Food Research International 44 (2011) 10441051

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Food Research International

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Squid gelatin hydrolysates with antihypertensive, anticancer and antioxidant activity

A. Alemn a, E. Prez-Santn a, S. Bordenave-Juchereau b, I. Arnaudin b, M.C. Gmez-Guilln a,, P. Montero a
a
b

Instituto de Ciencia y Tecnologa de Alimentos y Nutricin (ICTAN, CSIC) (formerly Instituto del Fro), C/ Jos Antonio Novais, 10, 28040, Madrid, Spain
Laboratoire Littoral ENvironnement et Societs (LIENSS), Universit de La Rochelle, CNR UMR 6250 UFR Sciences, Avenue M. Crepeau 17042, La Rochelle, France

a r t i c l e

i n f o

Article history:
Received 22 January 2011
Accepted 6 March 2011
Keywords:
Bioactive peptides
Gelatin hydrolysates
Antioxidant
ACE inhibition
Anticancer

a b s t r a c t
Gelatin obtained from giant squid (Dosidicus gigas) inner and outer tunics was hydrolyzed by seven commercial
proteases (Protamex, Trypsin, Neutrase, Savinase, NS37005, Esperase and Alcalase) to produce bioactive
hydrolysates. The Alcalase hydrolysate was the most potent angiotensin-converting enzyme (ACE) inhibitor
(IC50 = 0.34 mg/mL) while the Esperase hydrolysate showed the highest cytotoxic effect on cancer cells, with IC50
values of 0.13 and 0.10 mg/mL for MCF-7 (human breast carcinoma) and U87 (glioma) cell lines, respectively. The
radical scavenging capacity of gelatin increased approximately 3-fold for Protamex, Neutrase and NS37005
hydrolysates and between 7 and 10-fold for Trypsin, Savinase, Esperase and Alcalase hydrolysates. Trypsin,
Savinase, Esperase and Alcalase hydrolysates had a metal chelating capacity above 80% whereas Protamex,
Neutrase and NS37005 hydrolysates registered less than 25%. The antioxidant activity measured by FRAP (ferric
ion reducing power) was largely unaffected by the enzyme used, increasing approximately 2-fold for all
hydrolysates. The most active hydrolysates (Alcalase and Esperase) were comprised mostly of peptides with
molecular weights ranging from 500 to 1400 Da, however, a clear relationship between bioactive properties and
molecular weight distribution of all the hydrolysates was not fully established.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Hypertension is a multifactorial process and the main cause of
illness in industrialized countries. The angiotensin-I converting
enzyme (ACE) plays an important role in the regulation of blood
pressure and hypertension because it catalyses the conversion of
inactive angiotensin-I into angiotensin-II, a potent vasoconstrictor
(Goodfriend, Elliott, & Catt, 1996) and inactivates bradykinin, a
potent vasodilator (Witherow, Helmy, Webb, Fox, & Newby, 2001).
Consequently synthetic ACE inhibitors such as Captopril and Enalapril
are often used to treat hypertension and other cardio-related diseases.
However as these synthetic ACE inhibitors can cause adverse side
effects including cough, taste disturbance, rashes and angioedema
(Messerli, 1999), there is a growing interest in natural inhibitors.
Fish protein hydrolysates produced using commercially available
proteases have been shown to possess combined ACE-inhibitory and
antioxidant capacities (Je, Lee, Lee, & Ahn, 2009; Samaranayaka, Kitts,
& Li-Chan, 2010), and it has been postulated that these two activities
could contribute to their antihypertensive effect in vivo (Hou, Chen, &
Lin, 2003).
On the other hand, cancer is one of the largest single causes of death
in both men and women (Kaufman & Earnshaw, 2000). Frequently,
resistance to anticancer drugs has been observed (Lind et al., 2001).

Corresponding author. Tel.: +34 91 5492300; fax: +34 91 5493627.

E-mail address: cgomez@ictan.csic.es (M.C. Gmez-Guilln).
0963-9969/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2011.03.010

Therefore, the research and development of more effective and less toxic
drugs by the pharmaceutical industry has become necessary, prompting
a growing interest in the identication and characterization of natural
antitumor agents. The elimination of cancer in the early stages is an
integral part of chemoprevention, and measuring the cytotoxic
properties of a given compound against cancer cells provides useful
insight into its chemoprotective potential. Antioxidants have also been
proposed as potentially suitable candidates for the prevention and
treatment of diseases associated with active oxygen species, especially
applicable to cancer diseases (Leng, Liu, & Chen, 2005).
Fish or squid skin gelatins have been reported to give rise to
biologically active peptides with high ACE-inhibitory and antioxidant
activity, the latter due to its radical scavenging capacity, metal
chelating effects and reducing power or lipid peroxidation inhibition
(Zhao, Li, Liu, Dong, Zhao, & Zeng, 2007; Kim, Byun, Park, & Shahidi,
2001; Mendis, Rajapakse, Byun, & Kim, 2005; Mendis, Rajapakse, &
Kim, 2005; Lin & Li, 2006; Gimnez, Alemn, Montero, & GmezGuilln, 2009; Alemn, Gimnez, Montero, & Gmez-Guilln, 2011).
However, no reference has been made in previous reports of any
antiproliferative/cytotoxic effect of gelatin derived peptides or
hydrolysates on cancer cells. The biological properties of peptides
are to a large extent inuenced by their molecular weight and
conformational structure, which are in turn, greatly affected by
processing conditions. In contrast, the amino acid composition of the
hydrolysates resulting from the hydrolysis with different types of
enzymes is very similar to that of the parent gelatins, as reported
by Kim, Kim, Byun, Nam, Joo, and Shahidi (2001) with Alaska pollack

A. Alemn et al. / Food Research International 44 (2011) 10441051

skin gelatin or Gmez-Guilln et al. (2010) with tuna and giant squid
skin gelatins.
A number of commercial proteases have been used for the
production of antioxidant gelatin hydrolysates, including trypsin,
chymotrypsin, pepsin, Properase E, Pronase, collagenase, bromelain
and papain (Kim, Kim, et al., 2001; Mendis, Rajapakse, Byun, et al.,
2005; Mendis, Rajapakse, & Kim, 2005; Lin & Li, 2006; Yang, Ho, Chu, &
Chow, 2008). Alcalase, which is a commercial protease from a
microbial source (Bacillus licheniformis), has been shown to be one of
the most efcient in the hydrolysis of sh protein (Gurard, Dufoss,
De La Broise, & Binet, 2001). This enzyme preparation has also been
reported to produce high levels of hydrolysis in skin gelatin from
Alaska pollack, squid Todarodes pacicus or giant squid Dosidicus gigas
(Kim, Byun, et al., 2001; Nam, You, & Kim, 2008; Gimnez et al., 2009).
In a recent work, a peptide sequence Gly-Pro-X-Gly-X-X-Gly-Phe-XGly-Pro-X-Gly-X-Ser with the X positions occupied by either Hyp or
Leu, and a molecular mass of 1411 Da, together with a compound with
a molecular mass of 952 Da, associated with the carbohydrate fucose,
were identied as being responsible for most of the high anti-radical
and ACE-inhibitory activity in an Alcalase hydrolysate from giant squid
gelatin (Alemn, Gimnez, Prez-Santn, Gmez-Guilln, & Montero,
2011). Further work with giant squid gelatin revealed that Alcalase
also produced hydrolysates with much higher radical scavenging
and ferric reducing capacities than those of other single enzymes
such as collagenase, trypsin or pepsin (Alemn, Gimnez, Montero,
et al., 2011).
The aim of this study was to explore the potential health benets
which could be derived from giant squid tunic gelatin by investigating
the ACE-inhibitory, anticancer and antioxidant properties (radical
scavenging, metal chelating and reducing power) of hydrolysates
having different molecular weight distributions, obtained by using
several commercial proteases.
2. Materials and methods
2.1. Materials
Gelatin from inner and outer giant squid (D. gigas) tunics was
extracted using the method described by Gimnez et al. (2009).
Commercial proteases (Protamex, Trypsin, Neutrase 0.8 L, Savinase
16 L, NS37005, Esperase 0.8 L and Alcalase 2.4 L) were kindly supplied
by Novozymes (Bagsvaerd, Denmark). NaOH, HCl, NaCl, glycerol and
potassium persulfate were from Panreac (Barcelona, Spain). All other
reagents were from Sigma-Aldrich, Inc. (St. Louis, MO, USA).
2.2. Preparation of gelatin hydrolysates
The squid gelatin (2.5% w/v) was dissolved in water and subjected
to enzymatic hydrolysis for 3 h, under optimal temperature and pH
conditions. The optimal condition for each protease was as follows:
Protamex (pH 6.5, 60 C), Trypsin (pH 8, 55 C), Neutrase 0.8 L (pH
6.5, 55 C), Savinase 16 L (pH 9.5, 55 C), NS37005 (pH 7, 55 C),
Esperase 0.8 L (pH 8.5, 60 C) and Alcalase 2.4 L (pH 8, 50 C), with an
enzymesubstrate ratio of 1:20 (w:w). The pH of the reaction was
kept constant by adding a 1 N NaOH solution to the reaction medium
using a pH-stat (TIM 856, Radiometer Analytical, Villeurbanne Cedex,
France). The enzymes were inactivated by heating at 90 C for 10 min,
and the samples were centrifuged at 3000 g for 15 min. The supernatants comprised the hydrolysates and were lyophilized and stored
at 80 C for further assays.
2.3. Molecular weight distribution
The gelatin hydrolysates were analyzed by Tricine SDS-PAGE
according to Schagger and Von Jagow (1987), using a 5% stacking
gel and a 16.5% resolving gel. The hydrolysates were dissolved at

1045

a concentration of 10 mg/mL in the loading buffer (50 mM TrisHCl,

4% SDS, 12% glycerol, 2% mercaptoethanol and 0.01% SERVA Blue G),
heat-denatured at 90 C for 10 min, and run in a Mini Protean II unit
(Bio-Rad Laboratories, Hercules, CA, USA). Protein bands were stained
with Coomassie Brilliant Blue R250. The approximate molecular weight
(MW) of the hydrolysates was determined using a low molecular
weight marker kit (Amersham Pharmacia Biotech, Uppsala, Sweden)
consisting of triosephosphate isomerase (26.6 kDa), myoglobin
(16.9 kDa), -lactalbumin (14.4 kDa), aprotinin (6.5 kDa), oxidized
insulin chain (3.5 kDa) and bacitracin (1.4 kDa).
Hydrolysate molecular weight distribution was also evaluated
by size-exclusion HPLC (model SPE-MA10AVP, Shimadzu, Kyoto,
Japan) on a superdex peptide PC 3.2/30 column (GE Healthcare BioSciences, Barcelona, Spain), with a fractionation range between 7000
and 100 Da. The injection volume was 10 L and the ow rate was
0.1 mL/min using Milli-Q water in the mobile phase. Vitamin B12
(1355 Da) and hippuryl-L-histidyl-L-leucine (HHL, 502 Da), both
from Sigma-Aldrich, Inc. (St. Louis, MO, USA), were used as molecular
weight standards.
2.4. ACE-inhibition
Reversed-phase high performance liquid chromatography (RPHPLC) was used to determine ACE inhibition applying the method
used by Wu, Aluko, and Muir (2002) with slight modications. The
total reaction volume was 225 L, made up of 50 L of 5 mM HHL,
160 L of 25 mU/mL of ACE and 20 L of sample at six different
concentrations (from 0.1 to 1.5 mg/mL nal concentration), prepared
with 100 mM potassium-phosphate buffer, containing 300 mM NaCl,
pH 8.3. The mixture was incubated at 37 C for 120 min and the
reaction was quenched by adding 100 L of 0.1 M HCl. The released
hippuric acid (HA) was quantied by RP-HPLC (model SPE-MA10AVP,
Shimadzu, Kyoto, Japan) on a C18 column (Tracel excel 120 ODSA
5 m 25 0.46, Teknokroma, Barcelona, Spain). The injected volume
was 50 L and the ow rate was 0.8 mL/min using an acetonitrile
gradient of 20 to 60% in 0.1% triuoroacetic acid (TFA) for 26 min.
The HA and HHL were monitored at 228 nm and the retention times
were 8.30 and 15.70 min respectively. All determinations were
carried out at least in triplicate. The IC50 value of ACE inhibitory
activity was dened as the concentration of hydrolysate (mg/mL)
required to reduce the HA peak by 50%.
2.5. Cytotoxic and antiproliferative activities
The study of cytotoxic and antiproliferative activities of squid
gelatin hydrolysates was performed according to the methodology
proposed by Picot et al. (2006). MCF-7 human breast carcinoma
and U87 glioma cell lines were grown at 37 C in a 5% CO2, 95% air
humidied atmosphere, in DMEM-Ham's F12 medium (1:1, v:v,
Gibco). The medium was supplemented with 5% heat inactivated
Fetal Calf Serum (FCS, Dutscher) to which penicillin (100 U/mL) and
streptomycin (100 g/mL) had been added (Pan biotech GmbH). The
cells grown in asks were trypsinized, centrifuged at 800 rpm for
5 min and dissolved in fresh culture medium at 104 cells/90 L.
Subsequently a 90 L volume of suspension cells (10,000 cells) was
added to each well of a 96-well microplate and incubated at 37 C for
24 h. In order to perform cytotoxicity screening of the hydrolysates,
hydrolysate stock solution at a concentration of 10 mg/mL was
prepared in PBS 0.1 M (pH 7.4), and diluted 10-fold in cell culture
medium containing the cells. The microplate was then incubated at
37 C for 24, 48 and 72 h, changing the culture medium every 24 h and
adding the hydrolysate at a nal concentration of 1 mg/mL. At the end
of every incubation period, 15 L of 5 mg/mL tetrazolium salt (MTT)
solution was added to each well, and the plate was incubated for
3 h. To stop succinate-tetrazolium reductase activity and solubilize
formazan crystals, 200 L of dimethyl sulfoxide (DMSO) was then

1046

A. Alemn et al. / Food Research International 44 (2011) 10441051

added to each well and kept at 37 C for 1 h. Absorbance was read on

a plate reader (VERSAmax, Molecular Devices, Saint Gregoire, France)
at 570 nm. The data were analyzed to calculate the percentage
of viability inhibition induced by the presence of hydrolysate in
cell culture medium determined by the equation: Viability inhibition
(%) = 100 (AH 100 / AC) where AH and AC are the absorbencies
measured for cell viability in culture medium containing only
hydrolysate or PBS respectively.
Following the same procedure, the effect of addition of hydrolysates at the same time as the cancer cells at the beginning of the
experiment was also evaluated. For this evaluation, the initial
conditions of the culture medium and the added hydrolysates were
maintained for 72 h.
The viability inhibition of the Esperase and Alcalase hydrolysates
was also measured using six different concentrations ranging from
0.05 to 1.5 mg/mL, in order to determine the IC50. All experiments
were performed at least in triplicate.
2.6. Antioxidant activities
Different assays (ABTS, chelating ability and FRAP) were performed to study the antioxidant properties of the squid gelatin and
the corresponding hydrolysates, at a concentration of 10 mg/mL. The
ABTS radical [2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)]
scavenging assay was carried out according to the method described
by Re, Pellegrini, Proteggente, Pannala, Yang, and Rice-Evans (1999).
The stock solution of ABTS radical consisted of 7 mM ABTS in
potassium persulfate 2.45 mM, kept in the dark at room temperature
for 16 h. An aliquot of stock solution was diluted with Milli-Q water
in order to give the working solution of ABTS radical an absorbance
at 734 nm of 0.70 0.02. A 20 L aliquot sample was mixed with
980 L of ABTS reagent and then left to stand in the dark at 30 C
for 10 min. Absorbance values were read at 734 nm using a UV-1601
spectrophotometer (Model CPS-240, Shimadzu, Japan). Results
were expressed as mg Vitamin C Equivalent Antioxidant Capacity
(VCEAC)/g of sample (gelatin or hydrolysate) based on a standard
curve of vitamin C.
The Fe2+ chelating activity of the hydrolysates, at a concentration
of 10 mg/mL, was measured by the method introduced by Chung,
Chang, Chao, Lin, and Chou (2002). Briey, a test sample of 800 L was
mixed with 10 L of 2 mM FeCl2 and 20 L of 5 mM ferrozine. The
mixture was vortexed and kept at room temperature for 10 min prior
to measuring the absorbance at 562 nm using a UV-1601 spectrophotometer (Model CPS-240, Shimadzu, Japan). Chelating ability,
expressed as a percentage, was calculated as follows: Chelating
ability (%) = [1 (Abs. sample / Abs. control)] 100. The control was
a mixture composed of 800 L of Milli-Q water, 10 L of 2 mM FeCl2
and 20 L of 5 mM ferrozine.
FRAP, a measure of the reducing power of samples, was performed
according to the method described by Benzie and Strain (1996) with
some modications. A 60 L of dissolved sample (at a concentration of
10 mg/mL) was incubated at 37 C with 60 L of Milli-Q water and 900 L
of FRAP reagent, containing 10 mM of TPTZ (2,4,6-tripyridyl-s-triazine)
and 20 mM of FeCl3. Absorbance values were read at 595 nm after 30 min,
using a UV-1601 spectrophotometer (Model CPS-240, Shimadzu, Japan).
Results were expressed as mol Fe2+ equivalents/g of sample (gelatin or
hydrolysate) based on a standard curve of FeSO47H2O. All determinations were carried out at least in triplicate.

3. Results and discussion

3.1. Molecular weight distribution
Analysis of the overall molecular weight distribution of the
different hydrolysates (Fig. 1) was carried out by Tricine SDS-PAGE.
The Trypsin and NS37005 hydrolysates showed the highest quantity
of peptide bands in the MW range between 26 and 3.5 kDa. An
appreciable amount of protein with MW even higher than 26 kDa
was also present in the Trypsin hydrolysates, denoting the lowest
efciency in protein breakdown. All the hydrolysates were characterized by presenting an intense band at MW 1.4 kDa. However,
no peptide bands of more than 1.4 kDa could be observed in the
Alcalase hydrolysates, and hardly any in the Neutrase hydrolysate
and Esperase hydrolysate, indicating a considerably high degree of
proteolysis. Hydrolysates composed of a wide range of polydisperse
products, with varying degrees of hydrolysis and mostly small peptides
cannot usually be properly detected under analytical electrophoresis
conditions (Khantaphant & Benjakul, 2008; Gimnez et al., 2009).
Consequently, in order to know the differences in the molecular
weight distribution of the smallest peptides (MW 1.4 kDa), sizeexclusion HPLC (SE-HPLC) was performed in a fractionation range
between 7000 and 100 Da (Fig. 2). The varying MW proles
revealed considerable differences in the degree of protein breakdown
depending on the enzyme used. In accordance with the electrophoretic
prole, the Alcalase, Neutrase and Esperase hydrolysates showed
higher levels of peptides with MW below 1400 Da, whereas in Trypsin
and NS37005 hydrolysate peptides with MW above 1400 Da were
predominant.
3.2. ACE-inhibitory capacity
The amount of hydrolysate required to inhibit 50% of the ACE
activity (IC50) is shown in Table 1. Unlike the parent squid gelatin, all
the hydrolysates presented ACE inhibitory capacity, although it was
markedly different depending on the enzyme used. It was not possible
to determine the IC50 value in the NS37005 hydrolysate, whereas the
Alcalase hydrolysate showed the most potent effect with an IC50
of 0.34 mg/mL followed by the Neutrase hydrolysate (0.63 mg/mL)
(p 0.05). As revealed by SDS-PAGE and SE-HPLC, the peptides in
the Alcalase and Neutrase hydrolysates were mostly between ~ 1400
and 500 Da. In another study Zhao et al. (2007) found the highest
ACE-inhibitory capacity for a sea cucumber gelatin hydrolysate in the
fraction with MW b 1 kDa. Nevertheless, there are other studies where

2.7. Statistical analysis

Statistical tests were performed using the SPSS computer
programme (SPSS Statistical Software, Inc., Chicago, IL, USA). Data
were expressed as mean standard deviation of the mean (n = 3 or
n = 24). Tukey's test was used to determine the level of signicance
at p 0.05.

Fig. 1. Electrophoretic proles of gelatin hydrolysates at a concentration of 10 mg/mL

(Tricine SDS-PAGE). (m) molecular weight markers, (a) Protamex, (b) Trypsin,
(c) Neutrase, (d) Esperase, (e) NS37005, (f) Savinase and (g) Alcalase.

A. Alemn et al. / Food Research International 44 (2011) 10441051

mAU

Protamex

750

1047

mAU

Trypsin

750

> 1375 Da

500

1375-500 Da

< 500 Da

500
250

250
0

0
0

10

20

30

40

mAU

50

min

Neutrase

10

20

30

40

mAU

750

750

500

500

250

250

50

min

Esperase

0
0

10

20

30

40

mAU

50

min

NS37005

750

10

20

30

40

mAU

50

min

Savinase

750

500

500

250

250

0
0

10

20

30

40

mAU

50

min

10

20

30

40

50

min

Alcalase

750
500
250
0
0

10

20

30

40

50

min

Fig. 2. Molecular weight distribution of gelatin hydrolysates at a concentration of 10 mg/mL by SE-HPLC. The absorbance was monitored at 215 nm.

very small peptides did not appear to increase the activity of the
whole hydrolysate, but rather decreased it (Theodore & Kristinsson,
2007).
Bougatef et al. (2008) reported IC50 values in the concentration
range of 1.27.4 mg/mL for sardinelle by-product hydrolysates
prepared with different proteases, while the IC50 of a marine shrimp
hydrolysate digested by a crude protease was 0.97 mg/mL (Hai-lun,
Xiu-Lan, Cai-Yun, Yu-Zhong, & Bai-Cheing, 2006). Three peptides ValTyr-Ala-Pro, Val-Ile-Ile-Phe and Met-Ala-Trp with IC50 values of 6.1, 8.7
and 16.32 M, respectively, had been reported from cuttlesh muscle
protein hydrolysates (Balti, Nedjar-Arroume, Bougatef, Guillochon, &
Table 1
ACE-inhibitory capacity of squid gelatin hydrolysates.
IC50
Protamex
Trypsin
Neutrase
Esperase
NS37005
Savinase
Alcalase

1.06 0.01e
0.91 0.03d
0.63 0.01b
0.89 0.03c,d
nd
0.82 0.08c
0.34 0.02a

nd: not determined.

Different letters (a, b, c,) indicate signicant differences
(p 0.05).
IC50: concentration (mg/mL) required for inhibiting
50% of ACE activity.

Nasri, 2010). Although values of IC50 for ACE-inhibitory capacity of skin

gelatin whole hydrolysates had been previously reported by Kim, Kim,
et al. (2001) and Zhao et al. (2007), comparisons with IC50 values in the
present study were not fully possible due to the different methodology
employed for measuring activity.
Proline or aromatic amino acids existing at the C-terminal are suitable
for peptide binding to ACE (Cheung, Wang, Miguel, Emily, & Dvid, 1980).
Alcalase has broad specicity hydrolyzing most peptide bonds; preferentially those containing aromatic amino acid residues (Doucet, Otter,
Gauthier, & Foegeding, 2003), suggesting that this enzyme preparation
should generate peptides with the most favorable amino acids at the Cterminal position. Some hydrolysates prepared with Alcalase also exerted
a strong in vitro and in vivo ACE-inhibitory activity (Cheng et al., 2008).
Other studies demonstrated that Pro at the antepenultimate position in
the peptide sequence also enhanced binding to the ACE enzyme
(Contreras, Carrn, Montero, Ramos, & Recio, 2009). Most commercial
inhibitors (Captopril and Enalapril) also bear Pro residue in their structure.
The reasonably high ACE-inhibitory capacity (IC50 =0.341.06 mg/mL)
achieved with most of the hydrolysates tested may be favored by the
abundance of Pro residues (89 residues/1000 residues) in the parent squid
gelatin (Alemn, Gimnez, Montero, et al., 2011).
3.3. Cytotoxic and antiproliferative effects against cancer cells
Cytotoxic activity against cancer cells was considered to be the
effect produced when the hydrolysate was added to the culture

1048

A. Alemn et al. / Food Research International 44 (2011) 10441051

c/y
d/z

c/x
d/y
e/z

100

Neutrase

NS37005

Savinase

b/x

Trypsin

a/x
b/y
c/z

Protamex

a/x
ab/y
b/y

40

a/x
b/y
b/y

60

a/x
b/y
c/y

80

a/x
ab/y
b/y

Viability inhibition (%)

20
0
Esperase

24h

48h

Alcalase

72h

Trypsin

e/z

d/x
b/x
b/y
d/x

c/x
b/x
ab/x

Protamex

40

b/x
b/y
c/y

a/y
ab/y

60

b/x
a/x
a/x

80

20

e/y

d/y
f/z
e/x

100

a/x

viability inhibition (%)

NS37005

Savinase

0
Neutrase

Esperase

24h

48h

Alcalase

72h

Fig. 3. Effect of gelatin hydrolysates on the viability of cancer cells for 24, 48 and 72 h in cell culture medium containing a concentration of 1 mg/mL of hydrolysate. MCF-7 cell line
(A). U87 cell line (B). Different letters (a, b, c,) within the same time period indicate signicant differences (p 0.05). Different letters (x, y, z) within the same hydrolysate indicate
signicant differences (p 0.05).

activity on both cell lines (Fig. 4). Picot et al. (2006) evaluated the
antiproliferative activity of 18 sh protein hydrolysates and found
the highest effect on MCF-7 cell lines to be around 40% for a cod
hydrolysate obtained with Protamex and Alcalase. At the same
concentration (1 mg/mL) the Alcalase squid gelatin hydrolysate
caused a similar effect on MCF-7 cells (41.64 5.3% of growth
inhibition), however the Esperase hydrolysate had a much stronger
(p 0.05) effect (91.41 1.8% of inhibition), and remained similar for
a concentration reduction to 0.5 mg/mL.
The IC50 was determined for both MCF-7 and U87 cells in the case
of hydrolysates that had produced the strongest cytotoxic effects
(Esperase and Alcalase hydrolysates) at 24, 48 and 72 h (Table 2).
Both hydrolysates showed low IC50 values for both cell lines,
especially the Esperase hydrolysate. A pepsin hydrolysate from
algae protein waste showed an IC50 of 1.74 mg/mL against AGC cell
lines (Sheih, Fang, Wu, & Lin, 2010). To our knowledge, the IC50 values
for cytotoxic effect (0.13 and 0.10 mg/mL for MCF-7 and U87 cell lines,
respectively) of the Esperase hydrolysate, were the lowest found for
whole food protein hydrolysates, although similar IC50 values have
been reported for isolated peptides. The nonapeptide X-Met-Leu-ProSer-Tyr-Ser-Pro-Tyr, obtained by hydrolysis of defatted soy protein,

Growth inhibition (%)

medium after the cells had been growing for 24 h; antiproliferative

activity was considered to be the effect produced when the
hydrolysates were incorporated before cell growth started.
As expected, squid gelatin did not show cytotoxic or antiproliferative
effect on cancer cells. However, the hydrolysates prepared from squid
gelatin affected the viability of both cell lines differently depending on
the enzyme used (Fig. 3A, B). Cell viability was determined after 24, 48
and 72 h with a change of culture medium and the addition of 1 mg/mL
concentration of the hydrolysate on each occasion. Only Esperase,
Alcalase and Savinase hydrolysates intensied their effect on both
cancer cell lines after 48 and 72 h (p 0.05). The Esperase hydrolysate
had the highest (p 0.05) cytotoxic effect on both cancer cells (96.6
0.5 and 91.2 2.7% of viability inhibition on MCF-7 and U87 cells
respectively after 72 h). It is worth noting that the same results were
obtained for a concentration of 0.3 mg/mL for both cell lines. Alcalase
hydrolysate also showed relatively high cytotoxic activity (67.8 5.6
and 83.9 4.8% viability inhibition on both cell lines, respectively).
Jang, Jo, Kang, and Lee (2008) puried four peptides from beef protein
and caused a 20% viability inhibition on MCF-7 at 0.4 mg/mL.
Regarding antiproliferative activity, again Esperase hydrolysate
followed by Alcalase hydrolysate exhibited the highest (p 0.05)

100

80
60

40
20

c
d

bc

b
c

e d

a
a

0
Protamex

Trypsin

Neutrase

U87

Esperase

NS37005

Savinase

Alcalase

MCF-7

Fig. 4. Effect of gelatin hydrolysates on growth of MCF-7 and U87 human cells for 72 h in cell culture medium containing a concentration of 1 mg/mL of hydrolysate. Different letters
(a, b, c,) within the same cell cultures indicate signicant differences (p 0.05).

A. Alemn et al. / Food Research International 44 (2011) 10441051

1049

Table 2
Cytotoxic activity of Esperase and Alcalase squid gelatin hydrolysates.
IC50
MCF-7 cells

Esperase
Alcalase

U87 cells

24 h

48 h

72 h

24 h

48 h

72 h

0.15 0.01a
1.18 0.02c

0.14 0.01a
1.04 0.02b

0.13 0.01a
0.81 0.03a

0.31 0.01c
1.22 0.03c

0.13 0.01b
0.98 0.03b

0.10 0.01a
0.85 0.02a

Different letters (a, b) within the same hydrolysate and the same cell line indicate signicant differences (p 0.05).
IC50: concentration (mg/mL) required to induce 50% cells death.

had an IC50 of 0.16 mg/mL on a mouse monocyte macrophage cell line

(Kim et al., 2000). A peptide fraction from algae protein waste exerted
an IC50 value of 0.07 mg/mL against AGS cells (Sheih et al., 2010). The
cytotoxic effect of Esperase hydrolysate on MCF-7 cells increased
after 48 and 72 h but to a lesser extent than on U87 cells. After 24 h,
the IC50 for MCF-7 cells (0.15 mg/mL) was lower than that for U87
cells (0.31 mg/mL), however, after 72 h the Esperase hydrolysate was
more cytotoxic against U87 cells (IC50 = 0.10 mg/mL) than against
MCF-7 cells (IC50 = 0.13 mg/mL).
The Esperase and Alcalase hydrolysates had most of their peptides
in the range of 1400 to 500 Da. Sheih et al. (2010) fractioned a pepsin
hydrolysate from algae protein waste and found that the most
effective peptide fraction against AGS cells had a molecular weight
lower than 6500 Da and most of the peptides (N90%) were located
between the range of 2000 and 200 Da. Kannan, Hettiarachchy,
Johnson, and Nannapaneni (2008) observed the highest antiproliferative activity in human colon and liver cancer cells in the lower
molecular weight fraction (b5 kDa) of an Alcalase hydrolysate from
rice bran. However, molecular weight cannot be considered the most
important factor inuencing anticancer activity, since the Neutrase
hydrolysate which has relatively low cytotoxic and antiproliferative
activities is also made up mostly of peptides in the range of 1400
500 Da. Thus, the antiproliferative/cytotoxic effect against MCF-7
and U87 cells should be linked for the most part to the presence
of specic peptides that exert a direct cytotoxic effect on cancer
cells as previously reported for valorphin, a hemoglobin-derived
peptide (Blishchenko et al., 2005). Nevertheless, the hypothesis of a
binding competition between peptides and FCS growth factor on cell
membrane receptors cannot be excluded either. Three peptides from
a sh source have been described as having antitumor activity: (i) a
440.9 Da anchovy hydrophobic peptide, able to induce apoptosis in
human U937 lymphoma cells by increasing caspase-3 and caspase8 activity (Lee, Kim, Lee, Kim, & Lee, 2004), (ii) tilapia hepcidin TH2-3
that manifested signicant antitumor activity in human brosarcoma
cells (Chen, Lin, & Lin, 2009) and (iii) epinecidin-1, a peptide from
sh (Epinephelus coioides) which had an antitumor effect similar to
lytic peptides in human brosarcoma cells (Lin et al., 2009). However
none of these peptides originate from sh gelatin/collagen.
3.4. Antioxidant activity
The radical scavenging capacity of squid gelatin exhibited a 3-fold
increase when it was hydrolyzed with Protamex, Neutrase or
NS37005, whereas such an increase was between 7 and 10-fold
when the enzymes used were Trypsin, Savinase, Esperase or Alcalase
(Fig. 5A). However, radical scavenging capacity could not be well
correlated with the molecular weight distribution of the whole
hydrolysate, since Trypsin hydrolysate was the one with the highest
number of larger peptides. This nding suggested that the radical
scavenging activity of protein hydrolysates is dependent not only
on the peptide size, but also to a greater extent on their sequences,
as a result of the specicity of the enzyme used.
The metal chelating ability of a given compound may also serve
as a useful indicator of its potential antioxidant activity. As depicted

in Fig. 5B, the squid gelatin did not show any Fe2+ chelating ability at
10 mg/mL. However, when gelatin was hydrolyzed with Protamex,
Neutrase or NS37005 enzymes, the resulting hydrolysates did exert a
low chelating capacity (less than 25%). In contrast, the Trypsin,
Savinase or Esperase hydrolysates at the same concentration caused
~80% of Fe2+ chelation, which was near to 100% with the Alcalase
hydrolysate. Dong, Zeng, Wang, Liu, Zhao, and Huicheng (2008) found
a metal chelation activity of 92.97% with an Alcalase hydrolysate from
silver carp at 5 mg/mL.
Hydroxyl radicals can be formed from superoxide anion and
hydrogen peroxide in the presence of transition metal ions, such as
Fe2+ and Cu2+. Thus chelating metal ions may inhibit the formation of
hydroxyl radicals. Presumably, peptide cleavage leads to enhanced
metal ion binding because carboxyl and amino groups in the side
chains of the acidic (Glx and Asx) and basic (Lys, His, and Arg) amino
acids are thought to play an important role in chelating metal ions
(Saiga, Tanabe, & Nishimura, 2003).
The molecular weight of peptides is also believed to play a key role
in chelating metals. Klompong, Benjakul, Kantachote, and Shaidi
(2007) and Dong et al. (2008) pointed out that metal-chelating ability
and molecular weight had a linear relationship, and that metalchelating ability increased with decreasing molecular weight. Nevertheless, Megas et al. (2007) reported that if peptide length is too
short, chelation is unstable. In our study, hydrolysate with peptides
mostly between ~ 1400 and 500 Da (Alcalase hydrolysate) exerted
the highest metal chelating ability. However, it is worth noting the
considerably high metal chelating activity in the Trypsin hydrolysate,
as compared to other hydrolysates with higher proportions of smaller
peptides (Neutrase or Protamex), denoting once again the importance
of the presence of specic peptide sequences, rather than the whole
hydrolysate molecular weight distribution.
The behavior for Fe2+ chelation and ABTS+ scavenging followed a
similar trend suggesting that the two antioxidant mechanisms might
be related. Zhu, Chen, Tang, and Xiong (2008) also found a similar
trend between Cu2+ chelating capacity and ABTS radical scavenging
for an Alcalase hydrolysate from zein protein.
Another method for measuring antioxidant potential is the
ferric reducing antioxidant power (FRAP) assay. Reducing power is
a measurement that provides an estimation of a compound's ability
to reduce ferric iron (III) to ferrous iron (II), and is determined
using a redox-linked colorimetric reaction. Samples with higher
reducing power are more able to donate electrons or hydrogen.
Free radicals form stable substances by accepting donated electrons
and thus, the free radical chain reaction could be interrupted.
The reducing power of squid gelatin and hydrolysates is shown
in Fig. 5C. The ability of the hydrolysates to reduce Fe3+ to Fe2+
was approximately twice than that of the gelatin, with all the
hydrolysates showing a similar (p N 0.05) capacity independently of
the enzyme used, and consequently of molecular weight distribution too. In several studies involving protein hydrolysates, some
researchers reported that the reducing capacity improved as the
degree of protein hydrolysis increased (Thiansilakul, Benjakul,
& Shahidi, 2007), while some researchers reported the opposite
(Klompong et al., 2007).

1050

A. Alemn et al. / Food Research International 44 (2011) 10441051

50

VCEAC (mg/g)

40

Savinase

Alcalase

c
30
20
10

b
a

0
Gelatin

Protamex

Trypsin

Neutrase

Esperase NS37005

Chelating ability (%)

B
e
100

80

60
40
20

bc

0
Gelatin

moles FeEq/g

Protamex

Trypsin

Neutrase

Esperase

NS37005

Savinase

Protamex

Trypsin

Neutrase

Esperase

NS37005

Savinase

Alcalase

20
15
10

5
0
Gelatin

Alcalase

Fig. 5. Antioxidant activity of gelatin hydrolysates at a concentration of 10 mg/mL. ABTS radical scavenging capacity (A), Fe chelating ability (B) and Fe reducing power (FRAP) (C).
Different letters (a, b, c,) indicate signicant differences (p 0.05).

Alcalase, Esperase, Savinase and Trypsin signicantly improved

the antioxidant activity of the resulting hydrolysates via the enhancement of antiradical activity and metal chelation. Reducing power
was also increased by the action of these enzymes, but the degree
of enhancement was much lower. Consistent with our results the
hydrolysis of snapper gelatin with proteases from pyloric caeca
caused a 5.5 to 6.5-fold increase in ABTS radical scavenging capacity,
whereas ferric reducing power did not increase more than 2-fold
(Khantaphant & Benjakul, 2008). Free radical quenching was reported
to be the main antioxidative mechanism of some peptides derived
from squid gelatin hydrolysates (Mendis, Rajapakse, & Kim, 2005).
4. Conclusions
The Esperase hydrolysate exerted a potent cytotoxic and antiproliferative effects on the two cancer cell lines studied, with
signicant antioxidant activity especially with respect to metal
chelating and antiradical activities. The Alcalase hydrolysate was the
most potent ACE-inhibitor and metal chelator, while at the same time
exerting strong radical scavenging and cytotoxic activity against
cancer cells. These results suggest that squid gelatin hydrolysates
prepared with Esperase or Alcalase enzymes, could be utilized as new
materials in the development of functional foods with potential
anticancer, antihypertensive and antioxidant capacities.
Acknowledgments
Funding for this research was provided by: (i) the Interministerial
Scientic and Technical Commission of Spain's Ministry of Science

and Innovation under project AGL2008-02135/ALI and (ii) Xunta de

Galicia, Program ngeles Alvario (European Social Fund).
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