Journal of Food Engineering 90 (2009) 480–486

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Journal of Food Engineering

journal homepage: www.elsevier.com/locate/jfoodeng

Physico-chemical and film-forming properties of bovine-hide and tuna-skin gelatin:

A comparative study
J. Gómez-Estaca, P. Montero, F. Fernández-Martín, M.C. Gómez-Guillén *
Instituto del Frío (CSIC), Meat and Fish Science and Technology, José Antonio Novais, 10, 28040 Madrid, Spain

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

Article history: A bovine-hide gelatin and a tuna-skin gelatin, both characterized on the basis of their amino acid com-
Received 9 May 2008 position and molecular weight distribution, were used to prepare edible films by casting with glycerol
Received in revised form 16 July 2008 and sorbitol added as plasticizers. The molecular weight distribution of the tuna-skin gelatin exhibited
Accepted 19 July 2008
appreciably higher quantities of b-components (covalently linked a-chain dimers), whereas bovine-hide
Available online 31 July 2008
gelatin showed a certain degradation of a1-chains being indicative of a greater proteolysis. Intrinsic dif-
ferences in the gelatin attributes affected in diverse manner some of the physical properties of the films.
Keywords:
Thus, water vapour permeability was higher in the bovine-hide gelatin film, whereas deformability was
Bovine-hide gelatin
Tuna-skin gelatin
considerably higher (10 times higher) in the tuna-skin gelatin film. In contrast, breaking force and water
Biodegradable films solubility were basically unaffected by gelatin origin. Analysis of the thermal properties revealed both
Physico-chemical properties films to be wholly amorphous with similar glass transition temperature values thanks to the plasticizing
effects of the glycerol and sorbitol and the low moisture contents.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction
Gelatin is a protein with a wide range of industrial applications
employed worldwide. It enhances the functional properties of food
products by improving their elasticity, consistency, and stability,
and it may also be used as an outer film to protect food against dry-
ing, light, and oxygen, especially in those cases where oxidative
and microbiological deterioration occurs (Arvanitoyannis, 2002).
Gelatin is obtained by hydrolyzing the collagen present in the
bones and skin generated as waste during animal slaughtering
and processing. Bovine and porcine wastes are the most frequent
sources to obtain gelatin of good quality. Other sources of gelatin
are becoming increasingly relevant, such as fish bones and skins.
The waste produced by fish filleting can account for as much as
75% of the total weight of catches (Shahidi, 1994), and further pro-
cessing to yield gelatin can help offset harmful environmental
effects.
The quality of a fish gelatin is determined mainly by its bloom
strength and heat stability (melting and gelling temperatures). Cer-
tain uses, however, do not require these physical attributes to be as
high as those of mammalian gelatins, e.g., encapsulation. For a long
time, marine gelatins have been shown to present inferior rheolog-
ical properties as compared to mammalian gelatins, which is espe-
cially true in the case of gelatins from cold-water fish species
(Leuenberger, 1991; Gudmundsson and Hafsteinsson, 1997; Haug
et al., 2004). Nevertheless, recent studies showed that certain fish
* Corresponding author. Tel.: +34 91 5445607; fax: +34 91 5493627.
E-mail address: cgomez@if.csic.es (M.C. Gómez-Guillén).
gelatins might have not superior but similar quality characteristics
compared to mammalian gelatin, depending on the species gelatin
extracted from and the processing conditions (Choi and Regen-
stein, 2000; Cho et al., 2005; Zhou et al., 2006; Yang et al., 2007).
The lower values for the physical properties of gelatins from
cold-water fish species have largely been related to the consider-
ably lower number of proline and hydroxyproline-rich regions in
the collagen molecule (Ledward, 1986). The different physical
properties of gelatins are related not only to the amino acid com-
position but also to the relative a-chain, b- or c-component, and
higher-molecular-weight aggregate contents and to the presence
of lower-molecular-weight protein fragments (Johnston-Banks,
1990). For this reason, the extraction procedure greatly influences
the properties of the resulting gelatin. More severe treatment con-
ditions are widely agreed to be detrimental to a gelatin’s physical
properties. Nevertheless, high heat is commonly used to increase
yields of commercial mammalian gelatins. In the case of fish gela-
tins, the normally lower degree of crosslinking in the native colla-
gen (Montero et al., 1990; Bateman et al., 1996) allows milder acid
and heat treatment conditions, thus yielding gelatin preparations
of reasonably high quality (Gómez-Guillén et al., 2002).
In view of the growing interest in biodegradable films, any con-
sideration of the quality of a gelatin should take into account not
only its gel-forming properties but also its film-forming ability,
along with the physical properties of the resulting films. Both the
intrinsic differences between mammalian and fish gelatins and
the different extraction conditions employed may influence the
properties and the potential applications of films made from a gi-
ven gelatin.

0260-8774/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2008.07.022
 J. Gómez-Estaca et al. / Journal of Food Engineering 90 (2009) 480–486
There has been a review on gelatin films (Arvanitoyannis, 2002),
and a considerable body of recently published work on the use of
gelatin to obtain edible films is available in the literature (Mene-
galli et al., 1999; Sobral et al., 2001; Simon-Lukasik and Ludescher,
2004; Bertan et al., 2005). However, the bulk of this information
concerns commercial mammalian gelatins. Although researchers
are now increasingly turning their attention to fish gelatin films
(Muyonga et al., 2004; Jongjareonrak et al., 2006a,b; Gómez-Guil-
lén et al., 2007; Carvalho et al., 2008), the list of literature refer-
ences dealing with these latter films is considerably shorter. The
present literature seems to bear out that there are some differences
in the physical properties of films obtained from mammalian and
fish gelatins, the former being stronger and more permeable to
water vapour and the latter more elastic (Sobral et al., 2001; Thom-
azine et al., 2005; Avena-Bustillos et al., 2006; Gómez-Guillén
et al., 2007) although it remains somewhat unclear. Comparability
of the data is limited because of the wide range of different exper-
imental conditions employed for film producing, i.e., plasticizer
type and concentration, dehydration temperature, film thickness
and conditioning, etc. In addition, the recent knowledge about
extraction and characterization of gelatin from many fish species,
may difficult generalizing about fish gelatin properties.
The purposes of this study were: (i) to characterize the physico-
chemical attributes of two different origin gelatins (one from tuna-
skin, the other from bovine-hide) on the basis of the more distinct
parameters: aminoacid composition and molecular weight distri-
bution; as well as thermal and rheological properties in the pres-
ence of plasticizers and (ii) to determine how these attributes
affected the properties of the resulting films, produced in identical
conditions to provide a proper comparison.
2. Materials and methods
2.1. Characterization of the gelatins
The tuna-skin gelatin was prepared in our laboratory according
to the method described by Gómez-Guillén and Montero (2001).
The bovine-hide gelatin (Bloom 200/220) was commercially ob-
tained from Sancho de Borja S.L. (Saragossa, Spain).
2.2. Amino acid composition
An amount of 1 mg/ml of dry gelatin was dissolved in distilled
water, and samples (50 ll) were dried and hydrolyzed in vacuum-
sealed glass tubes at 108 °C for 18 h in the presence of continu-
ously boiling 5.7 N HCl containing 0.1% phenol with norleucine
as internal standard. After hydrolysis, samples were vacuum-dried,
dissolved in application buffer, and injected onto a Biochrom 20
amino acid analyser (Pharmacia, Barcelona, Spain). DL-5-Hydroxy-
lysine hydrochloride and cis-4-hydroxy-D-proline (Sigma, St. Louis,
MO, USA) were also used as standards to determine the amount of
hydroxylysine and hydroxyproline, respectively.
2.3. Molecular weight profile
The molecular weight distribution of the bovine-hide and tuna-
skin gelatins was determined by SDS–polyacrylamide gel electro-
phoresis. Gelatin (5 mg/ml) solutions were mixed with loading
buffer (2% SDS, 5% mercaptoethanol, and 0.002% bromophenol
blue) in a proportion of 1:4. Samples were heat-denatured at 90
°C for 5 min and analysed according to Laemmli (1970) in a Mini
Protean II unit (Bio-Rad Laboratories, Hercules, CA, USA) using 4%
stacking gels and 6% resolving gels at 25 mA/gel. Loading volume
was 15 ll in all lanes. Protein bands were stained with Coomassie
brilliant blue R-250. Type I collagen from foetal calf was used as a
481
marker for a-chain and b-component mobilities. Also a molecular
weight standard composed of myosin (212 kDa), a2-macroglobulin
(170 kDa), b-galactosidase (116 kDa), transferrin (76 kDa) and glu-
tamic dehydrogenase (53 kDa) (Amersham Pharmacia biotech,
Buckinghamshire, UK) was employed.
2.4. Film-forming solution and film preparation
The film-forming solutions were prepared using gelatin at a
concentration of 4 g/100 ml of distilled water. Sorbitol (0.15 g/g
gelatin) and glycerol (0.15 g/g gelatin) were employed as plasticiz-
ers. The mixtures were warmed and stirred at 40 °C for 15 min to
obtain a good blend, and the films were made by casting an
amount of 40 ml in square dishes (12  12 cm) and drying in a con-
vection oven at 45 °C for 15 h. Prior to determinations the films
were conditioned over a saturated solution of NaBr (58% relative
humidity) at 22 °C in desiccators for 2 d. The moisture content of
films, determined by AOAC method 24003 (AOAC, 1984) after the
conditioning period, was 10.23% (±0.30) for the bovine-hide gelatin
films and 13.46% (±0.34) for the tuna gelatin films. Film thickness
was measured using a digital micrometer (Mitutoyo, model
MDC-25M, Kanagawa, Japan), averaging nine different locations.
2.5. Characterization of the film-forming solutions
2.5.1. Dynamic viscoelastic properties
Dynamic viscoelastic analysis of the film-forming solutions was
carried out as described in Gómez-Guillén et al. (2007), on a Bohlin
CSR-10 rheometer rotary viscometer (Bohlin Instruments Ltd.,
Gloucestershire, UK) using a cone-plate geometry (cone angle =
4°, gap = 0.15 mm). Cooling and heating from 40 to 6 °C and back
to 40 °C took place at a scan rate of 1 °C/min, frequency of 1 Hz, and
a target strain of 0.2 mm. The elastic modulus (G0 ; Pa), viscous
modulus (G00 ; Pa) and phase angle (°) were determined as functions
of temperature. Several determinations were performed for each
sample, with an experimental error of less than 6% in all cases.
2.5.2. Gel strength
The film-forming solutions were poured into glasses 2.3 cm in
diameter and 3.6 cm in height and left to mature in a refrigerator
at 2 °C for 16–18 h. Gel strength at 8–9 °C was determined, as de-
scribed in Gómez-Guillén et al. (2002), on an Instron model 4501
Universal Testing Machine (Instron Co., Canton, MA, USA) with a
100-N load cell, a cross-head speed of 60 mm/min, and a flat-faced
cylindrical plunger 1.27 cm in diameter. The maximum force (g)
was determined when the plunger had penetrated 4 mm into the
gelatin gels.
2.6. Characterization of the films
2.6.1. Mechanical properties
A puncture test was performed to determine the breaking force
and deformation of films at the breaking point (Gómez-Guillén
et al. (2007). Films were placed in a cell 5.6 cm in diameter and
perforated to the breaking point using an Instron model 4501 Uni-
versal Testing Machine (Instron Co., Canton, MA, USA) with a
round-ended stainless-steel plunger (3 mm in diameter) at a
cross-head speed of 60 mm/min and a 100-N load-cell. Breaking
force was expressed in N and breaking deformation in %, according
to Sobral et al. (2001). All determinations are the means of at least
five measurements.
2.6.2. Thermal properties
Calorimetric analysis was performed with a model TA-Q1000
differential scanning calorimeter (DSC) (TA Instruments, New Cas-
tle, DE, USA) previously calibrated by running high purity indium
482 J. Gómez-Estaca et al. / Journal of Food Engineering 90 (2009) 480–486

(melting point 156.4 °C; enthalpy of melting 28.44 W/g). Samples Table 1
of approximately 10 mg (±0.002 mg) were weighed out using a Amino acid composition of the bovine-hide and the tuna-skin gelatines

model ME235S electronic balance (Sartorious, Goettingen, Ger- Number of residues/1000

many) and were tightly encapsulated in aluminium pans and Bovine-hide Tuna-skin
scanned under dry nitrogen purge (50 ml/min). Freshly condi-
Hyp 83 78
tioned films were rapidly cooled to 0 °C and scanned at between Asp 46 44
0 and 90 °C at a heating rate of 10 °C/min. Glass transition temper- Thr 33 21
atures, Tg (°C), were determined only on the first heating scans, val- Ser 39 48
ues obtained on the second scans being deemed not relevant Glu 74 71
Pro 127 107
because of the practical impossibility of reproducing the original
Gly 342 336
film conditioning. The glass transition temperature was estimated Ala 113 119
as the midpoint of the line drawn between the temperature at the Val 19 28
intersection of the initial tangent with the tangent through the Met 4 16
Ile 11 7
inflection point of the trace and the temperature of the intersection
Leu 24 21
of the tangent through the inflection point with the final tangent. Tyr 4 3
Tg data have been reported as the mean values of at least duplicate Phe 12 13
samples for each film, usually within ±1 °C. His 4 7
Lys 25 25
Arg 47 52
2.6.3. Water solubility
Hyl 5 6
Film portions measuring 4 cm2 were placed in aluminium cap-
sules with 15 ml of distilled water and shaken gently at 22 °C for
15 h. The solution was then filtered through Whatman no. 1 filter
paper to recover the remaining undissolved film, which was desic- and this promotes triple helix formation and stabilization of the
cated at 105 °C for 24 h. Film solubility was calculated by the equa- gelatin at low temperatures (Burjandze, 1979) thanks to the hydro-
tion FS (%) = ((W0  Wf)/W0)  100, where W0 was the initial weight gen bonding ability of the –OH group on the hydroxyproline. In the
of the film expressed as dry matter and Wf was the weight of the present work the Hyp content of the tuna-skin gelatin was rela-
undissolved desiccated film residue. All tests were carried out in tively high (78 residues/1000) although lower than the one for bo-
triplicate. vine-hide gelatin (83 residues/1000), due to the fact that this fish
species do not come from very cold waters. Similarly, a relatively
2.6.4. Water vapour permeability high content of hydroxyproline (74 residues/1000) has been re-
Water vapour permeability was determined following the meth- cently reported in gelatin extracted from tuna fin (Aewsiri et al.,
od described by Sobral et al. (2001). Films were attached over the 2008). Gelatins from warm-water fishes have a higher imino acid
openings of cells (permeation area = 15.9 cm2) containing silica content than gelatins from cold-water fish species, closer to mam-
gel, and the cells were placed in desiccators with distilled water at malian ones (Gilseman and Ross-Murphy, 2000a; Avena-Bustillos
22 °C. The cells were weighed daily for 7 d. Water vapour permeabil- et al., 2006). With respect to the hydrophobic nature of our gela-
ity was calculated using the equation WVP = w  x  t1  A1  DP1, tins, both of them presented a similar content of hydrophobic res-
where w was weight gain (g), x film thickness (mm), t elapsed time idues of around 310 per 1000 residues. This is again attributed to
for the weight gain (h), and DP the partial vapour pressure difference the relatively warm water in which tuna lives. Cold-water fish gel-
between the dry atmosphere and pure water (2642 Pa at 22 °C). Re- atins are typically more hydrophobic.
sults have been expressed as g mm h1 cm2 Pa1. All tests were car- The electrophoretic profile of the bovine-hide gelatin was con-
ried out in duplicate. siderably more heterogeneous than that of the tuna-skin gelatin,
thereby causing bands on the SDS gel to be more diffuse (Fig. 1).
2.7. Statistical analysis This is indicative of a broad range of molecular weights, probably
attributable to the normal use of more severe extraction conditions
Statistical tests were performed using the SPSSÒ computer pro- than for the fish gelatin, for which extraction was carried out at
gram (SPSS Statistical Software Inc., Chicago, IL, USA). One-way temperatures below 45 °C. Both types of gelatin contained a-
analysis of variance was carried out. Differences between pairs of chains weighing around 100 kDa and b-components weighing
means were assessed on the basis of confidence intervals using around 200 kDa, typical of type I collagen. Nevertheless, there were
the Tukey-b test. The level of significance was p 6 0.05. appreciable differences in a-chain yields during extraction. The
fish gelatin exhibited a a1/ a2 ratio of around 2, indicating that
the native structure is maintained. In contrast, intensities for the
3. Results and discussion bands for the a1 and a2 chains in the mammalian gelatin were sim-
ilar, indicating possible degradation of the a1-chains during the
3.1. Characterization of the gelatins extraction process. This is an interesting factor to bear in mind,
since, as reported by Sims et al. (1997), a1-chains have greater gel-
Table 1 presents the amino acid composition of the gelatins. ling ability than a2-chains. The amount of b-components was also
Both the bovine-hide and the tuna-skin gelatins exhibited typical considerably higher in the fish gelatin, suggesting that the milder
type I collagen Gly content, representing approximately 1/3 of extraction conditions were more conducive to extracting intact,
the total amino acids. As described by Asghar and Henrickson covalently linked a-chain dimers.
(1982), 50–60% of a-chains consist of tripeptides having the gen- Both gelatins had appreciable amounts of higher-molecular-
eral formula Gly-X-Y, where X is generally proline and Y is mainly weight (>200 kDa) fractions of several crosslinked a-chains, but
hydroxyproline. The proline plus hydroxyproline (imino acids) the bands were more distinct in the case of the fish gelatin, indicat-
content was higher in the bovine-hide gelatin than in the tuna-skin ing less dispersal of molecular weights in the gelatin. Very high-
one (210 vs. 185 residues/1000, respectively). Gelatins made from molecular-weight polymers resulting from residual heat-stable
warm-blooded animal tissues have been reported to have a higher crosslinks were also present in both types of gelatin, as revealed
imino acid content, hydroxyproline in particular (Norland, 1990), by the high-intensity bands at the tops of the polyacrylamide gels.
 J. Gómez-Estaca et al. / Journal of Food Engineering 90 (2009) 480–486 483

The <100 kDa polypeptide fraction appeared to be more abundant

in the bovine-hide gelatin, again largely attributable to more
extensive cleavage of the -chain peptide bonds during gelatin
HMW-a extraction. Moreover, the presence of a high-intensity band at
the bottom of the resolving gel in the mammalian gelatin indicated
the presence of extensively hydrolyzed fractions that did not ap-
pear in the fish gelatin.

β 3.2. Characterization of the film-forming solutions

α1 Fig. 2 shows the changes in the viscoelastic properties of the

α2 film-forming solutions during the cooling and subsequent heating
ramps. The bovine-hide gelatin showed higher elastic modulus (G0 )
values at low temperatures, indicative of enhanced ability to refold
into a triple helix (Ledward, 1986; Gómez-Guillén et al., 2002). The
phase angle exhibited higher thermal transition points in the bo-
vine-hide gelatin on both, the cooling and heating ramps, an indi-
cation that it was more heat stable. The average gelling
temperature and melting temperature for the bovine-hide gelatin
Tuna-skin were, respectively, 20 °C and 30 °C, whereas for the tuna-skin gel-
Bovine-hide Tuna-skin atin were 15 °C and 22.5 °C. These higher rheological properties
and thermostability are typical of mammalian gelatins (Leuenber-
Fig. 1. Electrophoretic profile of the bovine-hide and tuna-skin gelatins. HMW-a, ger, 1991; Gilseman and Ross-Murphy, 2000b) and are mainly re-
high-molecular-weight aggregates. lated to imino acid composition, hydroxyproline playing a

1750 a b
1500
1250
G' (Pa)

1000
750
500
250
0
25 20 15 10 5 5 10 15 20 25 30 35 40
Temperature (ºC) Temperature (ºC)

60
50
a b
40
G'' (Pa)

30
20
10
0
25 20 15 10 5 5 10 15 20 25 30 35 40
Temperature (ºC) Temperature (ºC)

90
80 a b
Phase angle (º)

70
60
50
40
30
20
10
0
25 20 15 10 5 5 10 15 20 25 30 35 40
Temperature (ºC) Temperature (ºC)

Bovine-hide Tuna-skin

Fig. 2. Changes in the viscoelastic properties (G0 , modulus of elasticity; G00 , modulus of viscosity; °, phase angle) of the film-forming solutions of bovine-hide and tuna-skin
gelatin during the cooling (a) and subsequent heating (b) ramps.
484 J. Gómez-Estaca et al. / Journal of Food Engineering 90 (2009) 480–486
singular role in stabilizing the triple-stranded helix thanks to the
hydrogen bonding ability of its –OH group (Burjandze, 1979). As
mentioned above, the total imino acid content was higher in the
bovine-hide gelatin.
As expected, gel strength was significantly (p 6 0.05) lower in
the tuna-skin gelatin (167 ± 4 g) than in the bovine-hide gelatin
(211 ± 6 g). However, the difference was less pronounced than
the difference observed in the elastic modulus. Triple helix forma-
tion takes place by association of the different a-chains during cold
maturation of the gel. This gives rise to differences according to
gelatin characteristics suggesting that the nucleation points ini-
tially formed in the tuna-skin gelatin are capable of considerable
growth during maturation under refrigeration. A possible explana-
tion for this is the molecular weight distribution of the tuna-skin
gelatin employed, with higher amount of native structures -mono-
mers and dimmers-. The tuna-skin gelatin had considerably higher
amounts of a1-chains and b components than the bovine-hide gel-
atin did, and according to Sims et al. (1997) this could be respon-
sible for its relatively high gel strength. This molecular weight
distribution, in which the two a-chains comprising the b-compo-
nent are already crosslinked, enables the chains to form triple heli-
ces upon cooling and contributes to helix growth during gel
maturation. The presence of glycerol and sorbitol in the film-form-
ing solutions, which were added as plasticizers in a relatively high
concentration (30 g/100 g gelatin) should be also taken into con-
sideration. Glycerol has been reported to exert a considerable gel
strength-enhancing effect in fish gelatin gels, largely attributed
to hydrogen bonding stabilization (Fernández-Díaz et al., 2001);
however, it is worth noting that in the study reported by those
authors, the glycerol concentration was approximately 7.5-fold
higher than in the present one.
3.3. Characterization of the films
Both the bovine-hide and the tuna-skin gelatin-based film-
forming solutions yielded flexible and transparent films, similar
(p 6 0.05) in thickness (109 ± 18 lm for the bovine-hide films
and 98 ± 15 for the tuna-skin films). The data from the physical
properties analysis are set out in Table 2. Both films presented a
similar (p 6 0.05) maximum load value at the breaking point
(10.5 N and 8.5 N for bovine-hide and tuna-skin, respectively).
On the contrary, the tuna-skin gelatin film had breaking deforma-
tion values around 10 times higher than the values for the bovine-
hide gelatin film. Data from bibliography are not easily comparable
because of the differences in film obtaining, type and concentration
of plasticizers, gelatin type, measurement methodology, etc. Hav-
ing this in mind there seems to be a trend to obtain stronger films
from mammalian gelatins whereas fish gelatins render more
deformable films (Sobral et al., 2001; Thomazine et al., 2005; Ave-
na-Bustillos et al., 2006; Gómez-Guillén et al., 2007; Pérez-Mateos
et al., 2009).
In the present work both films were elaborated with the same
type and concentration of plasticizers, so the differences in the
Table 2
Physical properties (puncture force and deformation at the break point, water vapour
permeability (WVP) and water solubility) of the bovine-hide and the tuna-skin
gelatine films
Bovine-hide Tuna-skin
Puncture force(N) 10.7 ± 2.2a 8.5 ± 1.6a
Puncture deformation(%) 14.1 ± 5a 154 ± 36b
WVP(108 g mm h1 cm2 Pa1) 2.20 ± 0.11a 1.65 ± 0.39b
Water solubility(%) 34.3 ± 0.6a 39.9 ± 1.3a
Tg (°C) 41.5 ± 1a 40.7 ± 1a
Results are expressed as mean value ± standard deviation. Different letters (a, b)
indicate significant differences.
mechanical properties should be linked to the physico-chemical
characteristics of the gelatin, especially regarding the aminoacid
composition, which is greatly a species-specific characteristic,
and the molecular weight distribution, which is mostly determined
by processing conditions. Regarding the amino acid composition,
mammal gelatins are well known to be richer in imino acids (Nor-
land, 1990). As discussed above, the higher imino acid content
(Pro + Hyp) strongly determine the superior rheological properties
and thermostability of bovine-hide gelatin gels. This is largely
attributable to the formation of junction zones by hydrogen bond-
ing, which in turn yield a multitude of intra- and inter-chain inter-
actions important to cold stabilization of the gel network. These
interactions are promoted by the availability of more -OH groups
to form hydrogen bonds with bridging water molecules inside
the triple helices and also between the chains of neighbouring tri-
ple helices (Asghar and Henrickson, 1982). Thus, water plays an
important role in augmenting the stability of the triple helical
structure. According to Djabourov et al. (1985), the structural
water fraction increases during cold gelation and is proportional
to helix formation. Water molecules are taken into the structure
of the triple helix and are hydrogen bonded to the -CO or -NH
groups. However, presumably the involvement of hydrogen bonds
is lower in dried films, in which most of the water has been re-
moved and the storage temperature is around 20 °C, which may
act to cause the differences in imino acid content between the
two gelatins to be less appreciable. In this connection, the mechan-
ical properties of gelatin films have been related to the triple helix
content of the gelatin employed (Bigi et al., 2004); however, it is
known to decrease upon isothermal dehydration of films (Wetzel
et al., 1987).
Regarding the molecular weight profile, it could be assumed that
the presumably more severe treatment conditions needed for the
commercial bovine-hide gelatin extraction led to a higher propor-
tion of protein fractions of low molecular weight (<100 kDa).
According to the works by Muyonga et al. (2004) and Carvalho
et al. (2008) this fact results in a reduction of film strength and an
increase in film extensibility. The role of gelatin molecular weight
distribution in the rheological properties of the films may be asso-
ciated to the presence and concentration of plasticizers in the for-
mulation, which in the present study was relatively high (30 g/
100 g gelatin). Thus, for the same mass concentration of plasticizer,
films made using a lower-molecular-weight biopolymer could be
more plasticized, because the molar plasticizer/biopolymer ratio
is higher (Thomazine et al., 2005). However, in our work both gela-
tins presented a considerable amount of high-molecular-weight
aggregates (Fig. 1) which would improve the film strength in both
cases. Moreover, the predominance of b-components in the fish gel-
atin could also act to noticeably strengthen the corresponding films,
despite the lower iminoacid content.
Regarding the differences in film deformation, which was 10-
fold higher for the tuna-skin films, data from literature show us
that fish gelatins produce more deformable films than mammal
ones (Sobral et al., 2001; Thomazine et al., 2005; Gómez-Guillén
et al., 2007; Pérez-Mateos et al., 2009). In the present work, this
finding is not possible to be explained in terms of a higher plasti-
cizing effect associated to the gelatin molecular weight distribu-
tion, since bovine-hide gelatin appeared to have a higher amount
of hydrolyzed peptide fractions which are prompt to interact with
plasticizer molecules. Thus, one possible explanation lies in the dif-
ferent imino acid content in both gelatins. The higher Pro + Hyp
content in the bovine-hide gelatin may impose conformational
constraints by imparting a higher degree of molecular rigidity to
the corresponding films, which would obviously lead to a lower
deformability.
Fig. 3 plots the DSC traces. Despite being encapsulated in non-
hermetically sealed pans, samples lost only 1–2% of their weight
 J. Gómez-Estaca et al. / Journal of Food Engineering 90 (2009) 480–486
after scanning, which indicated that the films had good water
retention capacity. This resulted in a certain bending of the trace
due to water vapourization, which somehow distorted the DSC
profiles at the glass transition point. On the other hand, glass tran-
sition seemed to overlap with an enthalpy relaxation phenomenon.
Both water vapourization and enthalpy relaxation made precise
measurement of the Tg values somewhat difficult. The films were
wholly amorphous, since they did not exhibit any melting event
in the temperature region that would otherwise be expected based
on the heat stability characteristics of the gelatins. The plasticizing
effects of the glycerol plus sorbitol (Sobral et al., 2001) and water
may inhibit gelatin crystallization. Additionally, moisture reduc-
tion up to the films typical levels (10–14% depending on the gela-
tin) caused the complex systems to move from regions of gel–sol–
gel transitions to vitreous domains. Tg values were similar in both
films, somewhat lower in the tuna-skin gelatin film, though the
difference was not significant (Table 2). Cold-water fish gelatins
have been reported (Gilseman and Ross-Murphy, 2000a) to be less
heat stable than mammalian gelatins, mostly due to the lower imi-
no acid content. In contrast, the properties of warm-water fish gel-
atins may be quite similar to those of mammalian gelatins
(Gilseman and Ross-Murphy, 2000b). Although tuna cannot be
considered a warm-water fish species it is not located at very
cold-water seas as for example cod or hake are.
Table 2 also lists the water vapour permeability (WVP) of the
films. The tuna-skin gelatin film was less permeable than the bo-
vine-hide gelatin film (p 6 0.05). One more time the explanation
could lie in the differences in the amino acid composition of the
two gelatins. Avena-Bustillos et al. (2006) observed that the hydro-
phobic nature of the amino acids of a gelatin highly determine the
water vapour permeability of the corresponding films, being the
fresh water fish species gelatin films the less permeable, followed
by the warm-water fish ones and finally the mammal gelatin films.
However in our work the differences recorded in water vapour per-
meability cannot be absolutely ascribed to amino acid composition
because the total amount of hydrophobic amino acids was quite
similar for both gelatins (310 for bovine-hide gelatin vs. 311 for
tuna-skin one). However Hyp, which is a hydrophilic amino acid,
was higher for the bovine-hide gelatin film, and this could have
contributed in part to its higher water vapour permeability. The
molecular weight distribution has also been thought to exert an ef-
fect. The bovine-hide gelatin presented a considerably higher
amount of protein fractions up to 100 kDa which could be more
susceptible to the plasticizing effects of glycerol and sorbitol than
a
Heat Flow (Endo Down)
b Tg
0.1 W/g
10 20 30 40 50 60 70
Temperature (ºC)
Fig. 3. DSC traces and Tg values for the bovine-hide (a) and tuna-skin (b) gelatin
films.
485
the bigger fragments. Both glycerol and sorbitol are well known to
cause an increase in the water vapour permeability of gelatin films
due to its hydrophilic –OH groups and this effect is directly related
to plasticizer concentration (Sobral et al., 2001; Jongjareonrak
et al., 2006a).
Contrary to expectation, the water solubility of the tuna-skin
gelatin film was not significantly different from that of the bo-
vine-hide gelatin film (Table 2). The bovine-hide gelatin may pre-
sumably present lower water solubility due to the straightening
effects of the hydrogen bonds promoted by imino acids. However,
as discussed for the mechanical properties, the lower water con-
tent seems to play a singular role reducing the straightening effects
of hydrogen bonding in the films. It seems to be more determinant
the nature and extent of the very high-molecular-weight fraction,
in which residual heat-stable crosslinks predominate. The electro-
phoretic analysis did not reveal any substantial differences in this
fraction in the two types of gelatin. The low extraction temperature
together with intrinsic attributes of the tuna-skin collagen used in
this study may help retain some heat-stable crosslinked fractions
in the resulting gelatin, leading to relatively low water solubility
of the film. Carvalho et al. (2008) elaborated films with two differ-
ently processed Atlantic halibut skin gelatins, which did not pres-
ent any fraction of very high-molecular-weight, and found them to
be totally soluble in water. Other study dealing with cod-skin gel-
atin film has yielded water solubility values as high as 90–100%
(Pérez-Mateos et al., 2009), compared with values of around 30%
for bovine gelatin film (Bertan et al., 2005). Unfortunately, these
works do report any data about neither the molecular weight dis-
tribution nor the amino acid profile of the gelatins.
4. Conclusions
As expected, the different origin and extraction process of the
bovine-hide and the tuna-skin gelatins influenced the physical
properties of the corresponding filmogenic solutions. However this
was not so evident in the films, presumably due to the lower
involvement of hydrogen bonding as a consequence of the reduc-
tion of the water content. The breaking force and the water solubil-
ity were scarcely affected by the gelatin origin (bovine-hide or
tuna-skin) and presented similar values in both films. On the con-
trary, the puncture deformation and the water vapour permeability
were quite different, being attributed to the amino acid profile and
the molecular weight distribution of the gelatins.
Acknowledgements
This research was financed by the ‘‘Ministerio de Educación y
Ciencia”, under Project AGL2005-02380.
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