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Food Hydrocolloids 19 (2005) 941–950

Use of lactic acid for extraction of fish skin gelatin

B. Giméneza, J. Turnayb, M.A. Lizarbeb, P. Monteroa, M.C. Gómez-Guilléna,*
Instituto del Frı́o (CSIC), Ciudad Universitaria, 28040 Madrid, Spain
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


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

(Tromp, Grotenhuis, & Olieman, 2002).
Gelatin is a protein compound derived from denatured The degree of collagen cross-linking is a key factor in
collagen. In order to obtain gelatin, a pre-treatment process order to decide the pretreatment process required for gelatin
is required to convert the tissue collagen into a suitable form manufacture, and is highly dependent on a number of
for extraction. This means the loss of the ordered structure factors, such as collagen type, tissue, animal species, age,
of the native insoluble collagen, which results in a swollen, etc. Fish skins represent an important source of highly
but still insoluble collagen (Stainsby, 1987). Subsequent soluble collagen, containing a low concentration of intra-
heating cleaves hydrogen and covalent bonds, leading to the and inter-chain non-reducible crosslinks (Ledward, 1986;
conversion into gelatin by a helix-to-coil transition Montero, Álvarez, Martı́, & Borderı́as, 1995; Montero,
(Djabourov, Lechaire, & Gaill, 1993). A sufficient number Borderı́as, Turnay, & Lizarbe, 1990). Therefore, a mild
of the covalent and non-covalent crosslinks in collagen must acid pre-treatment is usually used for gelatin production
be broken in order to enable the release of free a-chains and (Norland, 1990). Such treatment leads to a type-A gelatin
oligomers (Johnston-Banks, 1990). Oligomers composed of with an isoelectric point that can vary from 6.5 to 9
three a-chains may remain as intact triple helices, but a (Johnston-Banks, 1990). Increasing HC ions favours the
significant percentage of extended polymers a-chains access of water to collagen fibres. This water is held in by
bonded randomly by end-to-end or side-to-side-bonds. electrostatic forces between charged polar groups (electro-
Apart from covalent links, there may be additional static swelling) or by hydrogen bonding between uncharged
interactions of hydrophilic and hydrophobic nature between polar groups and negative atoms (lyotropic hydration)
the gelatin chains. Such interactions will promote gelatin (Gustavson, 1956). The type and concentration of acid used
self-aggregation above the gelling temperature by strongly influence swelling properties and solubilisation of
collagen, leading to variations in molecular weight
* Corresponding author. Tel: C34 91 549 2300; fax: C34 91 549 3627. distribution in the resultant gelatins, depending on the
E-mail address: (M.C. Gómez-Guillén). persistence of some of the cross-links between collagen
0268-005X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
942 B. Giménez et al. / Food Hydrocolloids 19 (2005) 941–950

chains. According to Asghar and Henrickson (1982), the 2. Materials and methods
lyotropic effect of carboxylic acids on collagen seems to
dominate the swelling capacity, rather than a specific ion 2.1. Gelatin extraction
effect, since it is the non-ionized acid that acts as the
swelling agent by competing with the peptide group Skins from Dover sole (S. vulgaris) were collected and
involved in intermolecular linking of the protein chain, stored frozen at K20 C until use. Cleaning of fish skins and
mainly because of the hydrogen bonding power of the acid. gelatin extraction procedure was carried out as previously
Citric acid is widely used for the manufacture of food- described (Gómez-Guillén & Montero, 2001). Briefly, the
grade gelatin from fish skin because it does not introduce method consists in a mild acid swelling step in 50 mM
undesirable colour or odour to the gelatin. The overall acetic acid and subsequent gelatin extraction in distilled
properties of gelatins obtained following this procedure water at 45 8C overnight (ACE-50). For the present work,
highly depend on the fish species used, largely attributed to the swelling step has been also carried out with 10, 25
differences in aminoacid composition. Thus, whereas (LAC-25) and 50 mM (LAC-50) lactic acid.
gelatin extracted from tilapia, a species from tropical
waters, presents good gelling properties (Grossman &
2.2. Amino acid analysis
Bergman, 1992), gelatin from cod skin produces very soft
and unstable gels (Gudmundsson & Hafsteinsson, 1997).
Dry gelatin was dissolved in distilled water at 1 mg/ml
Several organic acids have been tested for collagen
and 50 ml of each sample were dried and hydrolysed in
solubilisation from megrim skin (Lepidorhombus boscii)
vacuum-sealed glass tubes at 108 8C for 18 h in the presence
and the viscoelastic properties of the resulting gelatins have
of constant boiling 5.7N HCl containing 0.1% phenol and
been analyzed. Better gelling gelatins were obtained using
using norleucine as internal standard. After hydrolysis,
diluted acetic acid instead of citric acid (Gómez-Guillén &
samples were again vacuum-dried, dissolved in application
Montero, 2001; Montero & Gómez-Guillén, 2000). The
buffer and injected into a Beckman 6300 amino acid
small molecular size and low ionization constant of the
analyser (Beckman Instruments Inc., Palo Alto, CA).
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). 2.3. Electrophoretic analysis
However, the quality of the gelatins extracted following
this procedure strongly depends on the intrinsic character- Gelatin was first dissolved at 5 mg/ml in distilled water at
istics of the collagen molecules from each fish species. In a 60 8C and then three-fold-concentrated loading buffer
comparative study of gelatins extracted using acetic acid for containing b-mercaptoethanol was added. Protein samples
collagen swelling, gelatins from flat-fish species (sole and were heat-denatured 5 min at 90 8C and analysed by SDS-
megrim) presented the best gelling ability and the gels were PAGE according to Laemmli (1970) using 4% stacking gels
more thermostable than those from cold-adapted fish (cod and 5% resolving gels in a Mini Protean II unit (Bio-Rad
and hake). This different behaviour was explained by the Laboratories, Hercules, CA) at 25 mA/gel. The loading
amino acid composition, the a1/a2 collagen-chain ratio and volume was 15 ml in all lines. Protein bands were stained
the molecular weight distribution of the material extracted with Coomassie brilliant Blue R250. Type I and type III
from each skin (Gómez-Guillén et al., 2002). collagens from foetal calf skin were used as markers of a-
Lactic acid is a low molecular weight organic acid with a chain, b- and g-component mobilities.
significant number of food applications and with the great
advantage over other similar organic acids of being virtually 2.4. Gel strength
colourless, odourless and tasteless. In a preliminary study
using megrim skin, we have shown that lactic acid is also Gel strength was determined according to Gómez-Guil-
suitable for collagen extraction, although the resulting lén et al. (2002) using 6.67% gels (w/v) formed by
gelatin apparently presented slightly worse rheological dissolving the dry gelatin in distilled water at 60 8C and
properties than that extracted using acetic acid (Gómez-- cooling down the solution in a refrigerator at 7 8C
Guillén et al., 2001). For this reason, and taking into account (maturation temperature) for 16–18 h. The gel strength
the advantages that lactic acid presents over acetic acid was determined at 7 8C using samples with 3.3 cm diameter
concerning their organoleptic properties, in the present and 6 cm height on an Instron model 4501 Universal Testing
study we further analyze the use of lactic acid for gelatin Machine (Instron Co., Canton, MA, USA) with a load cell of
extraction and compare the rheological, chemical and 100N, cross-head speed 1 mm/s, and equipped with a
structural characteristics of the extracted material in 1.27 cm-diameter flat-faced cylindrical Teflonw plunger.
comparison with that extracted using acetic acid. For this Gel strength was expressed as maximum force (in g),
purpose, skins from Dover sole (Solea vulgaris), a species obtained when the plunger had penetrated 4 mm into the
which has been proved to offer a good potential for gelatin gelatin gels; data represent the average (GSD) of five
manufacture, were used. determinations.
B. Giménez et al. / Food Hydrocolloids 19 (2005) 941–950 943

2.5. Viscoelastic properties 2.7. Kinetics of aggregation at 314 nm

Dynamic studies were performed on a Bohlin CRS-10 Aggregation of gelatin molecular components during gel
controlled stress rheometer rotary viscometer (Bohlin formation was analysed by monitoring changes in absor-
Instrumments Ltd, Gloucestershire, UK) using a cone- bance at 314 nm during the cooling process. Dried gelatin
plate geometry (cone angle 48, gapZ0.15 mm). Solutions was dissolved in distilled water at 40 8C. In order to avoid
were prepared by dissolving dry gelatin in distilled water high initial absorbance values, it was necessary to dissolve
(6.67%, w/v) at 40 8C by mechanical stirring for 15–20 min. LAC-25 and LAC-50 gelatins at 0.5% (w/v). ACE-50
Some samples were then cooled down at 7 8C (maturation gelatin presented very low initial absorbance at this
temperature) for 16–18 h to analyse the effect of maturation concentration, and changes at 314 nm during the cooling
time on gelatin gel properties. Temperature ramps were process were almost negligible. Thus, a higher concen-
performed at a scan rate of 1 8C/min, frequency 1 Hz, and tration (2% w/v) was required in this case to monitor
oscillating applied stress of 3.0 Pa. The elastic modulus changes in absorbance. Samples were loaded into thermo-
(G 0 ), viscosity modulus (G 00 ) and the phase angle (d) were statized cuvettes at 40 8C and absorbance at 314 nm was
represented as a function of temperature. The ramps were monitored for 80 min using a UV-1601 spectrophotometer
implemented from 40 to 6 8C and back to 40 8C for (Model CPS-240, Shimadzu, Japan). Temperature was
dissolved gelatin, to study gelatin gelation and subsequent gradually lowered down to 7 8C at approximately
melting. Gelatin matured overnight (16–18 h) at 7 8C was 1 8C/min and maintained thereafter at this temperature.
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 3. Results and discussion
in different determinations of a single sample was 6%
or less. 3.1. Gelatin extraction

2.6. Circular dichroism Gelatin preparations were performed under identical

conditions with the exception of the acid pre-treatment. A
Gelatin samples were dissolved at 6.67% or 0.5% (w/v) similar yield, expressed as grams of dry gelatin per 100 g of
at 40 8C, and loaded into preheated 0.01-cm pathlength clean skin, were obtained by using a 50 mM concentration
thermostatized cuvettes. Circular dichroism (CD) spectra of acetic or lactic acid for collagen swelling (ACE-50:
were recorded between 200 and 260 nm in a Jasco J-715 21.35%; LAC-50: 20.39%), whereas a slightly lower yield
spectropolarimeter (Jasco Corp., Tokyo, Japan) equipped was obtained with 25 mM lactic acid (LAC-25: 18.14%).
with a Neslab RTE-111 thermostat. In order to evaluate the When 10 mM lactic acid was used for swelling, no gelatin
influence of temperature on triple helix formation, molar was extracted, which confirms that a suitable mild acid pre-
ellipticity of the gelatin preparations at 6.67% (w/v) was treatment is essential for gelatin extraction at such moderate
monitored at 229 nm due to the impossibility to use lower temperature (45 8C).
wavelengths at this protein concentration. When gelatin All the gelatin preparations presented a similar behaviour
preparations were analyzed at 0.5% (w/v), molar ellipticity concerning solubility in distilled water. However, prep-
changes were monitored at 220 nm (maximum of the arations obtained by pre-treatment with lactic acid showed
spectra and characteristic of the presence of triple helix). higher light scattering than those obtained with acetic acid.
Temperature was first decreased from 40 to 4 8C at 1 8C/min The higher turbidity of these preparations is not due to the
to allow gel formation in the cuvette. Once this temperature presence of traces of lactic acid in the gelatin preparations,
was reached, spectra were recorded at 4 8C, and afterwards as we have verified that addition of lactic acid to ACE-50
temperature was raised at the same rate up to 40 8C either gelatin does not induce light scattering. Thus, skin swelling
immediately afterwards or after no further apparent using lactic acid must induce the solubilization of material
variation in the molar ellipticity at 229 or 220 nm was showing a higher aggregation degree.
detected (around 1 h). Mid-point transition temperatures
were obtained from the maximum or minimum of the first 3.2. Amino acid composition
derivative from the temperature curves after noise reduction
using a Fourier-transformed-based utility included in the The amino acid composition of the three different gelatin
spectropolarimeter software package. Statistical analysis of preparations is shown in Table 1. In general, composition is
the differences between mean melting or folding tempera- quite similar to that of interstitial collagen, with a high
tures in the different gelatin preparations was evaluated by glycine (Gly) and imino acid content (34–36% and 17–18%,
using Student’s t-test (SigmaPlot 8.02, Systat Software Inc., respectively). Only small differences could be detected
Erkrath, Germany). Protein concentration was determined among these gelatins; a slightly higher hydroxyproline
by amino acid analysis of triplicate samples obtained from (Hyp) content was found in LAC-50 while the overall imino
gelatin preparations taken out directly from the CD cuvette. 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) 941–950 945

skin swelling. The highest strength was achieved in ACE-50 However, LAC-50 gelatin showed a two-fold-decrease in G 0
gels (158.7G11.23 g), followed by LAC-25 (118.17G and only a 1.8-fold increase in G 00 . Thus, cold maturation of
2.66 g). In contrast, a very soft gel was obtained when the gels is quite different in LAC-50 gelatin compared to the
using LAC-50 gelatin (11.70G0.14 g). These differences other two preparations. This fact is also reflected in a
could be explained by differences in molecular weight significantly higher thermal stabilisation of the LAC-25 and
distribution rather than in amino acid composition without ACE-50 gels, as deduced from the variation in the phase
disregarding the existence of additional factors that may angle upon heating the matured gels (Fig. 3C). Thus, the
influence this parameter. According to the gel in Fig. 1, melting temperature of matured gels is around 29–30 8C for
ACE-50 gelatin presents the larger amount of covalently- ACE-50 (8 8C higher) and 27–28 8C for LAC-25 (7 8C
linked polymers followed by LAC-25 preparation. The higher). Melting of cold-matured LAC-50 gels is charac-
existence of these oligomers may enhance the renaturation terised by a loss of sharpness in the phase angle transition
ability of these preparations versus LAC-50, allowing a (Fig. 3C), with an initial slow rise in this parameter from 10
better establishment of stabilising interactions and the up to 20–21 8C, followed by a sharper final increase; the
formation of more organised triple helical structures melting temperature is around 238C, only 4–5 8C higher
(Sims, Bailey, & Field, 1997; Stainsby, 1987). Moreover, than before cold-maturation.
the differences in gel strength between ACE-50 and LAC-25 The different behaviour observed in the variation of the
could be explained in terms of differences in molecular viscoelastic properties of LAC-50 gelatin with temperature
weight profile. On the other hand, the extremely low gel and with cold maturation in comparison with LAC-25 and
strength of LAC-50 gelatin preparation could be the ACE-50 could be explained by the higher fragmentation that
consequence of more extensive hydrolysation. This would this gelatin preparation presents. Even though nucleation
act hindering both, the formation of nucleation sites, and the sites probably exist in LAC-50 gelatin, the high fragmenta-
annealing of collagen chains by the growth of existing tion of a-chains probably impairs the growth of the existing
nucleation sites during overnight stabilisation (Ledward, ones by further annealing of collagen chains during cooling
1986; Normand, Muller, Ravey, & Parker, 2000). or maturation. This behaviour is also in good accordance
with the extremely low gel strength values observed for
3.5. Viscoelastic properties LAC-50 gels. On the other hand, LAC-25 and ACE-50
gelatins present a significantly higher percentage of intact
In order to further analyse the quality of the three gelatin a-chains and covalently-linked aggregates, allowing in both
preparations, their viscoelastic properties after dissolving cases not only the existence of nucleation sites, but also their
them at 6.67% (w/v) in distilled water were studied. Fig. 2 growth by further annealing or chain association during cold
represents the modulus of elasticity (G 0 ), modulus of maturation.
viscosity (G 00 ) and phase angle (d), during both cooling
(from 40 to 6 8C) and subsequent heating (from 6 to 40 8C). 3.6. Circular dichroism
Among the three gelatin preparations, LAC-50 presented
the least gelling ability, showing a significant lower increase The transition from random coil structure to collagen-
in G 0 and G 0 upon cooling than ACE-50 and LAC-25, which like triple helix has been proposed to be one of the major
presented quite similar behaviour (Fig. 2A and B). The events either in triggering or, at least, in the stabilisation of
analysis of the variation of the phase angle revealed that the gelatin gels. We have monitored the folding of gelatin
onset of gelling was also lower in LAC-50 gelatin (around chains into triple helical structures by CD spectroscopy in
12 8C) than in the other two preparations: gelling begins at the far UV region using gelatin solutions under identical
around 15 8C in LAC-25 and 16 8C in ACE-50. These conditions as those described for the analysis of
differences were also observed when the viscoelastic the viscoelastic properties (gelatin concentration 6.67%,
properties upon heating from 6 to 40 8C were analysed. w/v). Additionally, we have analysed the folding
Whereas LAC-25 and ACE-50 behaved in a similar way, and unfolding of these preparations at a lower protein
LAC-50 again showed lower G 0 and G 00 values than the concentration (0.5%, w/v).
other gelatins. The thermal stability of the gels was also Fig. 4 shows the CD spectra of the three gelatin
decreased in LAC-50, which showed a melting temperature preparations at 6.67% and 0.5% (w/v). Spectra are shown
between 18 and 19 8C, 2 and 3 8C lower than LAC-25 and in the unfolded state at 40 8C and after cooling down to 4 8C
ACE-50, respectively. and allowing stabilization of the molar ellipticity, a process
The viscoelastic properties of the gelatin preparations that was achieved in general after 1 h maturation at 4 8C.
was also analysed after cold maturation at 6 8C for 18 h and As observed in Fig. 4A, it is impossible to analyse the
further heating from 6 to 40 8C (Fig. 3). A striking fact is spectra below 225–228 nm at high protein concentration
that cold maturation induced a highly significant increase in even using a very small pathlength cuvette. However, at a
the modulus of elasticity (Fig. 3A; around two-fold lower concentration (0.5%, Fig. 4B) spectra can reach as
increase) and in the modulus of viscosity (Fig. 3B; around low as 200 nm without introducing artefacts. Spectra
five-fold increase) in LAC-25 and ACE-50 preparations. 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 gelatin concentration in this processes. Fig. 5 shows the
at 4 8C present a maximum at 220 nm that is characteristic variation in the molar ellipticity at 229 nm (6.67% gelatin
of triple helical conformation (Engel, 1987), as observed in solutions; left panels) and at 220 nm (0.5% gelatin
gelatin preparations at 0.5% (w/v). Preparations at 6.67% solutions; right panels) during cooling from 40 to 4 8C
(w/v) not only show a similar behaviour than those at 0.5%, (a curves) and subsequent heating (b curves). Unfolding
but even higher molar ellipticities are obtained in the 225– curves were also obtained after allowing stabilisation of the
250 nm region. This indicates that an increase in gelatin molar ellipticity at 4 8C (around 1 h; c curves). The different
concentration is paralleled by the achievement of a higher wavelength used is due to the impossibility to register
percentage of triple helical conformation, as we previously ellipticity at 220 nm (maximum of collagen triple helix
reported for fish-skin gelatins from several marine species spectrum) at 6.67% protein concentration.
(Gómez-Guillén et al., 2002). However, molar ellipticity in In all preparations, a slow gradual increase in molar
this region is always lower than that reported for intact ellipticity was observed upon cooling from 40 to 4 8C,
collagen molecules purified from fish skin (Ogawa et al., indicating that folding towards the formation of triple
2003) or from other origins (Engel, 1987), [q]MRW(220 nm) helical structures is taking place. In general, no highly
z10,000 deg cm2 dmolK1. When the different gelatin significant differences were detected during cooling of the
preparations are compared, no significant differences can three gelatins, even though the mid-point for the ellipticity
be found among them neither at 40 8C nor at 4 8C after change in LAC-50 was around 1 8C below that of ACE-50
stabilisation. Thus, the overall conformation of the different and LAC-25 (see Fig. 5, a curves, left panels). When this
gelatins analysed at the initial and final stages of the cooling process was analyzed at a 0.5% gelatin concentration, lower
and heating ramps are quite similar, even though the temperatures were required in all the preparations for the
transition from the unfolded to the folded state (and vice folding process to take place. This fact again confirms the
versa) may differ among them, as described below. importance of protein concentration for gelatin refolding.
We have analysed by CD the gelatin folding process When gelatin unfolding was studied, more significant
from random coil to a triple helical-rich structure in changes among the different preparations were observed.
identical conditions to those reported for the analysis of Whereas ACE-50 and LAC-25 gelatins presented a quite
the viscoelastic properties (6.67%, w/v). Additionally, we similar behaviour with a melting temperature close to 20 8C,
have also monitored folding and unfolding at a lower LAC-50 showed a 2 8C lower value. Surprisingly, no
concentration (0.5%, w/v) in order to verify the relevance of significant differences were found in the melting
B. Giménez et al. / Food Hydrocolloids 19 (2005) 941–950 947

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
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, folding process but not in the unfolding of the triple helical
cooled down to 7 8C and maintained at this temperature for 18 h. Changes structures, as observed in Fig. 5. Finally, after the
in the modulus of elasticity G 0 (A), modulus of viscosity G 00 (B) and phase establishment of trimeric nucleation sites, propagation of
angle (C), were monitorized during heating from 7 to 40 8C. ACE 50: pre- the triple helical structure takes place. Even though
treatment with 50 mM acetic acid; LAC 25: pre-treatment with 25 mM
lactic acid; LAC 50: pre-treatment with 50 mM lactic acid.
hysteresis was found in all cases, the folding and melting
processes were completely reversible as verified from the
temperatures obtained at 6.67% and 0.5% gelatin concen- quite identical curves obtained after at least three cooling
trations. Moreover, in all the cases analysed, the melting and heating cycles.
temperature was identical disregarding whether heating The lower temperature required for LAC-50 folding
began immediately after the cooling ramp or if stabilisation compared to ACE-50 and LAC-25 gelatins could originate

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).
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 3.7. Kinetics of aggregation at 314 nm
nucleation centres, as this gelatin preparation appears to be
more degraded and with a lower degree of covalently cross- Association of denatured collagen/gelatin polypeptide
linked polymers than the other preparations (Fig. 1). Thus, chains may be involved in the nucleation step required for
interactions between three different polypeptide chains may triple helix formation and gelatin gels stabilisation. As this
be more difficult and, consequently, lower temperatures are association could increase the size of the solubilized gelatin
required for this process to take place. This could also be moieties, determination of changes in absorbance at 314 nm
directly related to its inferior gelling ability and gel strength. could allow the study of this process. LAC-25 and LAC-50
On the other hand, unfolding of LAC-50 gelatin occurs at gelatin preparations show a significantly higher absorbance
lower temperatures; this probably is due to the establish- than ACE-50, even though the latter shows in PAGE-SDS a
ment of shorter and less stable triple helical regions in this higher degree of covalently linked aggregates (Fig. 1). Thus,
gelatin preparation due to increased chain fragmentation, considering that the chemical composition of the different
even though the total amount of peptide bonds showing gelatins is quite similar, non-covalent interactions must be
triple helical conformation must be quite similar among the taking place in LAC gelatins increasing the turbidity of
gelatin preparations as no significant differences in the final these preparations. As LAC-50 is the worst folding gelatin,
molar ellipticity at 4 8C is detected (Figs. 4 and 5). the aggregation responsible for the high absorbance at
When folding curves are compared with the phase angle 314 nm must be different from that required for the
variation upon cooling, a delay in the appearance of triple nucleation process, lacking probably the correct chain
helical structures is observed. The onset of gelation always organization.
precedes the increase in molar ellipticity; moreover, the Fig. 6 shows the behaviour of absorbance at 314 nm
folding process continues after gelling has been completed during cooling from 40 to 7 8C of the different
(Figs. 2 and 5). Thus, it could be suggested that additional gelatin preparations at 0.5% (w/v) and ACE-50 at 2%
interactions must take place among gelatin polypeptide (w/v). Initially, temperature was lowered at approximately
chains that trigger the gelling process and enhance the 1 8C/min, allowing afterwards around 1 h stabilisation at
nucleation step required for triple helix formation. 7 8C. The upper panel of Fig. 6 shows a detail of
B. Giménez et al. / Food Hydrocolloids 19 (2005) 941–950 949

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
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
using 50 mM acetic acid avoiding the negative organoleptic
loaded into pre-heated cuvettes. Changes in the absorbance at 314 nm were properties of that acid. Lactic acid seems to be a more
monitorized during cooling from 40 to 7 8C at approximately 1 8C/min and efficient disrupter of the skin tissue and collagenous
for a total time of 80 min. The upper panel shows a detail of the variation of structures probably due to its higher dissociation constant
A314 with temperature, showing the mid-point transition temperatures for and to the presence of hydroxyl group that may establish
each gelatin preparation.
additional hydrogen bonds that contribute to collagen
the variation in absorbance at 314 nm with temperature. A denaturation. Moreover, care must be taken to avoid the
sharp variation in absorbance is observed upon cooling extraction of highly fragmented material that may impair the
viscoelastic properties of the final gelatin preparation. We
between 9 and 8 8C in LAC-25 and LAC-50 gelatins at 0.5%
have performed a profound comparative analysis of the
(w/v) and in ACE-50 at 2% (w/v); the mid-point
viscoelastic properties of gelatin extracted by using 25 and
temperature for these transitions are shown in the upper
50 mM lactic acid and 50 mM acetic acid for skin swelling
panel of Fig. 6. Additionally, LAC-50 gelatin shows a
and also studied the molecular conformation changes that
further increase in A314 during incubation at 7 8C. Taking
take place during gelation and melting of gelatin gels. In
into account that this gelatin preparation is the one
conclusion, the use of 25 mM lactic acid for skin swelling has
presenting lower gel strength after overnight maturation at
proven to be an excellent substitute for acetic acid, yielding
7 8C, it can be suggested that the aggregation responsible for
gelatin preparations with quite similar properties to that
the increase in absorbance at 314 nm does not contribute to
obtained with 50 mM acetic acid.
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 Acknowledgements
absorbance values, which do not vary after temperature has
reached 7 8C. This research was financed by the Comunidad de Madrid
The increase in absorbance at 314 nm seems to take place under project 07G/0052/00 and by the Spanish Dirección
General de Investigación under project BMC02-1407.
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).
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