Food Hydrocolloids 19 (2005) 941–950

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Use of lactic acid for extraction of fish skin gelatin

B. Giméneza, J. Turnayb, M.A. Lizarbeb, P. Monteroa, M.C. Gómez-Guilléna,*
a
Instituto del Frı́o (CSIC), Ciudad Universitaria, 28040 Madrid, Spain
b
Dpto. Bioquı́mica y Biologı́a Molecular I, Facultad de Ciencias Quı́micas, Universidad Complutense, 28040 Madrid, Spain
Received 10 March 2004; revised 23 September 2004; accepted 29 September 2004

Abstract

The ability of lactic acid compared to acetic acid for Dover sole (Solea vulgaris) skin swelling and the subsequent gelatin extraction was
examined. The resultant gelatins were evaluated in terms of extraction yield, amino acid composition, molecular weight distribution, gel
strength, viscoelastic properties, ability to refold into triple helical structures, and aggregation phenomena. Lactic acid (25 mM) proved to be
an excellent substitute for acetic acid during the skin swelling process, as the gelatin preparation thus obtained presented quite similar
properties to that prepared by using 50 mM acetic acid without the negative organoleptic properties of this acid. However, the application of
50 mM lactic acid gave rise to a highly hydrolysed gelatin, with lower folding ability, gel strength and viscoelastic properties than those
obtained using 25 mM lactic acid or 50 mM acetic acid.
q 2004 Elsevier Ltd. All rights reserved.

Keywords: Circular dichroism; Gel strength; Gelatin extraction; Gelation; Viscoelastic properties

1. Introduction a mechanism different than triple helices formation

Gelatin is a protein compound derived from denatured
collagen. In order to obtain gelatin, a pre-treatment process
is required to convert the tissue collagen into a suitable form
for extraction. This means the loss of the ordered structure
of the native insoluble collagen, which results in a swollen,
but still insoluble collagen (Stainsby, 1987). Subsequent
heating cleaves hydrogen and covalent bonds, leading to the
conversion into gelatin by a helix-to-coil transition
(Djabourov, Lechaire, & Gaill, 1993). A sufficient number
of the covalent and non-covalent crosslinks in collagen must
be broken in order to enable the release of free a-chains and
oligomers (Johnston-Banks, 1990). Oligomers composed of
three a-chains may remain as intact triple helices, but a
significant percentage of extended polymers a-chains
bonded randomly by end-to-end or side-to-side-bonds.
Apart from covalent links, there may be additional
interactions of hydrophilic and hydrophobic nature between
the gelatin chains. Such interactions will promote gelatin
self-aggregation above the gelling temperature by
* Corresponding author. Tel: C34 91 549 2300; fax: C34 91 549 3627.
E-mail address: cgomez@if.csic.es (M.C. Gómez-Guillén).
0268-005X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2004.09.011
(Tromp, Grotenhuis, & Olieman, 2002).
The degree of collagen cross-linking is a key factor in
order to decide the pretreatment process required for gelatin
manufacture, and is highly dependent on a number of
factors, such as collagen type, tissue, animal species, age,
etc. Fish skins represent an important source of highly
soluble collagen, containing a low concentration of intra-
and inter-chain non-reducible crosslinks (Ledward, 1986;
Montero, Álvarez, Martı́, & Borderı́as, 1995; Montero,
Borderı́as, Turnay, & Lizarbe, 1990). Therefore, a mild
acid pre-treatment is usually used for gelatin production
(Norland, 1990). Such treatment leads to a type-A gelatin
with an isoelectric point that can vary from 6.5 to 9
(Johnston-Banks, 1990). Increasing HC ions favours the
access of water to collagen fibres. This water is held in by
electrostatic forces between charged polar groups (electro-
static swelling) or by hydrogen bonding between uncharged
polar groups and negative atoms (lyotropic hydration)
(Gustavson, 1956). The type and concentration of acid used
strongly influence swelling properties and solubilisation of
collagen, leading to variations in molecular weight
distribution in the resultant gelatins, depending on the
persistence of some of the cross-links between collagen
942 B. Giménez et al. / Food Hydrocolloids 19 (2005) 941–950
chains. According to Asghar and Henrickson (1982), the
lyotropic effect of carboxylic acids on collagen seems to
dominate the swelling capacity, rather than a specific ion
effect, since it is the non-ionized acid that acts as the
swelling agent by competing with the peptide group
involved in intermolecular linking of the protein chain,
mainly because of the hydrogen bonding power of the acid.
Citric acid is widely used for the manufacture of food-
grade gelatin from fish skin because it does not introduce
undesirable colour or odour to the gelatin. The overall
properties of gelatins obtained following this procedure
highly depend on the fish species used, largely attributed to
differences in aminoacid composition. Thus, whereas
gelatin extracted from tilapia, a species from tropical
waters, presents good gelling properties (Grossman &
Bergman, 1992), gelatin from cod skin produces very soft
and unstable gels (Gudmundsson & Hafsteinsson, 1997).
Several organic acids have been tested for collagen
solubilisation from megrim skin (Lepidorhombus boscii)
and the viscoelastic properties of the resulting gelatins have
been analyzed. Better gelling gelatins were obtained using
diluted acetic acid instead of citric acid (Gómez-Guillén &
Montero, 2001; Montero & Gómez-Guillén, 2000). The
small molecular size and low ionization constant of the
acetic acid molecule have been proposed as key factors for a
more suitable collagen swelling prior to conversion into
gelatin (Gómez-Guillén, Sarabia, & Montero, 2001).
However, the quality of the gelatins extracted following
this procedure strongly depends on the intrinsic character-
istics of the collagen molecules from each fish species. In a
comparative study of gelatins extracted using acetic acid for
collagen swelling, gelatins from flat-fish species (sole and
megrim) presented the best gelling ability and the gels were
more thermostable than those from cold-adapted fish (cod
and hake). This different behaviour was explained by the
amino acid composition, the a1/a2 collagen-chain ratio and
the molecular weight distribution of the material extracted
from each skin (Gómez-Guillén et al., 2002).
Lactic acid is a low molecular weight organic acid with a
significant number of food applications and with the great
advantage over other similar organic acids of being virtually
colourless, odourless and tasteless. In a preliminary study
using megrim skin, we have shown that lactic acid is also
suitable for collagen extraction, although the resulting
gelatin apparently presented slightly worse rheological
properties than that extracted using acetic acid (Gómez--
Guillén et al., 2001). For this reason, and taking into account
the advantages that lactic acid presents over acetic acid
concerning their organoleptic properties, in the present
study we further analyze the use of lactic acid for gelatin
extraction and compare the rheological, chemical and
structural characteristics of the extracted material in
comparison with that extracted using acetic acid. For this
purpose, skins from Dover sole (Solea vulgaris), a species
which has been proved to offer a good potential for gelatin
manufacture, were used.
2. Materials and methods
2.1. Gelatin extraction
Skins from Dover sole (S. vulgaris) were collected and
stored frozen at K20 C until use. Cleaning of fish skins and
gelatin extraction procedure was carried out as previously
described (Gómez-Guillén & Montero, 2001). Briefly, the
method consists in a mild acid swelling step in 50 mM
acetic acid and subsequent gelatin extraction in distilled
water at 45 8C overnight (ACE-50). For the present work,
the swelling step has been also carried out with 10, 25
(LAC-25) and 50 mM (LAC-50) lactic acid.
2.2. Amino acid analysis
Dry gelatin was dissolved in distilled water at 1 mg/ml
and 50 ml of each sample were dried and hydrolysed in
vacuum-sealed glass tubes at 108 8C for 18 h in the presence
of constant boiling 5.7N HCl containing 0.1% phenol and
using norleucine as internal standard. After hydrolysis,
samples were again vacuum-dried, dissolved in application
buffer and injected into a Beckman 6300 amino acid
analyser (Beckman Instruments Inc., Palo Alto, CA).
2.3. Electrophoretic analysis
Gelatin was first dissolved at 5 mg/ml in distilled water at
60 8C and then three-fold-concentrated loading buffer
containing b-mercaptoethanol was added. Protein samples
were heat-denatured 5 min at 90 8C and analysed by SDS-
PAGE according to Laemmli (1970) using 4% stacking gels
and 5% resolving gels in a Mini Protean II unit (Bio-Rad
Laboratories, Hercules, CA) at 25 mA/gel. The loading
volume was 15 ml in all lines. Protein bands were stained
with Coomassie brilliant Blue R250. Type I and type III
collagens from foetal calf skin were used as markers of a-
chain, b- and g-component mobilities.
2.4. Gel strength
Gel strength was determined according to Gómez-Guil-
lén et al. (2002) using 6.67% gels (w/v) formed by
dissolving the dry gelatin in distilled water at 60 8C and
cooling down the solution in a refrigerator at 7 8C
(maturation temperature) for 16–18 h. The gel strength
was determined at 7 8C using samples with 3.3 cm diameter
and 6 cm height on an Instron model 4501 Universal Testing
Machine (Instron Co., Canton, MA, USA) with a load cell of
100N, cross-head speed 1 mm/s, and equipped with a
1.27 cm-diameter flat-faced cylindrical Teflonw plunger.
Gel strength was expressed as maximum force (in g),
obtained when the plunger had penetrated 4 mm into the
gelatin gels; data represent the average (GSD) of five
determinations.
 B. Giménez et al. / Food Hydrocolloids 19 (2005) 941–950
2.5. Viscoelastic properties
Dynamic studies were performed on a Bohlin CRS-10
controlled stress rheometer rotary viscometer (Bohlin
Instrumments Ltd, Gloucestershire, UK) using a cone-
plate geometry (cone angle 48, gapZ0.15 mm). Solutions
were prepared by dissolving dry gelatin in distilled water
(6.67%, w/v) at 40 8C by mechanical stirring for 15–20 min.
Some samples were then cooled down at 7 8C (maturation
temperature) for 16–18 h to analyse the effect of maturation
time on gelatin gel properties. Temperature ramps were
performed at a scan rate of 1 8C/min, frequency 1 Hz, and
oscillating applied stress of 3.0 Pa. The elastic modulus
(G 0 ), viscosity modulus (G 00 ) and the phase angle (d) were
represented as a function of temperature. The ramps were
implemented from 40 to 6 8C and back to 40 8C for
dissolved gelatin, to study gelatin gelation and subsequent
melting. Gelatin matured overnight (16–18 h) at 7 8C was
also subjected to a temperature ramp from 6 to 40 8C, to
evaluate the effects of maturation time on the gelatin gel.
The error in the reproducibility of the parameters considered
in different determinations of a single sample was 6%
or less.
2.6. Circular dichroism
Gelatin samples were dissolved at 6.67% or 0.5% (w/v)
at 40 8C, and loaded into preheated 0.01-cm pathlength
thermostatized cuvettes. Circular dichroism (CD) spectra
were recorded between 200 and 260 nm in a Jasco J-715
spectropolarimeter (Jasco Corp., Tokyo, Japan) equipped
with a Neslab RTE-111 thermostat. In order to evaluate the
influence of temperature on triple helix formation, molar
ellipticity of the gelatin preparations at 6.67% (w/v) was
monitored at 229 nm due to the impossibility to use lower
wavelengths at this protein concentration. When gelatin
preparations were analyzed at 0.5% (w/v), molar ellipticity
changes were monitored at 220 nm (maximum of the
spectra and characteristic of the presence of triple helix).
Temperature was first decreased from 40 to 4 8C at 1 8C/min
to allow gel formation in the cuvette. Once this temperature
was reached, spectra were recorded at 4 8C, and afterwards
temperature was raised at the same rate up to 40 8C either
immediately afterwards or after no further apparent
variation in the molar ellipticity at 229 or 220 nm was
detected (around 1 h). Mid-point transition temperatures
were obtained from the maximum or minimum of the first
derivative from the temperature curves after noise reduction
using a Fourier-transformed-based utility included in the
spectropolarimeter software package. Statistical analysis of
the differences between mean melting or folding tempera-
tures in the different gelatin preparations was evaluated by
using Student’s t-test (SigmaPlot 8.02, Systat Software Inc.,
Erkrath, Germany). Protein concentration was determined
by amino acid analysis of triplicate samples obtained from
gelatin preparations taken out directly from the CD cuvette.
943
2.7. Kinetics of aggregation at 314 nm
Aggregation of gelatin molecular components during gel
formation was analysed by monitoring changes in absor-
bance at 314 nm during the cooling process. Dried gelatin
was dissolved in distilled water at 40 8C. In order to avoid
high initial absorbance values, it was necessary to dissolve
LAC-25 and LAC-50 gelatins at 0.5% (w/v). ACE-50
gelatin presented very low initial absorbance at this
concentration, and changes at 314 nm during the cooling
process were almost negligible. Thus, a higher concen-
tration (2% w/v) was required in this case to monitor
changes in absorbance. Samples were loaded into thermo-
statized cuvettes at 40 8C and absorbance at 314 nm was
monitored for 80 min using a UV-1601 spectrophotometer
(Model CPS-240, Shimadzu, Japan). Temperature was
gradually lowered down to 7 8C at approximately
1 8C/min and maintained thereafter at this temperature.
3. Results and discussion
3.1. Gelatin extraction
Gelatin preparations were performed under identical
conditions with the exception of the acid pre-treatment. A
similar yield, expressed as grams of dry gelatin per 100 g of
clean skin, were obtained by using a 50 mM concentration
of acetic or lactic acid for collagen swelling (ACE-50:
21.35%; LAC-50: 20.39%), whereas a slightly lower yield
was obtained with 25 mM lactic acid (LAC-25: 18.14%).
When 10 mM lactic acid was used for swelling, no gelatin
was extracted, which confirms that a suitable mild acid pre-
treatment is essential for gelatin extraction at such moderate
temperature (45 8C).
All the gelatin preparations presented a similar behaviour
concerning solubility in distilled water. However, prep-
arations obtained by pre-treatment with lactic acid showed
higher light scattering than those obtained with acetic acid.
The higher turbidity of these preparations is not due to the
presence of traces of lactic acid in the gelatin preparations,
as we have verified that addition of lactic acid to ACE-50
gelatin does not induce light scattering. Thus, skin swelling
using lactic acid must induce the solubilization of material
showing a higher aggregation degree.
3.2. Amino acid composition
The amino acid composition of the three different gelatin
preparations is shown in Table 1. In general, composition is
quite similar to that of interstitial collagen, with a high
glycine (Gly) and imino acid content (34–36% and 17–18%,
respectively). Only small differences could be detected
among these gelatins; a slightly higher hydroxyproline
(Hyp) content was found in LAC-50 while the overall imino
acid content was quite identical to that detected in ACE-50
944 B. Giménez et al. / Food Hydrocolloids 19 (2005) 941–950

Table 1 3.3. Molecular weight distribution

Amino acid composition of gelatins

Amino acid Number of residues/1000 Even though the amino acid composition of the gelatin
ACE-50 LAC-25 LAC-50 preparations was quite similar, differences among gelatin
Hyp 52 51 55 preparations may arise from the extraction of more or less
Asx 48 47 48 cross-linked material or from the possible degradation of the
Thr 20 20 20 collagen a-chains. Thus, we analysed the molecular weight
Ser 40 39 39 distribution of the collagenous material extracted, following
Glx 74 73 73
the three different protocols by polyacrylamide gel electro-
Pro 129 122 128
Gly 345 355 345 phoresis in the presence of SDS (Fig. 1). LAC-50 gelatin
Ala 119 119 116 showed a clearly different band pattern compared to LAC-25
Val 21 20 21 and ACE-50. In general, this preparation showed a very low
Met 14 15 15 content in intact a-chains and b-components, with a very low
Ile 8 8 8
amount of high molecular weight covalently-linked aggre-
Leu 22 22 23
Tyr 3 3 3 gates. No clear protein bands are observed, and staining
Phe 16 15 16 mainly corresponds to protein material below 100 kDa. Thus,
His 8 8 8 skin swelling with 50 mM lactic acid either only solubilizes
Hyl 1 1 1 degraded collagenous material or well induces degradation of
Lys 27 29 28
Arg 53 53 52
this material during gelatin extraction. Taking into account
Imino acids (ProCHyp) 181 173 183 that the use of lactic acid at a lower concentration allows the
Pro hydroxylation (%) 28.6G1.1 28.8G0.9 30.0G0.8 extraction of more intact material but with a lower yield, the
Lys hydroxylation (%) 3.7G0.3 3.7G0.4 3.9G0.4 second possibility seems more probable. Moreover, when skin
Amino acid composition was determined using hydrolysates from the three swelling is performed with a weaker acid (acetic acid) at the
different gelatins used throughout this study as described in Section 2. same concentration (50 mM), a similar gelatin yield is
Determinations were performed in triplicate and data correspond to mean obtained but the material is much less degraded. In fact, in a
values. Standard deviations were in all cases lower than 6%. Proline and previous study using megrim skin, we reported that collagen
lysine hydroxylation are also shown (meanGSD).
extraction using lactic acid in comparison with acetic acid
induced a more acidic environment that was finally reflected in
but higher than in LAC-25. Thus, the proline (Pro) lower gel strength of lactic acid-extracted material (Gómez--
hydroxylation degree in LAC-50 gelatin is around 1% Guillén et al., 2001).
higher than in ACE-50 and LAC-25 gelatins.
It has been reported that the stability of the triple helical 3.4. Gel strength
structures in renatured gelatins is dependent on the total
imino acid content, as regions rich in prolineChydroxypro- The gel strength after overnight maturation at 7 8C varied
line are likely involved in the formation of nucleation zones considerably depending on the acid treatment employed for
(Ledward, 1986). In these regions, hydroxyproline plays a
key role in the stabilization of triple helical structures by
establishment of water bridges through its hydroxyl group
(Burjandze, 1979; Ledward, 1986; Mizuno, Hayashi, &
Bächinger, 2003). These regions are rich in non-polar
sequences Gly-Pro-Y, where the third position is normally
occupied by hydroxyproline or by alanine (Ala). Thus, in
general, a gelatin preparation with high proline, hydro-
xyproline, and alanine content shows better viscoelastic
properties than others with a lower content in these amino
acids, as we have previously reported for fish skin-derived
gelatin preparations (Gómez-Guillén et al., 2002; Sarabia,
Gómez-Guillén, & Montero, 2000). However, the differ-
ences in amino acid compositions in these reported cases
was much higher than that herein reported, where hydro-
xyproline content is only slightly higher in the LAC-50 Fig. 1. Electrophoretic analysis of the gelatin preparations. Gelatin
preparation and no differences are found regarding Ala preparations extracted from sole skin pre-treated in 50 mM lactic acid
(a), 25 mM lactic acid (b) and 50 mM acetic acid (c) were analysed by
content. Thus, one could not expect differences in the
PAGE-SDS on 5% gels in the presence of b-mercaptoethanol. Bovine skin
viscoelastic properties of these gelatin preparations based type I and type III collagens were used as mobility markers for a-chains,
only on the analysis of the amino acid composition. and b- and g-components (unreduced type III collagen).
 B. Giménez et al. / Food Hydrocolloids 19 (2005) 941–950
skin swelling. The highest strength was achieved in ACE-50
gels (158.7G11.23 g), followed by LAC-25 (118.17G
2.66 g). In contrast, a very soft gel was obtained when
using LAC-50 gelatin (11.70G0.14 g). These differences
could be explained by differences in molecular weight
distribution rather than in amino acid composition without
disregarding the existence of additional factors that may
influence this parameter. According to the gel in Fig. 1,
ACE-50 gelatin presents the larger amount of covalently-
linked polymers followed by LAC-25 preparation. The
existence of these oligomers may enhance the renaturation
ability of these preparations versus LAC-50, allowing a
better establishment of stabilising interactions and the
formation of more organised triple helical structures
(Sims, Bailey, & Field, 1997; Stainsby, 1987). Moreover,
the differences in gel strength between ACE-50 and LAC-25
could be explained in terms of differences in molecular
weight profile. On the other hand, the extremely low gel
strength of LAC-50 gelatin preparation could be the
consequence of more extensive hydrolysation. This would
act hindering both, the formation of nucleation sites, and the
annealing of collagen chains by the growth of existing
nucleation sites during overnight stabilisation (Ledward,
1986; Normand, Muller, Ravey, & Parker, 2000).
3.5. Viscoelastic properties
In order to further analyse the quality of the three gelatin
preparations, their viscoelastic properties after dissolving
them at 6.67% (w/v) in distilled water were studied. Fig. 2
represents the modulus of elasticity (G 0 ), modulus of
viscosity (G 00 ) and phase angle (d), during both cooling
(from 40 to 6 8C) and subsequent heating (from 6 to 40 8C).
Among the three gelatin preparations, LAC-50 presented
the least gelling ability, showing a significant lower increase
in G 0 and G 0 upon cooling than ACE-50 and LAC-25, which
presented quite similar behaviour (Fig. 2A and B). The
analysis of the variation of the phase angle revealed that the
onset of gelling was also lower in LAC-50 gelatin (around
12 8C) than in the other two preparations: gelling begins at
around 15 8C in LAC-25 and 16 8C in ACE-50. These
differences were also observed when the viscoelastic
properties upon heating from 6 to 40 8C were analysed.
Whereas LAC-25 and ACE-50 behaved in a similar way,
LAC-50 again showed lower G 0 and G 00 values than the
other gelatins. The thermal stability of the gels was also
decreased in LAC-50, which showed a melting temperature
between 18 and 19 8C, 2 and 3 8C lower than LAC-25 and
ACE-50, respectively.
The viscoelastic properties of the gelatin preparations
was also analysed after cold maturation at 6 8C for 18 h and
further heating from 6 to 40 8C (Fig. 3). A striking fact is
that cold maturation induced a highly significant increase in
the modulus of elasticity (Fig. 3A; around two-fold
increase) and in the modulus of viscosity (Fig. 3B; around
five-fold increase) in LAC-25 and ACE-50 preparations.
945
However, LAC-50 gelatin showed a two-fold-decrease in G 0
and only a 1.8-fold increase in G 00 . Thus, cold maturation of
the gels is quite different in LAC-50 gelatin compared to the
other two preparations. This fact is also reflected in a
significantly higher thermal stabilisation of the LAC-25 and
ACE-50 gels, as deduced from the variation in the phase
angle upon heating the matured gels (Fig. 3C). Thus, the
melting temperature of matured gels is around 29–30 8C for
ACE-50 (8 8C higher) and 27–28 8C for LAC-25 (7 8C
higher). Melting of cold-matured LAC-50 gels is charac-
terised by a loss of sharpness in the phase angle transition
(Fig. 3C), with an initial slow rise in this parameter from 10
up to 20–21 8C, followed by a sharper final increase; the
melting temperature is around 238C, only 4–5 8C higher
than before cold-maturation.
The different behaviour observed in the variation of the
viscoelastic properties of LAC-50 gelatin with temperature
and with cold maturation in comparison with LAC-25 and
ACE-50 could be explained by the higher fragmentation that
this gelatin preparation presents. Even though nucleation
sites probably exist in LAC-50 gelatin, the high fragmenta-
tion of a-chains probably impairs the growth of the existing
ones by further annealing of collagen chains during cooling
or maturation. This behaviour is also in good accordance
with the extremely low gel strength values observed for
LAC-50 gels. On the other hand, LAC-25 and ACE-50
gelatins present a significantly higher percentage of intact
a-chains and covalently-linked aggregates, allowing in both
cases not only the existence of nucleation sites, but also their
growth by further annealing or chain association during cold
maturation.
3.6. Circular dichroism
The transition from random coil structure to collagen-
like triple helix has been proposed to be one of the major
events either in triggering or, at least, in the stabilisation of
gelatin gels. We have monitored the folding of gelatin
chains into triple helical structures by CD spectroscopy in
the far UV region using gelatin solutions under identical
conditions as those described for the analysis of
the viscoelastic properties (gelatin concentration 6.67%,
w/v). Additionally, we have analysed the folding
and unfolding of these preparations at a lower protein
concentration (0.5%, w/v).
Fig. 4 shows the CD spectra of the three gelatin
preparations at 6.67% and 0.5% (w/v). Spectra are shown
in the unfolded state at 40 8C and after cooling down to 4 8C
and allowing stabilization of the molar ellipticity, a process
that was achieved in general after 1 h maturation at 4 8C.
As observed in Fig. 4A, it is impossible to analyse the
spectra below 225–228 nm at high protein concentration
even using a very small pathlength cuvette. However, at a
lower concentration (0.5%, Fig. 4B) spectra can reach as
low as 200 nm without introducing artefacts. Spectra
obtained at 40 8C correspond in all cases to the typical
946 B. Giménez et al. / Food Hydrocolloids 19 (2005) 941–950

Fig. 2. Viscoelastic properties upon cooling and heating of gelatin preparations. Dry gelatins were dissolved at 6.67% (w/v) at 40 8C. Changes in the modulus of
elasticity G 0 (A,D), modulus of viscosity G 00 (B,E) and phase angle (C,F), were monitorized during cooling from 40 8C to 7 8C (A–C) and subsequent heating
from 7 to 40 8C (D–F). ACE-50: pre-treatment with 50 mM acetic acid; LAC-25: pre-treatment with 25 mM lactic acid; LAC-50: pre-treatment with 50 mM
lactic acid.

spectrum of denatured collagen; on the other hand, gelatins
at 4 8C present a maximum at 220 nm that is characteristic
of triple helical conformation (Engel, 1987), as observed in
gelatin preparations at 0.5% (w/v). Preparations at 6.67%
(w/v) not only show a similar behaviour than those at 0.5%,
but even higher molar ellipticities are obtained in the 225–
250 nm region. This indicates that an increase in gelatin
concentration is paralleled by the achievement of a higher
percentage of triple helical conformation, as we previously
reported for fish-skin gelatins from several marine species
(Gómez-Guillén et al., 2002). However, molar ellipticity in
this region is always lower than that reported for intact
collagen molecules purified from fish skin (Ogawa et al.,
2003) or from other origins (Engel, 1987), [q]MRW(220 nm)
z10,000 deg cm2 dmolK1. When the different gelatin
preparations are compared, no significant differences can
be found among them neither at 40 8C nor at 4 8C after
stabilisation. Thus, the overall conformation of the different
gelatins analysed at the initial and final stages of the cooling
and heating ramps are quite similar, even though the
transition from the unfolded to the folded state (and vice
versa) may differ among them, as described below.
We have analysed by CD the gelatin folding process
from random coil to a triple helical-rich structure in
identical conditions to those reported for the analysis of
the viscoelastic properties (6.67%, w/v). Additionally, we
have also monitored folding and unfolding at a lower
concentration (0.5%, w/v) in order to verify the relevance of
gelatin concentration in this processes. Fig. 5 shows the
variation in the molar ellipticity at 229 nm (6.67% gelatin
solutions; left panels) and at 220 nm (0.5% gelatin
solutions; right panels) during cooling from 40 to 4 8C
(a curves) and subsequent heating (b curves). Unfolding
curves were also obtained after allowing stabilisation of the
molar ellipticity at 4 8C (around 1 h; c curves). The different
wavelength used is due to the impossibility to register
ellipticity at 220 nm (maximum of collagen triple helix
spectrum) at 6.67% protein concentration.
In all preparations, a slow gradual increase in molar
ellipticity was observed upon cooling from 40 to 4 8C,
indicating that folding towards the formation of triple
helical structures is taking place. In general, no highly
significant differences were detected during cooling of the
three gelatins, even though the mid-point for the ellipticity
change in LAC-50 was around 1 8C below that of ACE-50
and LAC-25 (see Fig. 5, a curves, left panels). When this
process was analyzed at a 0.5% gelatin concentration, lower
temperatures were required in all the preparations for the
folding process to take place. This fact again confirms the
importance of protein concentration for gelatin refolding.
When gelatin unfolding was studied, more significant
changes among the different preparations were observed.
Whereas ACE-50 and LAC-25 gelatins presented a quite
similar behaviour with a melting temperature close to 20 8C,
LAC-50 showed a 2 8C lower value. Surprisingly, no
significant differences were found in the melting
 B. Giménez et al. / Food Hydrocolloids 19 (2005) 941–950
Fig. 3. Viscoelastic properties upon heating of gelatin gels after overnight
cold maturation. Dry gelatins were dissolved at 6.67% (w/v) at 40 8C,
cooled down to 7 8C and maintained at this temperature for 18 h. Changes
in the modulus of elasticity G 0 (A), modulus of viscosity G 00 (B) and phase
angle (C), were monitorized during heating from 7 to 40 8C. ACE 50: pre-
treatment with 50 mM acetic acid; LAC 25: pre-treatment with 25 mM
lactic acid; LAC 50: pre-treatment with 50 mM lactic acid.
temperatures obtained at 6.67% and 0.5% gelatin concen-
trations. Moreover, in all the cases analysed, the melting
temperature was identical disregarding whether heating
began immediately after the cooling ramp or if stabilisation
Fig. 4. Circular dichroism spectra of sole-skin gelatin. Spectra were recorded at 6.67% (A; 225–250 nm) or 0.5% (w/v) (B; 200–250 nm) at 40 8C and after
cooling down to 4 8C and allowing to molar ellipticity to stabilize (around 1 h).
947
of ellipticity was allowed at 4 8C (Fig. 5, b and c curves),
even though initial ellipticity values were always higher
after stabilisation.
We have verified that refolding and unfolding of gelatins
presents hysteresis as also described for intact collagen type
III molecules (Davis & Bächinger, 1993; Engel &
Bächinger, 2000). This process is characterized in the
three gelatin preparations by a shift in the unfolding curves
to higher temperatures by around 10 8C at 6.67% and
12–13 8C at 0.5%. Hysteresis in collagen models has been
suggested to appear due to a slow refolding kinetics
compared to the fast unfolding process. During collagen
denaturation, a partial isomerization of trans to cis peptide
bonds take place. Cis isomers are sterically inconsistent
with the triple helical structure but are possible and
entropically favoured in the unfolded state, mainly in
imino acid rich molecules as collagen. Thus, during
refolding of collagen or gelatin, the relatively slow process
of cis–trans isomerization must take place Engel &
Bächinger, 2000; Xu et al., 2003). The nucleation step is
an additional energetic barrier for the folding process, but
not for unfolding, and strongly depends on denatured
collagen concentration (Xu, Bhate, & Brodsky, 2002). This
could explain the influence of gelatin concentration in the
folding process but not in the unfolding of the triple helical
structures, as observed in Fig. 5. Finally, after the
establishment of trimeric nucleation sites, propagation of
the triple helical structure takes place. Even though
hysteresis was found in all cases, the folding and melting
processes were completely reversible as verified from the
quite identical curves obtained after at least three cooling
and heating cycles.
The lower temperature required for LAC-50 folding
compared to ACE-50 and LAC-25 gelatins could originate
948 B. Giménez et al. / Food Hydrocolloids 19 (2005) 941–950

Fig. 5. Changes in molar ellipticity at 229 or 220 nm upon cooling and heating of gelatin preparations. Dry gelatins were dissolved at 6.67% (left panels) or
0.5% (w/v) (right panels) at 40 8C and loaded into preheated cuvettes. Changes in the molar ellipciticy at 229 nm (gelatins at 6.67%) or 220 nm (gelatins at
0.5%) were monitorized during cooling from 40 to 4 8C (a) and subsequent heating from 4 to 40 8C either immediately afterwards the cooling process (b) of
after allowing stabilization of the molar ellipticity at 4 8C (c). Mid-point transition-temperatures are included.

from impairment in the establishment of triple helical
nucleation centres, as this gelatin preparation appears to be
more degraded and with a lower degree of covalently cross-
linked polymers than the other preparations (Fig. 1). Thus,
interactions between three different polypeptide chains may
be more difficult and, consequently, lower temperatures are
required for this process to take place. This could also be
directly related to its inferior gelling ability and gel strength.
On the other hand, unfolding of LAC-50 gelatin occurs at
lower temperatures; this probably is due to the establish-
ment of shorter and less stable triple helical regions in this
gelatin preparation due to increased chain fragmentation,
even though the total amount of peptide bonds showing
triple helical conformation must be quite similar among the
gelatin preparations as no significant differences in the final
molar ellipticity at 4 8C is detected (Figs. 4 and 5).
When folding curves are compared with the phase angle
variation upon cooling, a delay in the appearance of triple
helical structures is observed. The onset of gelation always
precedes the increase in molar ellipticity; moreover, the
folding process continues after gelling has been completed
(Figs. 2 and 5). Thus, it could be suggested that additional
interactions must take place among gelatin polypeptide
chains that trigger the gelling process and enhance the
nucleation step required for triple helix formation.
3.7. Kinetics of aggregation at 314 nm
Association of denatured collagen/gelatin polypeptide
chains may be involved in the nucleation step required for
triple helix formation and gelatin gels stabilisation. As this
association could increase the size of the solubilized gelatin
moieties, determination of changes in absorbance at 314 nm
could allow the study of this process. LAC-25 and LAC-50
gelatin preparations show a significantly higher absorbance
than ACE-50, even though the latter shows in PAGE-SDS a
higher degree of covalently linked aggregates (Fig. 1). Thus,
considering that the chemical composition of the different
gelatins is quite similar, non-covalent interactions must be
taking place in LAC gelatins increasing the turbidity of
these preparations. As LAC-50 is the worst folding gelatin,
the aggregation responsible for the high absorbance at
314 nm must be different from that required for the
nucleation process, lacking probably the correct chain
organization.
Fig. 6 shows the behaviour of absorbance at 314 nm
during cooling from 40 to 7 8C of the different
gelatin preparations at 0.5% (w/v) and ACE-50 at 2%
(w/v). Initially, temperature was lowered at approximately
1 8C/min, allowing afterwards around 1 h stabilisation at
7 8C. The upper panel of Fig. 6 shows a detail of
 B. Giménez et al. / Food Hydrocolloids 19 (2005) 941–950
Fig. 6. Changes in absorbance at 314 nm upon cooling of gelatin
preparations. Dry gelatins were dissolved at 0.5 or 2% (w/v) at 40 8C and
loaded into pre-heated cuvettes. Changes in the absorbance at 314 nm were
monitorized during cooling from 40 to 7 8C at approximately 1 8C/min and
for a total time of 80 min. The upper panel shows a detail of the variation of
A314 with temperature, showing the mid-point transition temperatures for
each gelatin preparation.
the variation in absorbance at 314 nm with temperature. A
sharp variation in absorbance is observed upon cooling
between 9 and 8 8C in LAC-25 and LAC-50 gelatins at 0.5%
(w/v) and in ACE-50 at 2% (w/v); the mid-point
temperature for these transitions are shown in the upper
panel of Fig. 6. Additionally, LAC-50 gelatin shows a
further increase in A314 during incubation at 7 8C. Taking
into account that this gelatin preparation is the one
presenting lower gel strength after overnight maturation at
7 8C, it can be suggested that the aggregation responsible for
the increase in absorbance at 314 nm does not contribute to
gel stability but, on the contrary, may diminish it. This
hypothesis is also supported by the fact that the gelatin
preparation that reaches a higher gel strength after cold
maturation, ACE-50, is the one presenting the lowest
absorbance values, which do not vary after temperature has
reached 7 8C.
The increase in absorbance at 314 nm seems to take place
after gelatin gels are completely formed, as changes in
absorbance are only detected at temperatures lower than
those required for complete gelling (Figs. 2C and 6).
However, as gelatin concentrations for this analysis are
lower than those used in the measurement of the viscoelastic
properties of the different preparations, this observation
should be considered with caution. In general, when gelatin
concentration is lowered, gelation is slightly impaired. The
main absorbance change takes place simultaneously to the
formation of triple helical structures at this gelatin concen-
tration (Fig. 5); thus, it could be associated to changes in the
aggregation of gelatin chains and triple helical structures
during gelation, similar to the microfibrilar assembly of triple
949
helices described during the lateral association of collagen
subunits (Djabourov et al., 1993). On the other hand, further
changes in absorbance during cold maturation must be
associated to a different type of aggregation that impairs gel
stability, as occurs in LAC-50 gelatin gels in contrast to the
much stronger ones obtained in LAC-25 and ACE-50 gels,
which do not show a significant increase in absorbance
during gel maturation. On this idea, Tromp et al. (2002) have
described that under some circumstances, additional inter-
actions different from those promoting the additional
formation or stabilisation of triple helical structures may
take place below the gelling temperature.
4. Conclusions
We have developed a method for extracting gelatin from
fish skin that resembles the high quality material obtained by
using 50 mM acetic acid avoiding the negative organoleptic
properties of that acid. Lactic acid seems to be a more
efficient disrupter of the skin tissue and collagenous
structures probably due to its higher dissociation constant
and to the presence of hydroxyl group that may establish
additional hydrogen bonds that contribute to collagen
denaturation. Moreover, care must be taken to avoid the
extraction of highly fragmented material that may impair the
viscoelastic properties of the final gelatin preparation. We
have performed a profound comparative analysis of the
viscoelastic properties of gelatin extracted by using 25 and
50 mM lactic acid and 50 mM acetic acid for skin swelling
and also studied the molecular conformation changes that
take place during gelation and melting of gelatin gels. In
conclusion, the use of 25 mM lactic acid for skin swelling has
proven to be an excellent substitute for acetic acid, yielding
gelatin preparations with quite similar properties to that
obtained with 50 mM acetic acid.
Acknowledgements
This research was financed by the Comunidad de Madrid
under project 07G/0052/00 and by the Spanish Dirección
General de Investigación under project BMC02-1407.
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