Food Research International 43 (2010) 902–906

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Food Research International

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Impact of processing on stability of angiotensin I-converting enzyme (ACE)

inhibitory peptides obtained from tuna cooking juice
Jyh-Sheng Hwang *
Department of Food Science and Applied Biotechnology, Hungkuang University, 34 Chung-Chie Road, Sha-Lu, Taichung 433, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history: In this study, angiotensin I-converting enzyme (ACE) inhibitory peptides, which had previously been
Received 12 September 2009 identified in an active gelfiltration fraction from tuna cooking juice, were examined for the stability of
Accepted 15 December 2009 their inhibitory properties and composition changes during processing and in the presence of gastroin-
testinal proteases. Results indicated that ACE inhibitory peptides reserved almost the same composition
before and after various temperatures (20–100 °C), levels of pressure (50–300 MPa) and pH (2–10) treat-
Keywords: ments. ACE inhibitory peptides retained 95–99% activity after simulated digestion. High Performance
ACE inhibitory peptides
Liquid Chromatography (HPLC) chromatograph peptide mappings exhibited slight differences before
Processing
High pressure
and after temperature (100 °C), pressure (300 MPa) and pH (2, 10) treatments. Our results indicate that
tuna cooking juice-derived ACE inhibitory peptides possess some degree resistance to the influence of
temperature, pressure, pH treatments, and gastrointestinal proteases.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction
In recent years, peptides from partial enzymatic hydrolysates of
food proteins have received greater attention from food scientists
than ever before. Many biological peptides, with health benefits
such as opioid activity, antihypertensive activity, antibacterial
activity, mineral-binding activity and enhancement of intestinal
activity have been classified and identified from food protein
hydrolysates (Ariyoshi, 1993; Clare & Swaisgood, 2000; Schlimme
& Meisel, 1995; Steijns, 1996). Peptides possess specific biological
properties which make these components potential ingredients of
functional or health-promoting foods. Technological processes
used in food manufacture may affect the functional, nutritional
and biological properties of peptides. Hannu, Anne, Pirjo, and Tuo-
mo (1998) reported that functional properties of peptides in the
food matrix are highly influenced by molecular structure, interac-
tions with other components and the conditions of processing. On
the other hand, it is essential to apply or develop technologies
which retain or even enhance the activity of bioactive peptides in
food systems. The influence of food processing and preparation
on the bioactivity of ACE (angiotensin-I-converting enzyme)-inhib-
itory and antihypertensive peptides is not well documented.
Therefore, study on the stability of peptides during processing
and the effects on their bioactivity are crucial issues.
* Tel.: +886 4 26318652 5062; fax: +886 4 26319176.
E-mail address: jyh3478@yahoo.com.tw
Moreover, once ACE inhibitory peptides are released from food
proteins they have to be able to survive gastrointestinal digestion,
to be absorbed, and to reach the cardiovascular system in an active
form. In this respect, several studies have demonstrated the impor-
tant role of gastrointestinal digestion on ACE inhibitory peptide
formation. For example, several authors have reported an increase
in ACE inhibitory activity by the action of digestive enzymes on fer-
mented casein solutions (Pihlanto-Lepp, Rokka, & Korhonen, 1998;
Vermeirssen, Van Camp, Decroos, Van Wijmelbeke, & Verstraete,
2003). Maeno, Yamamoto, and Takano (1996) found a potent
in vivo antihypertensive peptide with unexpectedly low in vitro
ACE inhibitory activity, as the active form of the peptide was re-
leased by pancreatic digestion of the precursor peptide. In contrast,
peptides that exhibited apparent in vitro ACE inhibitory activity
can fail to show in vivo antihypertensive activity if these peptides
are substrates of ACE instead of true enzyme inhibitors (Fujita,
Yokoyama, & Yoshikawa, 2000). These authors classified peptides
that showed in vitro ACE inhibitory activity into three groups: true
inhibitor type, substrate type, and pro-drug type depending on
their behavior after incubation with ACE.
Antihypertensive peptides inhibit angiotensin I-converting en-
zyme (ACE) and thus reduce blood pressure in vivo. Our previous
study indicated that orientase (a commercial protease derived
from Bacillus subtillis) effectively released ACE inhibitory peptides
from tuna cooking juice protein and exhibited a hypotensive effect
in SHRs (Hwang & Ko, 2004). In this study, the strongest ACE inhib-
iting fraction (OA3 oligopeptides) from orientase hydrolysate was
used to study the impact of processing on its stability and in the
presence of gastrointestinal proteases.

0963-9969/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2009.12.012
 J.-S. Hwang / Food Research International 43 (2010) 902–906
2. Materials and methods
2.1. Materials and reagents
Tuna cooking juice containing 4% proteins was obtained from a
tuna canning plant in Chiayi County, Taiwan. Pepsin and pancrea-
tin were purchased from Sigma Chemicals (MO 63178, USA). Ori-
entase, an endo-peptidase prepared from Bacillus subtilis, was
purchased from Hankyu Bioindustry Co. (Osaka, Japan). All other
chemicals were analytical grade products.
2.2. Hydrolysis and fractionation of tuna cooking juice
Orientase (1.0 wt.%) was added to the tuna cooking juice with a
ratio of substrate:enzyme = 25:1 v/v. Enzymatic hydrolysis was
performed at 50 °C for 3 h; then the juice was boiled for 10 min
to inactivate the protease. The post-reaction mixture was centri-
fuged at 10,000g for 10 min, and the supernatant was collected
as orientase hydrolysate (OAH). Lyophilized OAH 280 mg was dis-
solved in 1 ml of distilled water and applied to a Sephadex G-25
column (2.5  70 cm), then eluted with a 0.05 M phosphate buffer
(pH 6.5). Each fraction of 5 ml eluted was collected at a flow rate of
45 ml/h. The absorbance at 280 nm and the ACE inhibitory activity
of all fractions were measured.
Fractionation was completed by SDS–PAGE and MW was esti-
mated by using Endothein (MW 2,573), Neurotensin (MW
1672.9), Bradykinin (MW 1060), angiotensin-II (MW 926), N-for-
myl-Met-Leu-Phe-Lys (MW 565.7) and Tryptophan (MW 204) as
standards. These standards were purchased from Sigma Chemicals
(MO 63178, USA).
The yield of collected OA3 fraction (204 < MW < 565) is about
2.7% from lyophilized OAH. Lyophilized OA3 was used in the fol-
lowing experiments.
2.3. Characterization of the inhibition of ACE
ACE inhibitory activity was measured spectrophotometrically
using hippurl-L-histidyl-L-leucine (Hip-His-Leu) as substrate, fol-
lowing the method of Cushman and Cheung (1971). Fifty microlitres
of sample solution and 100 ll of 2.5 mU ACE solution were added to
100 ll of 12.5 mM Hip-His-Leu solution in a 1.0 M NaCl–borate buf-
fer at pH 8.3. After incubation at 37 °C for 1 h, the reaction was
stopped by adding 250 ll of 0.5 N HCl. The liberated hippuric acid
was extracted with 1.5 ml of ethyl acetate, and absorbance at
228 nm was determined to evaluate the ACE inhibitory activity.
The inhibition rate (%) is shown as {(Ec – Es)/(Ec – Eb)}  100, where
Es is the absorbance when the sample is added to the post-reaction
mixture; Ec is the absorbance with a buffer (instead of the sample)
added, and Eb is the absorbance when a stop solution was added be-
fore the reaction occurred. The IC50 value is the concentration asso-
ciated with 50% ACE inhibition in the reaction mixture. The
experiments were performed in three determinations.
2.4. HPLC chromatograph of ACE inhibitory peptides
The amount of the single peptides was evaluated by HPLC as fol-
lows: each isolated peptide was analyzed on an RP-18(e) column
(4 mm i.d.  250 mml, Merck, USA) equilibrated with 0.1% of triflu-
oroacetic acid (TFA) and eluted using a linear gradient (0–20% ace-
tonitrile/50 min) under a flow rate of 0.7 ml/min.
2.5. Stability of tuna cooking juice-derived ACE inhibitory peptides
Tuna cooking juice-derived ACE inhibitory peptide solutions
(25 mg/ml) were incubated at various temperatures, 20, 40, 60, 80,
903
and 100 °C. The peptide solutions were also incubated at 40 °C and
pH values of 2, 4, 6, 8, and 10 for 2 h. After the solutions were accli-
mated to room temperature, pH was adjusted to 8.3 before the ACE
inhibitory activity was determined as described above. In all the
experiments, each treatment was carried out as triplicate samples.
2.6. Stability against high hydrostatic pressure
Tuna cooking juice-derived ACE inhibitory peptide solutions
(25 mg/ml) were incubated at various pressures, 50, 100, 150,
200, 250, and 300 MPa for 30 min at room temperature.
A high-pressure apparatus (CIP UNIT, Mitsubishi Heavy Indus-
tries Ltd., Japan) with an oil-pressure generator and a compressing
vessel, in which the internal portion (diameter: 50 mm; height:
120 mm) was a flat-bottomed cylinder, was employed. The pres-
sure-raising rate was 200 MPa/min, and the pressure-decreasing
rate was 400 MPa/min. The ACE inhibitory activities were deter-
mined before and after high pressure treatment.
2.7. Stability against in vitro gastric proteases
Stability against in vitro gastric proteases was assessed by treat-
ing a 1% (w/v) peptide solution in 0.1 M KCl–HCl (pH 2.0) buffer,
with pepsin (20 lg/ml) for 4 h in a water-circle bath at 37 °C,
and was stopped by boiling in a water bath for 15 min and neutral-
ized to pH 7.0 with the addition of a 2 N NaOH solution. Neutral-
ized suspension (1 ml) was centrifuged (10,000g, 40 min) with
the supernatant used for ACE inhibitory activity determination.
The remaining neutralized suspension was digested further by 2%
(w/w) pancreatin (Sigma Chemical Co.) at 37 °C for 4 h. The en-
zyme was inactivated by boiling for 15 min followed by centrifuga-
tion (10,000g, 40 min) with the supernatant used for ACE
inhibitory activity determination.
2.8. Protein determination
Protein concentrations were determined by the Lowry method
(1951). Triplicate assays were performed for each sample and bo-
vine serum albumin was used as the protein standard.
3. Results and discussion
3.1. Impact of temperature, pH and pressure on the activity of ACE
inhibitory peptides
The bioactive peptides derived from tuna cooking juice proteins
can be delivered in the form of functional ingredients, such as
hydrolysates, per se or incorporated into other food products. De-
spite the way of delivery, the product—and in particular the ACE
inhibitory peptides therein—must be stable during the final pro-
cessing. As shown in Fig. 1, these peptides retained ACE inhibitory
activity after various temperatures, pH levels and pressure treat-
ments, which indicate that tuna cooking juice-derived ACE inhibi-
tory peptides have satisfactory heat, pH and pressure resistance/
stability. These results are consistent with ACE inhibitory peptides
derived from soy-protein which exhibited good resistance to tem-
perature and pH treatments (Wu & Ding, 2002). In addition, the use
of high pressure to partially unfold the whey proteins, before or
during proteolysis, might also increase the rate of proteolysis and
alter the relative proportion of peptides (Knudsen, Otte, Olsen, &
Skibsted, 2002). In this study, ACE inhibitory oligopeptides OA3
(204 < MW < 565) retained relatively high activity after various
pressure treatments (0–300 MPa), which represent an excellent
stability of inhibitory properties. Heremans, Van Camp, and Huyg-
hebaert (1997) reported that low pressures usually induce revers-
904 J.-S. Hwang / Food Research International 43 (2010) 902–906

120 1000
Relative ACE inhibition index (%)

100 800

80
600

MV
60
400

40
200
20

0
0 0 10 20 30 40 50
0 20 40 60 80 100 120
Temperature ( ) Retention time (min)

1000
120
Relative ACE inhibition index (%)

100 800

80 600
MV
60
400

40
200

20
0
0 0 10 20 30 40 50
0 2 4 6 8 10 12 Retention time (min)
pH
Fig. 2. HPLC chromatograph of ACE inhibitory peptides derived from tuna cooking
120 juice. (Control) and heat treatment (100 °C 2 h). Column: RP-18(e) (4 mm
id  250 mm l), elutent: 0–20% acetonitrile/0.1%, trichloroacetic acid (0–50 min),
Relative ACE inhibition index (%)

flow rate: 0.7 ml/min.

100

80
60
40
20
0 peptides, especially in can sterilization, may lead to losses of avail-
0 50 100 150 200
Pressure (MPa)
Fig. 1. Stability of tuna cooking juice-derived ACE inhibitory peptides after 2 h
incubation at various temperature, pH treatments and 30 min incubation at various
pressures. The relative ACE inhibitory percent was calculated as the ratio of ACE
inhibitory activity between the control and treatments. Bars represent means ± SD.

Bars with different letters are significantly different at P < 0.05.
ible changes such as dissociation of protein–protein complexes, the
binding of ligands and conformational changes. Pressures higher
than 500 MPa induce, in most cases, irreversible denaturation.
3.2. HPLC chromatograph ACE inhibitory peptides before and after
temperature, pH and pressure treatment
In order to examine the stability of the inhibitory properties of
the oligopeptides OA3 during processing, ACE inhibitory activities
were detected before and after various temperature, pH and pres-
sure treatments. However, it is essential to elucidate changes in the
composition of ACE inhibitory peptides after these treatments.
Fig. 2 exhibited that severe thermal processing (100 °C, 30 min)
can lead to little changes in the components of peptides. This com-
pares favorably with the soy-protein-derived ACE inhibitory pep-
tides whose resistance was found at 100 °C, 30 min (Wu & Ding,
2002). However, more severe heat treatments of ACE inhibitory
250 300 350 able peptides compared to this treatment.
pH changes may affect functional properties by modifying spe-
cifically one or more amino acids. For example, acidic treatments
destroy glutamine and asparagine, whereas alkaline treatments
destroy cystine, serine and threonine, and produce lysinoalanine
and D-amino acids (Anantharaman & Finot, 1993; Finot, 1997).
Nevertheless, Fig. 3 shows that ACE inhibitory peptides possessed
resistance to pH 2 and pH 10 incubation for 2 h. This proves that
ACE inhibitory peptides retained almost intact activity after pH
treatment is attributed to little changes in the composition. Simi-
larly, ACE inhibitory peptides also reserved almost the same com-
position before and after high isostatic pressure of 300 MPa for
30 min treatment (Fig. 4). High pressure processing can affect pro-
tein conformation and lead to protein denaturation, aggregation or
gelation, depending on the protein system, the applied pressure,
the temperature and the duration of the pressure treatment (Han-
nu et al., 1998). However, blood plasma and egg white proteins
 J.-S. Hwang / Food Research International 43 (2010) 902–906 905

pH 2.0 2h 30
1000

Peptide concentration (mg/mL)

25

800
20

600
15
MV

400 10

200 5

0
0 -20 0 20 40 60 80 100 120
0 10 20 30 40 50 60
Temperature ( )
Retention time (min)
30
pH 10.0 2h

Peptide concentration (mg/mL)

1000 25

20
800

15
600
MV

10
400
5

200
0
0 2 4 6 8 10 12
0 pH
0 10 20 30 40 50
30
Retention time (min)
Peptide concentration (mg/mL)

Fig. 3. HPLC chromatograph of tuna cooking juice-derived ACE inhibitory peptides 25

after pH treatment (pH 2.0, 2 h or pH 10.0, 2 h). Column: RP-18(e) (4 mm id
250 mm l), elutent: 0–20% acetonitrile/0.1%, trichloroacetic acid (0–50 min), flow
rate: 0.7 ml/min. 20

800 15

10
600
5

400
MV

0
0 50 100 150 200 250 300 350
Pressure (MPa)
200
Fig. 5. Peptide concentration of the ACE inhibitory peptide solutions after 2 h
incubation at various temperature, pH treatments, and 30 min incubation at various
pressure. Bars represent means ± SD. Bars with different letters are significantly
0 different at P < 0.05.
0 10 20 30 40 50
Retention time (min)
compositional stability of ACE inhibitory oligopeptides OA3
Fig. 4. HPLC chromatograph of tuna cooking juice-derived ACE inhibitory peptides
(204 < MW < 565) may be correlated with the short chain confor-
after high pressure treatment (300 MPa, 30 min). Column: RP-18(e) (4 mm id
250 mml), elutent: 0–20% acetonitrile/0.1%, trichloroacetic acid (0–50 min), flow mation which had difficulty in forming gel networks at 300 MPa
rate: 0.7 ml/min. of pressure. In addition, as the results shown in Fig. 5, a slight dif-
ference was found among these peptide concentrations before and
after various treatments. Therefore, it is obvious that ACE inhibi-
exhibited no gelation occurrence when pressurized for 30 min at a tory oligopeptides are considerably stable during the above
pressure of 400 MPa (Heremans et al., 1997). In this study, the treatments.
906 J.-S. Hwang / Food Research International 43 (2010) 902–906
Table 1
Activity of OA3 oligopeptides following digestion by gastrointestinal proteases.
Enzyme ACE inhibition rate (%) IC50 (mg/ml)
Control 78.13 ± 2.3 0.21 ± 0.01
Pepsin 77.50 ± 2.1 0.22 ± 0.02
Pepsin + pancreatin 74.20 ± 3.1 0.25 ± 0.03
All values are mean ± standard deviation for triplicate experiments.
Values with different superscripts are significantly different at P < 0.05.
3.3. Impact of in vitro gastric proteases on ACE inhibitory peptides
Gastric in vitro incubation provides a practical and easy process,
imitating the fate of these peptides under oral administration.
Some food-protein-derived ACE inhibitors failed to show hypoten-
sive activity after oral administration in vivo due to the fact that
they are substrates of ACE or hydrolyzed in the gastrointestinal
tract to peptides with reduced activity (Fujita et al., 2000). The
ACE inhibitory activity of OA3 oligopeptides showed little change
after in vitro incubation with gastric enzymes (Table 1) suggesting
that these peptides may be resistant to digestion in the gastroin-
testinal tract. Previous reports have also shown that small peptides
similar to those obtained in the present work have low susceptibil-
ity to hydrolysis by gastric proteases (Grimble et al., 1987; Matt-
ews, 1977). Little difference was found among these HPLC
chromatography peptide mappings before and after gastric prote-
ases treatments (data not shown), which suggests the composi-
tional stability of the peptides.
4. Conclusion
The influence of food processing, heat, high pressure and pH
treatments in this study, on the bioactivity of ACE inhibitory pep-
tides is not significant. Peptides also show resistance to in vitro
digestion by gastrointestinal proteases. However, the interactions
of ACE inhibitory peptides with other food components and
changes in amino acid composition and sequence during process-
ing are needed to clarify by more research in our future study.
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