LWT - Food Science and Technology 60 (2015) 881e889

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LWT - Food Science and Technology

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Chemical and biophysical properties of gelatins extracted from the

skin of octopus (Octopus vulgaris)
Mourad Jridi a, *, 1, Rim Nasri a, 1, Rabeb Ben Slama-Ben Salem a, Imen Lassoued a,
Ahmed Barkia a, Moncef Nasri a, Nabil Souissi b
a
Laboratoire de Genie Enzymatique et de Microbiologie, Universit e de Sfax, Ecole Nationale d'Ing
enieurs de Sfax, B.P. 1173, 3038 Sfax, Tunisia
b
Laboratoire de Biodiversit
e et Biotechnologie Marine, Institut National des Sciences et technologies de la Mer. Centre de Sfax, B.P. 1035, 3018 Sfax, Tunisia

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

Article history: Gelatins from alkali-pre-treated skin of Octopus (Octopus vulgaris) were extracted with different con-
Received 11 March 2014 centrations of pepsin at pH 2.0. The resulting octopus skin gelatins OSG0, OSG5, OSG10 and OSG15,
Received in revised form extracted, respectively, without enzyme treatment or with 5, 10 and 15 U of pepsin/g alkaline treated
10 July 2014
skin were evaluated for gel strength, textural parameters, thermal and gelling properties. The yield of
Accepted 23 October 2014
Available online 31 October 2014
gelatin extracted without enzymatic pretreatment was only 3.21% and the addition of pepsin (15 U/g)
increased the yield of gelatin extraction to 7.78%. Molecular weight distribution of gelatins indicates that
OSG10 and OSG15 contain peptides with molecular weights less than 10 kDa (>40%). In addition, Fourier
Keywords:
Octopus vulgaris
transform infrared (FTIR) spectra of extracted gelatins were slightly different, indicating that the triple
Gelatin helical structure of gelatins was affected by pepsin treatment. Compared to OSG0, pepsin gelatins
Gel strength exhibited lower gel strength, hardness, adhesiveness, transition, gelling and melting temperatures and
Pepsin treatment all of the values decreased with increasing enzyme concentration. In addition foam expansion (FE) was
Fourier transform infrared affected by enzyme treatment, and the values decreased as pepsin increased. However, the emulsifying
activity index (EAI) values of all gelatins were the same. Further, FE and EAI increased with increasing
concentrations (1e3%, w/v). The results showed that octopus skin can be a good source for gelatin.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction (Uriarte-Montoya et al., 2011), splendid squid (Nagarajan, Benjakul,

Gelatin is an important functional biopolymer widely used in
food, pharmaceutical and cosmetic industries as stabilizing, thick-
ening and gelling agent (Boran, Mulvaney, & Regenstein, 2010;
Go mez-Guille n, Gime nez, Lo pez-Caballero, & Montero, 2011;
Kittiphattanabawon, Benjakul, Visessanguan, & Shahidi, 2010).
Gelatin is commercially made from the skin or bone of porcine and
bovine by alkaline or acidic treatment (Go  mez-Guille n et al. 2002).
Waste from fish processing are generally considered as low-
value resources with negligible market value. It has been re-
ported that about 30% of such wastes comprise of bone and skin are
very rich in collagen, the precursor of gelatin (Go  mez-Guille n et al.
2002). In this context, fish gelatin has gained increasing interest as
the potential alternative of animal gelatins (Kittiphattanabawon
et al. 2010). Several studies reported the extraction and character-
ization of skin gelatins from several fish species such as silver carp
(Boran et al., 2010), grey triggerfish (Jellouli et al., 2011), giant squid
* Corresponding author. Tel.: þ216 28 142 818; fax: þ216 74 275 595.
E-mail address: jridimourad@gmail.com (M. Jridi).
1
Authors with equal contributions of the first.
Prodpran, Songtipya, & Kishimura, 2012), unicorn leatherjacket
(Ahmad & Benjakul, 2011; Kaewruang, Benjakul, & Prodpran,
2013), cuttlefish (Jridi et al., 2013), zebra blenny (Ktari et al.,
2014) and thornback ray (Lassoued et al., 2014).
The quality of fish gelatin is largely determined by its gelling
strength, rheological properties and thermal stability. The amino
acid composition of gelatin is indeed species-specific, but the mo-
lecular weight distribution mainly depends on the extraction pro-
cess (Aewsiri, Benjakul, & Visessanguan, 2009; Boran et al., 2010;
Go mez-Guillen et al. 2002; Nagarajan et al., 2012). Nalinanon,
Benjakul, Visessanguan, and Kishimura (2008) and Jridi et al.
(2013) demonstrated that enzymatic extraction increases the
yield of gelatin extraction. Furthermore, pepsin has been reported
to solubilize the native collagen in the skin matrix during the acid-
swelling process, by cleaving some peptide bonds, resulting in a
higher content of peptides with lower molecular weight.
Octopus (Octopus vulgaris) has been used for industry trans-
formation in Tunisia and the skin, generated as by-products with a
low market value, could be a source of many value-added products,
especially gelatin. So far no gelatin has been extracted from the skin
of this species and no information on its properties is available.

http://dx.doi.org/10.1016/j.lwt.2014.10.057
0023-6438/© 2014 Elsevier Ltd. All rights reserved.
882 M. Jridi et al. / LWT - Food Science and Technology 60 (2015) 881e889
Therefore, the objective of this work was to extract gelatin from
skin of Octopus (O. vulgaris) using different concentrations of
pepsin, and to study their physicochemical characteristics and
functional properties.
2. Materials and methods
2.1. Chemicals
Bovine hemoglobin, trichloroacetic acid (TCA), glycine and
ammonium sulphate were purchased from Sigma Chemical Co. (St.
Louis MO, USA). Sodium dodecyl sulphate (SDS), acrylamide,
ammonium persulphate, N,N,N0 ,N0 -tetramethyl ethylene diamine
(TEMED), Coomassie Brilliant Blue R-250 were from Bio-Rad Lab-
oratories (Hercules, CA, USA). Porcine pepsin (Lyophilized powder,
3000 units/mg protein) was purchased from MP Biomedicals
(France). Other chemicals and reagents used were of analytical
grade.
2.2. Preparation of octopus skin
Skin from Octopus (O. vulgaris) was obtained from CALEMBO
Company, Sfax City Tunisia. The samples were placed in ice with a
skin/ice ratio of approximately 1:3 (w/w) and transported to the
laboratory within 20 min. Upon arrival, skins were immediately
washed with cold tap water. Cleaned skins were then placed in
polyethylene bags and stored at 20  C until use. The storage time
was less than one week.
2.3. Extraction of gelatin
Gelatins were extracted from the prepared Octopus skin ac-
cording to the method of Jridi et al. (2013). Frozen skins were cut
into small pieces (1 cm  1 cm) and then mixed with 0.05 M NaOH
for 1 h at 4  C with continuous stirring at a sample/alkaline solution
ratio of 1:5 (w/v), to remove non-collagenous proteins, solution
was changed every 20 min. The alkaline-treated skins were then
washed with cold distilled water until the neutral pH of wash water
was obtained. The alkaline-treated and washed skins were then
soaked in 100 mM glycine-HCl buffer (pH 2), with a solid/solvent
ratio of 1:5 (w/v) for 10 h at 4  C with continuous stirring in the
absence or presence of pepsin at different levels 0, 5, 10 and 15
units/g of alkaline treated skin. The pH of the mixtures was then
raised to 7.0 using 10 M NaOH, and stirred gently for 1 h at 4  C. To
extract the gelatin, treated skin mixtures were then incubated at
40  C for 4 h with continuous stirring. The mixtures were centri-
fuged at 10,000  g for 30 min at 4  C, using a refrigerated
centrifuge to remove insoluble material. Finally, the supernatants
were freeze-dried (Moduloyd Freeze dryer, Thermo Fisher, USA).
Gelatins obtained using different levels of pepsin 0, 5, 10, and 15 U/g
of skin were referred to as OSG0, OSG5, OSG10 and OSG15,
respectively.
2.4. Analysis of gelatin
2.4.1. Calculation of gelatin yield and proximate analysis
The gelatin yield was calculated as follows:
Yieldð%Þ ¼ weight of dried gelatinðgÞ
 100=weight of dry matter of Octopus skinðgÞ:
Gelatins were subjected to proximate analysis, including mois-
ture, protein, lipid and ash contents, according to the AOAC (2000)
method No: 950.64, 928.08, 963.39 and 920.153, respectively.
2.4.2. Color evaluation
The color of gelatin samples was determined using a ColorFlex
spectrocolorimeter (Hunter Associates Laboratory, Inc., Reston, VA,
USA). The instrument was standardized using standard white
plates. CIE lightness (L*), redness (a*) and yellowness (b*) were
recorded. The sample was filled in a 64 mm glass sample cup with
three readings in the same place and triplicate determinations were
taken per sample.
2.4.3. SDS- polyacrylamide gel electrophoresis (SDS-PAGE)
Gelatins were analyzed by SDS-PAGE according to the method of
Laemmli (1970). Gelatin solutions were prepared by dissolving the
dried gelatins in distilled water for 30 min at 60  C at final con-
centration of 1 mg/ml, and then mixed with the loading buffer (2%
SDS, 5% b-mercaptoethanol, and 0.002% bromophenol blue) in a
proportion of 1:4 (v/v). Sample (30 mg of gelatin) was heat-
denatured for 5 min at 90  C and loaded onto an SDS-PAGE using
4% (w/v) stacking and 7% (w/v) resolving gels. After electrophoresis,
the gel was stained with 0.1% Coomassie brilliant blue R-250 dis-
solved in (15%) methanol and (5%) trichloroacetic acid, and finally
destained with methanol: distilled water: acetic acid, solution at a
ratio of 5:4:1. High molecular weight markers (GE Healthcare UK
Limited, Buckinghamshire, UK) were used to estimate the molec-
ular weight of gelatin samples.
2.4.4. Molecular weight analysis by FPLC
The molecular weight analysis of gelatin was carried out by Fast
Protein Liquid Chromatography (FPLC), using a Silica gel packed in a
TSKgel G2000SWXL column (7.8 mm I.D  30 cm L). The eluant
used was phosphate buffer (0.1 M) containing 0.2 M sodium chlo-
ride filtered through whatman cellulose acetate membrane
(0.2 mm). The flow rate was adjusted to 0.6 ml/min. Gelatin was
loaded to the column at a concentration of 6 mg/ml. Standard
molecular weight markers supplied by Sigma Chemicals (Bovine
serum albumin (66 kDa), b-amylase (200 kDa), apoferritin
(443 kDa) and Blue Dextran (2000 kDa)) were loaded separately at
a concentration of 4 mg/ml. A calibration curve was obtained by
plotting log molecular weight vs peak elution time. The average
molecular weight of gelatin was determined from the standard
curve.
2.4.5. Determination of amino acid composition
Gelatins were totally hydrolyzed with 6 N HCl (at final con-
centration of 5 mg/ml) at 110  C for 24 h on a heating block, and
then filtered through a 0.45 mm membrane filter prior to analysis.
10 mL of the treated sample was derivatized using 6-aminoquinolyl-
N-hydroxysuccinimidyl carbamate Waters AccQ Fluor Reagent Kit
(according to Waters AccQ Tag Chemistry Package Instruction
Manual).
The amino acid composition of the gelatins was analysed on a
Waters 2996 Separation Module (Photodiode Array Detector)
equipped with a Waters 2475 multi-wavelength fluorescence de-
tector and amino acids were separated on a Waters AccQ Tag amino
acid analyzing Column (Nova-Pak C18, 150  3.9 mm). The amount
of amino acids was calculated, based on the peak area in compar-
ison with that of amino acids standard (Accq tag chemistry kits
(WAT088122)). The amino acid content was expressed as amino
acid/1000 amino acids in the sample. All analyses were performed
in duplicate.
2.4.6. Determination of gel strength and textural parameter
analysis (TPA)
Gel strengths of Octopus skin gelatin gels were determined ac-
cording to the method of Go mez-Guillen et al. (2002), using 6.67%
gels (w/v) prepared by dissolving the dried gelatin in distilled water
 M. Jridi et al. / LWT - Food Science and Technology 60 (2015) 881e889
for 30 min at 60  C, using a water bath with occasional stirring
using a spatula. The samples were refrigerated at 7  C for 16e18 h,
for gel maturation. The Bloom of gelatin gels were determined at
7  C using a Model TA-TX2 texture analyzer with a 5 kN load cell
equipped with a 1.27 cm diameter flat-faced cylindrical Teflon
plunger. The dimensions of the sample were 3.8 cm in diameter and
2.7 cm in height. Gel strength was expressed as maximum force (in
grams), required for the plunger to press the gel by 4 mm depres-
sion at a rate of 0.5 mm/s. For, TPA analysis, gels prepared (3 cm in
diameter and 2 cm in length) were compressed twice at 0.5 mm/s
to 30% of their original height. The results are reported as the means
of triplicate tests. Textural parameters such as hardness, elasticity,
cohesiveness and chewiness were obtained from the TPA curve. The
measurement was performed in triplicate.
2.4.7. Viscoelastic properties
Dynamic studies were performed on an AR1000 Rheometer
(Physica MCR-301, Anton Paar, Germany) using a cone-plate ge-
ometry (cone angle 2 ). Gelatin solutions at 6.67% (w/v) were pre-
pared by dissolving dried protein in distilled water. The mixture was
then stirred at 45  C for 20 min. The viscosity measurement of
gelatin solutions were performed at a scan rate of 1  C/min, fre-
quency 1 Hz, oscillating applied stress of 3.0 Pa and gap 0.15 mm
(Montero, Ferna ndez-Díaz, & Go mez-Guille n, 2002). During the
testing, the gelatin solutions were firstly cooled from 50 to 5  C, kept
at 5  C for 10 min and then heated from 5 to 50  C. The tan d was
calculated from the ratio of G00 (loss modulus) and G0 (storage
modulus) obtained from frequency sweep tests. All the experiments
were performed in the linear viscoelastic region of gels. The phase
angle (d) values were represented as a function of temperature.
2.4.8. X-ray diffraction analysis
X-ray diffraction patterns of gelatins were obtained using a
SIEMENS D501 diffractometer equipped with a back mono-
chromator using the CuK radiation from a rotating anode generator
operated at 35 kV and 20 mA in the range of 2q ¼ 5e50 . The
scanning step was 0.02 while the scanning speed was 2 /min.
From equation d(Å) ¼ l/2sinq (l ¼ 1.54 Å), the minimum values (d)
of the repeat spacing were calculated.
2.4.9. Fourier transform infrared spectroscopy
Gelatins samples were subjected to FTIR analysis using a Nicolet
FTIR spectrometer equipped with an attenuated total reflection
(ATR) accessory. The horizontal attenuated total reflectance (HATR)
accessory was mounted into the sample compartment. The internal
reflection crystal made of zinc selenide, had a 45 angle of incidence
to the IR beam. Spectra were acquired at a resolution of 4 cm1 and
the measurement range was 4000e500 cm1 (mid-IR region) at
room temperature. Automatic signals were collected in 32 scans at a
resolution of 4 cm1 against a background spectrum recorded from
the clean, empty cell at 25  C. Analysis of spectral data was carried
out using the ORIGIN 8.0 data collection software programmer.
2.4.10. Emulsifying and foaming properties
The emulsion activity index (EAI), the emulsion stability index
(ESI), foam expansion (FE) and foam stability (FS) of gelatins at
different concentrations solutions (1%, 2% and 3% (w/v)) were
determined as previously reported (Jridi et al., 2013).
2.5. Statistical analysis
All analytical determinations were performed at least in tripli-
cate. Values were expressed as the mean ± standard deviation
(n ¼ 3). Analysis of variance was conducted, and differences be-
tween variables were tested for significance by one-way analysis of
883
variance using SPSS software, 17.0. A difference was considered
statistically significant when p < 0.05.
3. Results and discussion
3.1. Yield, proximate composition and color of gelatins
The gelatin extraction yields obtained with or without pepsin
treatment are shown in Table 1. The yield of gelatin extracted
without pepsin was about 3.21% (wet weight basis). The result was
similar to that obtained for cuttlefish (2.8%) (Jridi et al., 2013), but
lower than those of gelatins extracted from the skins of tilapia
(5.0%) (Jamilah & Harvinder, 2002), Red tilapia, Walking catfish and
Striped catfish (Jamilah, Tan, Umi Hartina, & Azizah, 2011).
It is interesting to note that the addition of pepsin, during
extraction process at pH 2.0, resulted in an increase of gelatin yield.
Furthermore, the yield of gelatin increased with increasing pepsin
concentration. The yields of gelatins OSG5, OSG10 and OSG15, were
5.45%, 6.87% and 7.78% (wet weight basis), respectively. The ob-
tained results are in line with those of Nalinanon et al. (2008) and
Lassoued et al. (2014), who reported an increase in the yield of
gelatins extracted from the skins of bigeye snapper and thornback
ray, respectively, when pepsin was added during the extraction
process. Pepsin has been reported to solubilize collagen in the
telopeptide region, by cleaving some peptide bonds resulting a
higher efficacy of gelatin extraction (Nalinanon et al., 2008).
The proximate composition of Octopus skin gelatins is shown in
Table 1. The extracted dried gelatins had high protein
(86.25e90.38%), low fat (0.78e1.23%) and ash (0.06e0.07%) con-
tents. Furthermore, the extracted gelatins had lower levels of
moisture content (5.8e10.42%) than the limit prescribed for edible
gelatin, i.e., 15 g/100 g (GME, 2008). The obtained results indicated
that there was an efficient removal of fat from the processed skin. In
addition, the relatively low ash content suggests that the extracted
gelatin are of good quality, considering that the maximum limit
recommended for gelatin is 2.5 g/100 g (Jones, 1977).
Color of gelatin from Octopus skin was expressed in terms of L*, a*
and b* (Table 2). The lightness and yellowness values of the Octopus
gelatins decrease with increasing pepsin concentration (p < 0.05). No
significant difference was observed in a* values. The decrease of L*
and b* values may be due to the increase in darkness caused by the
Maillard reaction of free amino acid released by pepsin in contact
with C]O component and this confirms the visual observations on
the gelatins. It can be concluded that factors such as molecular
weight distribution influence the color characteristics of extracted
gelatin. Gelatin extracted without pepsin was more yellowish than
gelatin extracted from cuttlefish skin (Jridi et al., 2013). Both color
and clarity of a gelatin gel are important aesthetic properties,
depending on the application for which the gelatin is intended.
Meanwhile, color did not affect functional properties of gelatin.
3.2. Electrophoretic analysis
The electrophoretic (SDS-PAGE) profiles of gelatins are shown in
Fig. 1. All gelatins displayed one b band and two a bands, which
were the characteristic polypeptide chains of gelatin. For OSG10
and OSG15, a decrease in a-band intensity and the apparition of
peptides with lower molecular weights (LMW) were observed
(Fig. 1). The apparition of peptides with LMW during gelatin
manufacturing process is attributed to the action of pepsin, which
undergo partial hydrolysis of inter-chain covalent crosslink and
intra-chain peptide linkages (Zhou, Mulvaney, & Regenstein, 2006).
The obtained results are in line with several works reported that
protein patterns of gelatins are influenced by the extraction con-
ditions, including pretreatment and extraction temperatures.
884 M. Jridi et al. / LWT - Food Science and Technology 60 (2015) 881e889

Table 1
Yield and proximate composition of gelatins extracted from Octopus skin at various pepsin concentrations.

Gelatin Yield* Moisture** Protein** Fat** Ash** pH***

Octopus skin e 79.26 ± 0.52 15.32 ± 0.21 3.50 ± 0.2 0.50 ± 0.05 e
OSG0 3.21 8.62 ± 0.62b 86.25 ± 0.24c 0.97 ± 0.10b 0.07 ± 0.01a 6.84 ± 0.01a
OSG5 5.45 10.42 ± 0.02a 87.68 ± 0.12b 0.97 ± 0.02b 0.07 ± 0.01a 6.57 ± 0.01b
OSG10 6.87 5.9 ± 0.01c 90.38 ± 0.4a 0.78 ± 0.01c 0.06 ± 0.00b 6.43 ± 0.02c
OSG15 7.78 5.78 ± 0.06d 89.32 ± 0.9a 1.23 ± 0.01a 0.069 ± 0.01a 6.21 ± 0.01d

OSG5, OSG10 and OSG15 denote gelatins extracted by 5, 10 and 15 U of pepsin/g of skin, respectively. OSG0: gelatin extracted without pepsin.
aed
Different letters in the same column indicate significant differences (p < 0.05).
*
g of gelatin per 100 g og octopus skin.
**
g per 100 g of gelatin.
***
1 g of gelatin per 100 ml of distilled water.

Nalinanon et al., (2008) reported that pepsin-extracted collagen consistent with that obtained by the SDS-PAGE which showed the
showed slightly lower molecular weight of a1 and a2 than did acid- decrease of the intensity of a-chain bands (Fig. 1). The content of
extracted collagen. Jridi et al. (2013), also reported that gelatin from polypeptide chains of gelatin obtained by treatment with 5 U of
cuttlefish skin extracted at high pepsin concentration contained pepsin is very close to that of gelatin control, suggesting that
more peptides with low molecular weights. treatment with low enzyme concentration did not highly affect the
structure of the extracted gelatin. However, no significant difference
3.3. Molecular weight distribution in the distribution peptide's molecular weight was found when
compared OSG10 and OSG15 (Table 3). The degradation of gelatin to
The molecular weight distribution profiles of the extracted gel- lower molecular weight fractions was reported to be responsible for
atins are shown in Fig. 2. MW of extracted gelatins was affected by low viscosity, low melting and setting points, high setting time, and
pepsin treatment, since there was a significant difference in MW low bloom strength of gelatin (Go mez-Guillen et al. 2002).
distribution between OSG0-OSG5 and OSG10-OSG15. In the case of
OSG0 and OSG5, the major MW fraction was located between 10 3.4. Amino acid composition of the extracted gelatins
and 120 kDa, which corresponds to the molecular weight of the a-
chain. On the other hand, the level of this fraction in OSG10-OSG15 The amino acid compositions of the extracted gelatins expressed
decreased with the increase of pepsin concentration, while the level as residues per 1000 total amino acid residues are shown in Table 4.
of molecules with molecular weight lower than 10 kDa increased, The amino acid composition of gelatins showed low content of
which might be resulted from the excessive cleavage of a1 and a2- methionine and tyrosine, and no content of cysteine, which is a
chain of gelatin by pepsin during gelatin preparation. This result is characteristic of all gelatins. Glycine (310e413 residues per 1000
residues) was the major amino acid in all the extracted gelatins. This
Table 2
result is in accordance with that of Jamilah et al., (2011) who re-
Color values of gelatin extracted from the skin of Octopus at various pepsin
concentrations.
ported that glycine, was the most dominant amino acids in gelatins
obtained from different fish species. In addition, all Octopus gelatins
Color OSG0 OSG5 OSG10 OSG15
possessed high content of His (164e183 residues/1000 residues).
L* 69.3 ± 0.60a 67.66 ± 0.20c 68.35 ± 0.26b 60.94 ± 0.06d The imino acids (proline and hydroxyproline) content of the control
a* 9.21 ± 0.10ab 9.04 ± 0.16b 9.24 ± 0.13ab 10.13 ± 1.07a gelatin was higher than those found in all gelatins extracted with
b* 11.62 ± 0.32a 10.09 ± 0.15b 10.23 ± 0.04b 7.63 ± 0.06c
pepsin (p > 0.05). It was noted that the imino acids content
OSG5, OSG10 and OSG15 denote gelatins extracted by 5, 10 and 15 U of pepsin/g of decreased as pepsin level increased. The numbers of imino acids
skin, respectively. OSG0: gelatin extracted without pepsin.
aed (proline and hydroxyproline) content, in OSG5, OSG10 and OSG15
Different letters within a line mean significant difference at p < 0.05.
were about 101, 50 and 29 residues per 1000 residues. Pepsin
preferentially cleaves peptide bonds after aromatic amino acids.
Because of the richness of native collagen telopeptide regions with
Tyr (Shoulders & Raines, 2009) and imino acids (Pro and Hyp),
planying a binding role in the crosslinks regions, the excessive
cleavage of those regions with pepsin may leads to the important
liberation of imino acids which may be eliminated during extraction
process. The imino acid content of octopus skin gelatin treated with
low concentration of pepsin (5 U/g) was similar to that of grey
triggerfish gelatin (Jellouli et al., 2011), but lower than those in giant
squid (Alema n, Gimenez, Montero, & Go mez-Guillen, 2011), bigeye
snapper and brownstripe red snapper skin gelatins (Jongjareonrak,
Benjakul, Visessanguan, & Tanaka, 2006). Hydroxyproline is
believed to play a key role in the stabilization of the triple-stranded
collagen helix due to its hydrogen bonding ability through its hy-
droxyl group (Go mez-Guille n et al., 2011; Karim & Bhat, 2009). In
general, the amino acid composition of pepsin gelatins is very close
to that obtained without pepsin treatment with minor exception.

3.5. Gel strength and textural parameter analysis

Fig. 1. SDS-PAGE pattern of gelatin from the skin of Octopus. OSG5, OSG10 and OSG15
denote gelatins extracted by 5, 10 and 15 U of pepsin/g of skin, respectively. OSG0: Bloom strengths of Octopus skin gelatin gels are reported in
gelatin extracted without pepsin. Table 5. Gelatin control exhibited higher bloom strength (283.6 g)
 M. Jridi et al. / LWT - Food Science and Technology 60 (2015) 881e889 885

Fig. 2. The molecular weight distribution profiles of gelatins from the skin of Octopus. OSG5, OSG10 and OSG15 denote gelatins extracted by 5, 10 and 15 U of pepsin/g of skin,
respectively. OSG0: gelatin extracted without pepsin.

than did pepsin gelatins, suggesting the difference in gel-forming
ability of the different extracted gelatins. According to Holzer
(1996), the gel strength of commercial gelatins, expressed as
bloom value, ranges from 100 to 300 g, but gelatins with bloom
values of 250e300 are most desired. Thus, it is interesting to note
the bloom strength of gelatin control was 283.6 g, which was
slightly higher than that of Bovin gelatin (260 g). Gel strength of
gelatin gels decreased significantly as the level of pepsin concen-
tration increased (p < 0.05). The bloom strengths of OSG5, OSG10
and OSG15 were 201.6, 142.32 and 62.44 g, respectively. Several
studies also reported that gelatins extracted with enzymatic
treatment exhibited lower bloom strength than control gelatin
(Jridi et al., 2013; Nalinanon et al., 2008).
The significant difference of gel strengths between gelatins
could be attributed to the differences in intrinsic characteristics,
such as the molecular weight distribution of gelatins as demon-
strated in Table 3. Indeed, hydrolysis of gelatin using high con-
Indeed, hydroxyl groups of imino acids, especially hydroxyproline,
play a part in inter-chain hydrogen binding via a bridging water
molecule as well as direct hydrogen binding to the carboxyl group.
The decrease in bloom strength as the level of pepsin concentration
increased was correlated with the decrease in imino acids content
(Table 4). Typically, fish gelatin had a gel strength ranging from as
low as zero to 426 g, compared to 200e300 g for bovine or porcine
gelatine (Karim & Bhat, 2009).
Textural parameter values of Octopus gelatins are illustrated in
Table 5. Gelatin with high molecular weight polymers showed,
hardness, adhesivity and cohesiveness, this is the case of OSG0.
However, gelatins, which contain shorter chains, such as OSG10 and
OSGG15, have lower textural parameters. Further, all textural pa-
rameters decreased with increasing pepsin concentration used
Table 4
Amino acid composition of OSGs (Number of residues/1000 residues).

centration of pepsin promotes cleavage of a-chain in the Amino acid OSG5 OSG10 OSG15
telopeptide region and generates shorter peptides. Therefore Hyp 45.2 25.9 16
gelatin with low molecular weight polymers showed lower gel Asx 35.1 31.8 29.3
strength. Badii and Howell (2006) also reported that gelatin with Ser 32.6 30.6 22.4
low molecular weight distribution had lower gelling properties Glx 54.3 48.9 45.2
Gly 310.2 347.4 412.6
compared to high molecular weight gelatin. In addition, the dif-
His 164.5 181.2 183.3
ferences among these gelatins in gel strength could be attributed to Arg 20 31 37.1
difference between Octopus gelatins in the imino acid (proline and Thr 34.2 45.3 34.2
hydroxyproline) contents, which could result in less organized Ala 51.6 52.1 37.2
Pro 56 27.8 13.2
triple helix structures. Both imino acids are responsible for the
Cys 0 0 0
stability the triple helix of gelatin structure (Karim & Bhat, 2009). Tyr 6.3 6.8 6.2
Val 39.3 32 28.4
Table 3 Met 6.6 5.4 5.8
Molecular weight distribution (FPLC) of gelatin extracted from the skin of Octopus Lys 14.3 11.2 13.2
using different pepsin concentration. Leu 30.8 35 30.6
Ileu 67.6 59.2 61.2
Molecular weight OSG0 OSG5 OSG10 OSG15
Phe 31.4 28.4 24.1
>200 kDa 5.9 3.7 1.78 1.09 TIA 101.2 53.7 29.2
120e200 kDa 20.1 18.2 16.2 15.9 THAA 593.5 587.3 613.1
10e120 kDa 70.18 72.8 41.97 42.48 TAA 1000 1000 1000
<10 kDa 3.82 5.3 40.05 40.53
The aspartic and glutamic acid contents include, respectively, asparagines and
OSG5, OSG10 and OSG15 denote gelatins extracted by 5, 10 and 15 U of pepsin/g of glutamine, Asx ¼ Asp þ Asn; Glx ¼ Glu þ Gln; TAA ¼ total amino acids; THAA ¼ total
skin, respectively. OSG0: gelatin extracted without pepsin. hydrophibic amino acids; TIA ¼ total imino acids.
886 M. Jridi et al. / LWT - Food Science and Technology 60 (2015) 881e889

Table 5
Viscoelastic properties, gel strength and textural properties of gelatins extracted from Octopus skin at various pepsin concentrations.

Gelatin Melting point ( C) Gelling point ( C) Gel strength (g) Textural properties

Hardness (g) Elasticity (mm) Adhesiveness (g) Cohesiveness index

OSG0 24.5 19.6 283.6 560 18.45 38.35 1.73

OSG5 22.4 17.5 201.6 497 17.62 25.75 0.89
OSG10 21.7 16.2 142.32 362 18.5 21.22 0.86
OSG15 19.4 13.7 62.44 220 17.93 19.36 0.66

OSG5, OSG10 and OSG15 denote gelatins extracted by 5, 10 and 15 U of pepsin/g of skin, respectively. OSG0: gelatin extracted without pepsin.

during the swelling process. The results are also in agreement with
those published by Go  mez-Guille
n et al. (2002), who reported that
a weak gelatin gel is associated with the formation of small frag-
ments. The results demonstrated that treatment with pepsin,
particularly with high level affected the bloom strength and the
textural properties of gelatins.
3.6. Melting and gelling temperatures
Evolution of the phase angle during cooling from 50  C to 5  C
and heating from 5  C to 50  C of 66.7 g/l gelatin solutions are re-
ported in Fig. 3. Gelling and melting points of gelatin solutions
(6.67% w/v) were measured through the programs of cooling and
heating. With further cooling of the sol, the formation of triple helix
starts. Gelling point is defined as a point where the viscosity begins
to increase sharply with decreasing temperature. During cooling
ramp, a large amount of helices are formed and network formation
occurred. This process is dominated by intermolecular interactions
(Zandi, Mirzadeh, & Mayer, 2007).
As shown in Table 5, melting and gelling temperatures of pepsin
gelatins are lower than those of control gelatin (OSG0); and values
decreased with increasing pepsin concentration. The solution of
OSG0 became gel at 19.6  C and subsequently melted at 24.5  C. The
melting and gelling points of OSG5 are 22.4  C, and 17.5  C,
respectively. However, gelatin extracted with 15 U of pepsin/g of
alkali pretreated skin melt at 19.4  C and became gel at 13.7  C.
OSG0 contained more content of a-chain and fewer amounts of low
molecular weight peptides, compared with pepsin gelatins. The
differences in gelling and melting temperatures of the extracted
gelatins could be attributed to pepsin treatment, which might
affect gelatin structure, especially triple helical structure. In this
context, it has been shown that the thermal stability of gelatin is
proportional to the number and stability of proline-rich regions in
collagen and gelatin molecules (Go  mez-Guillen et al. 2002).
Gelling and melting points of gelatin extracted with different
concentration of pepsin are in accordance with result obtained in
SDS-PAGE, molecular weight distribution and gel strength.

3.7. Fourier transform infrared (FTIR) spectra

FTIR spectroscopy has been used to study the changes in the

Fig. 3. Evolution of the phase angle during cooling from 50  C to 5  C (A) and heating
from 5  C to 50  C (B) of 66.7 g/l gelatin solutions. OSG5, OSG10 and OSG15 denote
gelatins extracted by 5, 10 and 15 U of pepsin/g of skin, respectively. OSG0: gelatin
extracted without pepsin.
functional groups and secondary structure of gelatins (Muyonga,
Cole, & Duodu, 2004). FTIR spectra of gelatins extracted from the
skin of Octopus in the absence or presence of pepsin at different
concentrations are reported in Fig. 4. Spectra of the different gel-
atins displayed the major bands in Amide region. OSG0 and OSG5
showed similar FTIR spectra suggesting that treatment with only
5 U of pepsin/g skin did not affect the secondary structure of gel-
atins. Some spectral differences on unique region of band stretch-
ing and amide regions were observed in the case of OSG10 and
OSG15. The spectral differences of the different Octopus gelatins
may be related to the different MW distribution.
The Amide-A bands appeared at wavenumbers of 3241.12,
3268.5 3274.1 and 3300.0 cm1 for OSG0, OSG5, OSG10 and OSG15,
respectively. At amide A region, spectra of OSG10 and OSG15 pre-
sent the lowest amplitudes when compared with other gelatins.
This result indicates that OSG10 and OSG15 contain more NeH
groups of shorter peptide fragments. The amide B bands of the
gelatins extracted without pepsin or with 5, 10, and 15 U of pepsin/g
of alkali pretreated skin, were observed at wavenumbers of 2966.2,
2959.5, 2946.39, and 2906.25 cm1, respectively. Lower wave-
numbers of amide B peak indicate higher interaction of NH3 group
between peptide chains in pepsin gelatin.
OSG0, OSG5 and OSG10 exhibited the amide I bands at wave-
numbers of 1628, 1627 and 1640 cm1, respectively. OSG10
 M. Jridi et al. / LWT - Food Science and Technology 60 (2015) 881e889
Fig. 4. Fourier transform infrared spectra of gelatin from the skin of Octopus. OSG5,
OSG10 and OSG15 denote gelatins extracted by 5, 10 and 15 U of pepsin/g of skin,
respectively. OSG0: gelatin extracted without pepsin.
exhibited the lowest amplitude of amide I compared to OSG0 and
OSG5 indicating the loss of triple helix due to excessive hydrolysis
of intermolecular cross-links of collagen by pepsin during the
extraction. In addition, lower amplitude of OSG10 indicates modi-
fication of the secondary structure, while aggregation might occur
to some extent during hydrolysis. Since, OSG10 and OSG15 con-
tained a higher amount of low molecular weight peptides due to
higher concentration of pepsin used during extraction, the reactive
groups (C]O) were more exposed and became more reactive be-
tween a-chains.
In amide II region, the wavenumber of bands increased with
increasing pepsin concentration. Muyonga et al., (2004) reported
that a disorder of molecular structure (increase of wavenumber of
bands in amide II region) due to the transformation of a a-helical to
a random coil structure obtained during enzymatic hydrolysis and
these changes were associated with loss of triple-helix state as a
result of denaturation of collagen to gelatin.
OSG0, OSG5 OSG10 and OSG15 exhibited the amide III bands at
the wavenumbers of 1233, 1232, 1234 and 1200 cm1, respectively.
In addition, OSG10 and OSG15 have high intensity peaks at
wavenumbers of 1092.15, 1010.36 and 942.72 cm1. These bands
were associated with the C]O stretching vibrations of the short
peptide chains (Kittiphattanabawon et al., 2010). The occurrence of
those peaks was coincidental with more degradation of peptide
chains in OSG15 sample (Fig. 1). In addition, the absorption bands in
OSG15 at wavenumbers of 715.26 cm1, most likely arose from
asymmetric stretching vibrations of phosphate groups of phos-
phorylated proteins coupled to ]CH2 of the amino acid residues
(Kittiphattanabawon et al., 2010). These changes were indicative of
a greater disorder from an helical to a random coil structure (Friess
& Lee, 1996) and were associated with loss of triple helix state as a
result of denaturation of collagen to gelatin (Muyonga et al., 2004).
Thus, it can be concluded that the secondary structure of gelatins
obtained from the skin of Octopus was affected by enzymatic
hydrolysis.
3.8. X-ray diffraction analysis
Collagen has a uniform triple-helical conformation with semi
crystalline structure, which is determined by its repeating (Gly-X-
Y)n sequence pattern and high imino acid content. Polypeptides
with these sequence features also adopt this conformation
(Brodsky, Eikenberry, Belbruno, & Sterling, 2004). Generally, X-ray
diffraction is used to investigate the collagen fibril distribution and
887
orientation (Bigi et al., 2001). Fig. 5 shows the X-ray spectra of
gelatin extracted without pepsin (OSG0) and that of gelatin
extracted using 15 U/g of pepsin (OSG15). As reported in Fig. 5,
there are two diffraction peaks in these X-ray spectra, which are in
accordance with the characteristic diffraction peaks of gelatin. For
the first peak, the diffraction angle values (2q) of OSG0 and OSG15
were about 12.5 and 9.82 and for the second peak of OSG0 and
OSG15 were 26.97 and 28.09 , respectively. The d values of the
OSG0 of the two peaks were 7.07 and 3.08 Å, and for OSG15 were
8.99 and 3.30 Å. Gioffre , Torricelli, Panzavolta, Rubini, and Bigi
(2012) reported that these peaks are related to the diameter of
the triple helix and their intensity would be associated with the
triple-helix content of the sample. Therefore, the decrease of peaks
intensity indicates that the triple-helix was reduced during
extraction.
3.9. Foaming properties
FE and FS values recorded for various concentrations of gelatins
from octopus skin are presented in Table 6. Control gelatin had a
slightly higher FE and FS values than gelatin extracted with pepsin
treatment. Further, FE and FS decreased with increasing pepsin
concentration, due to the apparition of smaller size peptides, which
are known to possess poor foam formation properties. This is in line
with previous findings reporting that good film cohesiveness is
reached with high molecular-weight peptides or partially hydro-
lyzed proteins (Kong, Zhou, & Qian, 2007).
The difference in foam properties of Octopus gelatins might be
influenced by intrinsic properties of protein, by the hydrophobic
amino acids (587e613 residues per 1000 residues), the conforma-
tions of the protein in solution and at the airewater interface
(Zayas, 1997). Kristinsson and Rasco (2000) stated that hydropho-
bicity of unfolded proteins has been shown to correlate with
foaming characteristics.
On the other hand, FE and FS values of all gelatins increased with
the increase of gelatin concentrations (p < 0.05). This is in line with
the work of Kaewruang et al. (2013) who reported that increasing
concentrations of gelatin extracted at different temperatures from
the skin of leatherjacket lead to the increase of FE and FS levels.
Further in our previous work (Jridi et al., 2013) we have observed
that FE and FS of cuttlefish skin gelatin increased with increasing
protein concentrations. Foams with higher proteins concentrations
were denser and more stable, presumably because of an increase in
the thickness of interfacial films (Zayas, 1997).
Fig. 5. X-ray spectra of the Octopus skin gelatins. OSG15 denote gelatin extracted by
15 U/g of pepsin/g of skin. OSG0: gelatin extracted without pepsin.
888 M. Jridi et al. / LWT - Food Science and Technology 60 (2015) 881e889

Table 6
Emulsifying and foaming properties of gelatins extracted from Octopus skin at various pepsin levels.

Ca FE FS EAI (m2/g) ESI (min)

15 min 30 min 45 min

OSG0 1 174 ± 1.32cA 35.0 ± 0.6cA 31.2 ± 0.62cA 31.5 ± 6.95cA 21.26 ± 1.3cA 25.21 ± 0.15aB
2 171.2 ± 0.8bA 48.6 ± 1.8bB 36.3 ± 1.2bA 34.0 ± 0.62bB 25.71 ± 0.16bA 21.62 ± 0.62bC
3 190.0 ± 1.2aA 76.8 ± 0.15aA 71.32 ± 2.6aA 52.0 ± 1.43aA 32.6 ± 1.60aA 15.32 ± 0.95cC
OSG5 1 163 ± 1.59cB 33.3 ± 1.2cA 26.3 ± 1.6cB 26.5 ± 0.45cB 20.29 ± 0.23cAB 25.32 ± 0.24aB
2 170 ± 1.68bA 48.9 ± 2.6bB 36.3 ± 1.0bA 33.3 ± 0.35bB 25.05 ± 0.68bA 20.01 ± 0.69bC
3 186 ± 2.02aB 73.3 ± 2.6aB 66.6 ± 2.34aA 43.6 ± 1.20aB 30.7 ± 0.70aA 15.23 ± 0.36cC
OSG10 1 143 ± 2.05cC 30.1 ± 0.98cB 20.0 ± 2.34cC 20.1 ± 1.68cC 22.21 ± 0.78cA 39.89 ± 0.41aA
2 160 ± 0.16bB 63.9 ± 1.97bA 33.3 ± 1.69bB 36.6 ± 1.00bA 23.78 ± 0.79bB 32.13 ± 0.34bA
3 169 ± 0.26aC 66.1 ± 1.96aC 53.9 ± 1.57aB 46.9 ± 2.0aB 31.02 ± 0.39aAA 26.21 ± 0.10cA
OSG15 1 140 ± 0.41cC 30.4 ± 1.97cB 26.9 ± 1.48cB 16.9 ± 1.20cD 21.93 ± 1.24cA 40.52 ± 0.35aA
2 143 ± 0.93bC 30.6 ± 1.47bC 33.4 ± 1.67bB 28.01 ± 2.36bC 25.05 ± 0.36bA 24.72 ± 0.14bB
3 159 ± 1.37aD 65.6 ± 6.32aC 50.2 ± 3.02aB 37.0 ± 2.64aC 29.79 ± 0.71aA 22.98 ± 0.36cB

OSG5, OSG10 and OSG15 denote gelatins extracted by 5, 10 and 15 U of pepsin/g of skin, respectively. OSG0: gelatin extracted without pepsin. aeb Different letters in the same
column within the same gelatin indicate significant differences (p < 0.05). AeD Different capital letters in the same column within the same concentration indicate significant
differences (p < 0.05).
a
Gelatin concentration g per 100 ml.

3.10. Emulsifying properties
The EAI and ESI of Octopus skin gelatins extracted at different
levels of pepsin in comparison with control gelatin are shown in
Table 6. At the same concentrations used, no differences in the EAI
values of the different gelatins were observed (p < 0.05). Further,
the EAI values of gelatins extracted with pepsin aided process,
increased with increasing concentrations (p < 0.05), suggested that
high level of polypeptide chains were adsorbed at the oil/water
interface.
The ESI values of gelatin control and OSG5 were lower than
those of OSG10 and OSG15. This suggests that emulsions of the
control gelatin and OSG5 were less stable than those of pepsin
gelatins. Low molecular weight peptides, generated upon pepsin
treatment, could stabilize the protein film at the interface more
effectively than high molecular weight peptides. Nevertheless, ESI
values of OSG significantly decreased with increasing concentration
(p < 0.05), which may be due to an increase in proteineprotein
interactions with increasing concentration, resulting in a lower
protein concentration at the oil-water interface (Lawal, 2004). At
low protein concentrations, protein adsorption at the interface is
diffusion-controlled. However, at high concentrations, the activa-
tion energy barrier does not allow protein migration to take place in
a diffusion-dependent manner, leading to the accumulation of
proteins in the aqueous phase (Kinsella, 1976).
However, a decrease in emulsifying ability with increasing
protein concentration was previously reported for gelatin from
zebra blenny skin (Ktari et al., 2014). Therefore, emulsifying sta-
bility of gelatins was governed by the molecular properties,
particularly the size of peptides and the concentration employed
(Kristinsson & Rasco, 2000). Mutilangi, Panyam, and Kilara (1996)
postulated that higher contents of higher molecular weight pep-
tides contribute to the stability of the emulsion.
4. Conclusion
Gelatins extraction from O. vulgaris alkali-pretreated skin was
improved by treatment with pepsin. Fourier transform infrared
(FTIR) and X ray diffraction analysis, indicates that the secondary
structure of Octopus gelatins was affected by enzymatic hydrolysis.
Further, gelling and melting points of gelatins extracted were found
to decrease with the increase of pepsin level. The functional
properties, including foam capacity, foam stability and gel strength
gelatin were affected by pepsin pretreatment. The differences in
functional and physical properties among all extracted gelatins
might be influenced by the intrinsic properties of gelatin amino
acids composition. Therefore, this study indicates that OSG could be
a useful as an additive in food materials with different properties
for different applications.
Acknowledgment
This work was funded by the Ministry of Higher Education and
Scientific Research, Tunisia.
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