitrile containing 0.05%
trifluoroacetic acid (TFA)] and B (10% acetonitrile containing 0.05%
TFA); mobile phase, elution A 100% to elution B 100% within 30 min;
and at a flow rate of 0.5 ml/min under 40°C and detection at 220 nm.

peaks were collected by the repetition of RP-HPLC.

Four components (A, B, F, and G) were purified and
gave single peaks; these were subjected to further puri-
fication by GPC-HPLC to remove minor contaminants.
Finally, CE confirmed the purities of four peptides (A,
B, F, and G) (Figure 4), and they were then subjected
to structural analyses.

Structural Analysis of Antihypertensive Peptides

Four peptides (A, B, F, and G) were sequenced by
automated Edman degradation with a pulsed-liquid
phase protein sequencer. The N-terminal amino acids
of four peptides (A, B, F, and G) were Arg, Arg, Tyr,
and Met, respectively, but the C-terminals of peptides
(B and G) were not clearly identified. The molecular
weights of four peptides (A, B, F, and G) were deter-
mined by fast atom bombardment-mass spectrometry
to be 1141, 1537, 1002, and 1352 (m/z, M++H), respec-
tively (mass spectrum not shown). We analyzed the
amino acid composition of these four peptides. Peptide
A contained six amino acids and, by molar ratios, was
also considered to be the nonapeptide [Arg, (Pro)2,
(Lys)2, (His)2, Ile, and Gln] by the calculation of molecu-
lar weight = 1140 (M+). The combination of these analy-
ses enabled accurate determination of the sequence of
Figure 4. Profiles of four peptides by capillary electrophoresis
each peptide, including the C-terminal end. In this isolated by reversed-phase and gel permeation HPLC from the pep-
manner, the C-termini of peptides A, B, F, and G were tide fraction G8-45. The data were recorded by using a BioFocus 3000
confirmed to be Gln, Gln, Asn, and Phe, respectively. (Bio-Rad Laboratories, Richmond, VA) equipped with a coated column
(25 µm × 24 cm) in 0.1 M phosphate buffer (pH 2.5) at 10.00 kV for
Finally, the origin of each peptide was determined 15 min. Each peptide was monitored at 200 nm, and the carousel
from protein sequence analysis with a protein-data was maintained at 20°C. Numbers in parentheses represent elution
base. Table 3 summarizes the primary structures, ori- time (min).

Journal of Dairy Science Vol. 83, No. 7, 2000

 ANTIHYPERTENSIVE PEPTIDES IN GOUDA CHEESE
Table 3. The primary structure, origin, and molecular mass of four peptides isolated from Gouda (8 mo)
cheese.
Sample Sequence
A NH2-Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-COOH
B Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln
F Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn
G Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val-Gln-Pro-Phe
1
αs1-CN, B-8P (f 1–9) is bovine αs1-CN, genetic variant type B, combining eight residues of the phosphate
group (f 1–9).
gins, and molecular weights of the four peptides. Pep-
tides A (nonapeptide) and B (tridecapeptide) were found
to originate from αs1-CN,B-8P, and peptides F (nona-
peptide) and G (undecapeptide) originated from β-CN,
A2-5P.
Bioactivity of Synthetic Peptides
To determine the IC50 values in ACE inhibitory activ-
ity and antihypertensive effects, two peptides (A and F)
were chemically synthesized according to information
obtained from structural analyses. Peptides A and F
gave very low IC50 values (13.4 and 14.8 µM, respec-
tively), and the antihypertensive effects on SHR were
−9.3 ± 4.8 and −7.0 ± 3.8 mm Hg, respectively.
DISCUSSION
The ACE inhibitory and antihypertensive activities
of water-soluble peptides from seven representative
bacterial (Gouda, Emmental, Edam, and Havarti) and
moldy (Blue and Camembert) types of ripened cheeses
were evaluated by in vitro and in vivo experiments. In
ripened-type cheeses, proteolysis of milk proteins by
various proteases and peptidases from lactic acid bacte-
ria, mold, and added chymosin is the most important
process that determines cheese flavor, taste, and tex-
ture. Smacchi and Gobbetti (17) have reported that pep-
tides isolated from Italian cheeses (Mozzarella, Italico,
Crescenza, and Gorgonzola) showed inhibitory activity
to amino- and endopeptidases from some Lactobacillus
bulgaricus, Streptococcus thermophilus, and Lactococ-
cus cremoris strains and that most of them were also
effective in reducing the activity of ACE. Recently, the
antihypertensive effects of sour milk and isolated pep-
tides were investigated in SHR before and after oral
administration (2, 6), and chronologic change in blood
pressure was observed (20).
In this study, we isolated four peptides from 8-mo-
aged Gouda cheese. Peptide A (αs1-CN, B-8P; f 1–9),
which has been isolated from Cheddar cheese (16), and
peptide B (αs1-CN, B-8P; f 1–13) are major peptides
1439
Molecular
mass (Da) Origin
1140 αs1-CN, B-8P (f 1–9)1
1536 αs1-CN, B-8P (f 1–13)
1001 β-CN, A2-5P (f 60–68)
1351 β-CN, A2-5P (f 109–119)
produced during ripening (5, 16). Various water-soluble
peptides that mainly originated from N-terminal por-
tions of αs1-CN or internal portions of β-CN have been
reported in Cheddar (15) and Feta cheeses (9). Although
the same sequenced peptides, such as peptides A (16),
B (5, 16), and F(15) in this report, have been already
reported, the peptide G (β-CN, A2-5P; f 109–119) is a
novel peptide that was isolated from cheeses. All four
peptides contained more than two residues of Pro at an
internal position.
Chemically synthesized peptides A and F showed po-
tent ACE inhibitory activity of 13.4 and 14.8 µM (IC50
values), respectively. Cheung et al. (4) have reported
binding of ACE and synthesized peptides and showed
the importance of hydrophobic (aromatic or branched-
chain aliphatic) amino acid residues at each of the three
C-terminal positions. We previously reported that aro-
matic amino acids such as Trp, Tyr, and Phe or imino
acid (Pro) at the C-terminus generally contribute to
enhancement of ACE inhibitory activity (2). The potent
inhibitory effect of peptide F containing Ile and Pro at
the C-terminus corresponded to this rule, but peptide
A, which has such amino acids at the C-terminal and
the internal position, also showed potent activity.
Peptides A and F did not have strong antihyperten-
sivity (−9.3 ± 4.8 and −7.0 ± 3.8 mm Hg, respectively)
and that of p-peptosin C (undecapeptide, β-CN:f 80–90)
in our previous report (2) was also weak (−8.0 ± 2.6 mm
Hg) in spite of their low IC50 values. These peptides
have rather large molecules that require further diges-
tion by intestinal protease or peptidase before absorp-
tion. The antihypertensive activity might be generated
after additional digestion in SHR. The deletion study,
which makes derivatives from the N-terminus or C-
terminus of the two peptides, would be also very im-
portant to understanding the ACE inhibitory mecha-
nism of oligopeptides in vivo. Further studies on the
purification and structural analysis on antihyperten-
sive peptides in the fraction G8-45 are now in progress.
ACKNOWLEDGMENTS
We are grateful to Y. Furukawa and M. Komai of
Tohoku University (Laboratory of Nutrition, Graduate
Journal of Dairy Science Vol. 83, No. 7, 2000
1440 SAITO ET AL.
School of Agricultural Science) for technical assistance
in SBP measurements of SHR. We also thank Amhar
Abubakar of Syiah Kuala University (Indonesia) for
measurement of ACE inhibitory activity. This research
was partially supported by Meiji Milk Products Co., Ltd.
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