Food Hydrocolloids 18 (2004) 203–213
www.elsevier.com/locate/foodhyd
Physical and rheological properties of fish gelatin compared
to mammalian gelatin
Ingvild J. Haug*, Kurt I. Draget, Olav Smidsrød
Norwegian Biopolymer Laboratory (NOBIPOL), Institute of Biotechnology, Norwegian University
of Science and Technology (NTNU), Sem Sælandsvei 6/8, N-7491 Trondheim, Norway
Received 14 November 2002; accepted 11 April 2003
Abstract
This study comprises characterisation of fish gelatin (FG) from cold water fish species, including rheological and optical rotation
measurements. SEC-MALLS analysis revealed that fish gelatin is heterogeneous in molecular compositions and that it mainly contains aand b-chains. Fish gelatin gave gels with a considerably lower storage modulus, G0 ; gelling (4– 5 8C) and melting temperature (12– 13 8C)
compared to mammalian gelatin gels. This is probably due the lower content of proline and hydroxyproline in fish gelatin. Recording the
storage modulus for 10 (w/w)% FG at various ionic strengths showed that G0 increased at low ionic strengths, while decreasing at medium to
high salt concentrations. This suggests that electrostatic interactions are important in the stabilisation of the gelatin gel network. This
suggestion was further supported by a partly reversible lowering of the gel modulus by the neutralisation of the carboxyl and amino groups at
low and high pH, respectively. Optical rotation experiments clearly showed the importance of the amount of Pro and Hyp present in the
gelatin. The degree of chain segment ordering at the gelling temperature in fish gelatin (at 5 8C) and mammalian gelatin (20 8C) was almost
identical. This clearly showed the importance of the content of imino acids for the formation of some ordered structures and stabilisation of
the gelatin gel network.
q 2003 Elsevier Ltd. All rights reserved.
Keywords: Fish gelatin; Rheology; SEC-MALLS; Electrostatic interaction; Optical rotation
1. Introduction
Gelatin is one of the most versatile and utilised gelling
agents in food applications due to its special texture and the
‘melt-in-mouth’ perception. In addition to foodstuffs,
gelatin has found a variety of applications in the
pharmaceutical and photographic industry. Generally,
gelatin is produced from skin or bone collagen by acid or
alkali treatment to give type A and type B gelatins,
respectively (Veis, 1964; Ward & Courts, 1977).
Collagen is a fibrous protein and the most abundant
protein in animals. The collagen molecule is a right
handed helical rod made up from three parallel a-chains,
which are intertwined. One turn in the collagen superhelix is made up from three amino acid residues. The
general amino acid sequence in the a-chain is Gly-X-Y
where X often is proline and Y often is hydroxyproline.
* Corresponding author. Tel.: þ 47-73-59-1689; fax: þ47-73-59-1283.
E-mail address: ingvild.haug@chembio.ntnu.no (I.J. Haug).
0268-005X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0268-005X(03)00065-1
The a-chain forms a left-handed helix due to the
presence of Pro and Hyp, which give the chain its
kinks and turns and help stabilising the secondary
structure of the single helix. The collagen super-helix
takes up a trans poly-L -proline II conformation and is
believed to be stabilised through inter-chain hydrogen
binding between amide groups of glycine and carbonyl
oxygen in the neighbouring chains.
It has been discussed if there are one or two hydrogen
bonds involved per amino acid triplet and whether water
participates in these hydrogen bonds. It is generally believed
that there is one direct hydrogen bond involved per triplet.
Several publications support that water is an integral part of
the hydrogen bond, but that it could be involved in a second
hydrogen bond, which may also be an integral part of the
collagen structure. The OH-group on the Hyp can
additionally stabilise the triple helical areas (supposed to
be the junction zones) through inter-chain hydrogen bond
(Ledward, 1986; Piez & Gross, 1960; Veis, 1964; Ward &
Courts, 1977).
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In gelatin, parts of the collagen structure are regenerated
when the temperature is lowered below the coil – helix
transition temperature. The pyrrolidine-rich regions act as
nucleation sites for formation of potential junction zones
(Harrington & Rao, 1967; Harrington & Rao, 1970) and the
length of a junction zone has been proposed to be at least
20 – 30 amino acids (Harrington & Rao, 1967). It is
generally believed that the junction zones in gelatin are
stabilised by hydrogen bonds similar to those in native
collagen. The junction zones are interconnected through
flexible peptide chains (elastic segments).
Theoretical treatments of a gelatin gel mostly assume
that the junction zones are individual triple helices
(Ledward, 1986), but at least some of the junctions can
be composed of several triple helices aggregated together
(Boedtker & Doty, 1954; Veis, 1964). The aggregation of
gelatin molecules is, however, not universally accepted
(Busnel, Morris, & Ross-Murphy, 1989; Ross-Murphy,
1997). Ross-Murphy (1997) reports that inter-chain
aggregation, which frequently occurs in other biopolymer
gelling systems, is of minor importance for gelatin. The
melting and gelling temperature of gelatin have been
found to correlate with the proportion of Pro and Hyp in
the original collagen (Ledward, 1986; Piez & Gross,
1960; Veis, 1964).
Gelatin from marine sources (fish skin, bone and fins)
has been looked upon as a possible alternative to bovine
and porcine gelatin, especially since the outbreak of the
BSE (‘mad cow disease’) in the 80s. Search for new
gelling agents to replace mammalian gelatin led to
patents for fish gelatin production (Grossman & Bergman
1992; Holzer, 1996) as well as several published methods
for fish gelatin production (Gómez-Guillén & Montero,
2001; Gudmundsson & Hafsteinsson, 1997; Nagai &
Suzuki, 2000). Recently, harp seal also has been
considered as raw material for gelatin production
(Arnesen & Gildberg, 2002).
The commercial interest in fish gelatin has this far,
however, been relatively low. This is due to sub-optimal
physical properties compared to mammalian gelatin.
Common problems connected with fish gelatin from cold
water species, representing the majority of the industrial
fisheries, are low gelling and melting temperature and low
gel modulus (Leuenberger, 1991). This makes these gelatins
unsuited as mammalian gelatin replacements, especially
since they typically gel below 8 8C (Leuenberger, 1991;
Norland, 1990). The differences in the physical properties
between mammalian gelatin and gelatin from cold water
species are due to a lower content of the imino acids Pro and
Hyp. Calf skin gelatin contains approximately 94 Hyp and
138 Pro residues per 1000 total amino acid residues, while
cod skin gelatin contains approximately 53 and 102 amino
acids of Hyp and Pro, respectively, per 1000 total residues
(Piez & Gross, 1960). Gelatins from warm water fish
species, like fish gelatin from tilapia, contains circa 70 and
119 residues of Hyp and Pro, respectively, per 1000 total
residues, and have physical properties more equal to those
of mammalian gelatins (Sarabia, Gómez-Guillén, & Montero, 2000). Harp seal gelatin also contains almost the same
amounts of Hyp and Pro as bovine gelatin, and therefore has
properties very similar to those of bovine origin (Arnesen &
Gildberg, 2002). Quantitatively, however, fish gelatin from
cold water fish species is still preferred due to the greater
availability of by-products (e.g. skin and bone) from which
it can be manufactured. Collagen from fish has just recently
been identified as a potential allergen and could possible
become a problem for the use of fish gelatin in commercial
products (Hamada, Nagashima, & Shiomi, 2001; Sakaguchi
et al., 1999).
The scope of the present paper was to take on a
comparative study between gelatin from cold water fish
species and published data on fish and mammalian gelatin.
This is in order to reveal differences in physical and
rheological properties between mammalian gelatin and fish
gelatin and to decide whether these differences can be
explained by the lower content of imino acids in fish gelatin.
The object of the present paper is also to study the gelling
mechanism of fish gelatin at different concentrations, pH
and ionic strengths.
2. Materials and methods
2.1. Fish gelatins
The fish gelatin samples were kindly provided by
Norland Inc., USA. Norland HMW fish gelatin is a type A
gelatin, produced from skins of cold-water fish species such
as cod, haddock and pollack. The gelatins have a high
degree of purity and high molecular weight (Norland Prod.
Inc., 1999– 2001). Three different fish gelatin batches were
used and labelled FG1, FG2 and FG3. The batch numbers
and the isoelectric point (IEP) for the gelatins are given in
Table 1.
2.2. Mammalian gelatin
The mammalian gelatin from cattle hide (DGF Stoess, lot
232635) was a type B gelatin. The IEP was typical 4.9– 5.2.
The weight average molecular weight was 171 kDa (DGF
Stoess, 1999).
Table 1
Isoelectric point and batch number for applied fish gelatins (Norland Prod.
Inc., 1999–2001)
Sample
Batch
IEP
FG1
FG2
FG3
7394
9187
0101
7.8
8.7
7.8
I.J. Haug et al. / Food Hydrocolloids 18 (2004) 203–213
205
The amount of the ions Naþ, Kþ and Ca2þ were
quantified on a Perkin Elmer 560 Atomic Absorption
Spectrophotometer. Standard curves were prepared from
absorption at five different concentrations, and accepted if
R2 . 0:98: Solutions of 10 (w/v)% FG were made and
diluted to give a series of solutions (1, 0.1 and 0.01 (w/v)%).
the frequency was 1 Hz. The shear stress varied depending
on the test sample, and was chosen from stress sweep
experiments. The start and end temperature was 25 8C,
while the curing temperature was 4 8C. A sample of 2.5 ml
was applied to the serrated plate/plate geometry, and 1.3 ml
to the cone and plate geometry. The sample was covered
with low viscosity silicone oil (BDH Silicone Products,
KeboLab—10cSt at 20 8C) to prevent evaporation.
2.4. Ash and water content in fish gelatin powder
2.8. pH adjustments
Ceramic beakers were dried at 104 8C over night to
remove water. The beakers were placed in a vacuum
incubator to reach room temperature and the precise weights
were recorded. Approximately 1.0 g of FG sample was
applied to the ceramic beakers, the beaker with the sample
was dried over night at 104 8C, cooled down in a vacuum
incubator and the exact weight was registered. Subsequently, the beakers with sample were combusted at
530 8C over night, cooled in vacuum incubator and the
amount of ash evaluated.
The pH in the FG solution was adjusted with HCl or
NaOH. The added amounts of acid or alkali were less than
120 ml in 10 ml, imposing a negligible reduction in the
original FG concentration.
2.3. Atomic absorption
2.5. SEC-MALLS
The molecular weight distribution and polydispersity
index were determined from light scattering, SEC-MALLS
(TSK pre-column þ 4000 PWXL-DAWN DSP/Optilab
DSP). The eluting buffer was 0.05 M Na2SO4, 0.01 M
Na2EDTA and 0.025 M tris-base. The pH of the buffer was
approximately one pH-unit above IEP. A 10 mg/ml fish
gelatin (FG) sample was mixed with equally amounts of
double concentrated buffer to obtain a fish gelatin solution at
correct ionic content.
Two of the samples were ultra-centrifuged on a Sorvall
Ultraspeed Centrifuge at 40,000 rpm. (TY70.1) for 1.5 h to
remove aggregates and injected into the SEC-MALLS.
2.6. Intrinsic viscosity
A 0.2 (w/v)% FG solution was made and mixed with
equal amounts of 0.2 M NaCl. During the measurements the
FG solution was diluted with 0.1 M NaCl to keep the ionic
strength constant while reducing the fish gelatin concentration, and the intrinsic viscosity was determined by
extrapolating to zero concentration. The viscometer was a
type Schott-Geräte 531 01/0a. The temperature was kept
stable at 30.0 ^ 0.1 8C during the measurements.
2.9. Ionic strength
Different ionic strengths in the FG solutions were
obtained by adjusting with 1.0 M NaCl and keeping a
constant FG concentration. After addition of salt, the
solutions were applied to the rheometer and G0 ; G00 and
phase angle were recorded as described above.
2.10. Optical rotation
FG was dissolved in MQ-water until the desired
concentration; 2, 4, and 6 (w/v)%. The solutions were
filtered (pore size of 0.8 mm), applied in a micro cell
(l ¼ 10 cm; d ¼ 5 mm) and the optical rotation spectra were
collected at 436 nm. The optical rotation apparatus (Perkin
Elmer 241) was connected to a temperature bath (Haake)
filled with silicone oil (Haake Bath Liquid SIL 180, stable
from þ 200 to 2 40 8C).
The temperature was quenched down to a temperature
where fish gelatin is known to undergo coil –helix transition
and then kept constant for 5 h. By repeating the experiments
at increased temperatures it could be possible to identify the
coil –helix transition temperature for FG. A 6 (w/v)% FG3
solution was held at 5, 6 and 11 8C. After maturing for 5 h
the temperature was increased to 25 8C, and the helix – coil
transition was followed by recording the optical rotation at
each degree Celsius until 25 8C. The optical rotation value at
25 8C was assumed to be the rotation of 100% random coil
conformation.
3. Results and discussion
2.7. Small-strain oscillatory measurements
3.1. Chemical composition
The small-strain oscillatory measurements were performed on a StressTech Rheometer from Reologica, Lund,
Sweden. Measurements on FG were carried out on a 40 mm
serrated plate/plate geometry with 1 mm gap and with the
cone and plate geometry (d ¼ 40 mm). The temperature
gradient was 0.5 8C/min both on cooling and heating, while
The ionic content of potassium, sodium and calcium in
the fish gelatins are given in Table 2 and are, as excepted,
low. It was not possible to detect potassium ions in the FG
solutions, and sodium was the most abundant ion in both fish
gelatin samples. It was assumed that since the excess
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Table 2
Ionic content in 10 (w/v)% fish gelatin solutions
Sample
Naþ (mM)
Ca2þ (mM)
Kþ (mM)
FG2
FG3
8.0
8.2
1.1
0.52
,0.005
,0.005
content of ions was minimal, the ionic content would not
contribute significantly to the ionic strength of the fish
gelatin solutions.
The content of ash and water in FG3 were found to be
0.82 and 12.9 (w/w)%, respectively. From the atomic
absorption values the amount of metal oxides left after
combustion could be calculated and for FG3 the values
became 5.08 mg Na2O and 0.29 mg CaO in 1.0 g fish
gelatin. In addition the producer reports traces of heavy
metals and chromium, which have not been included in the
calculations. The calculated mass of metal oxides seemed to
be in good agreement with the ash content, which was
totally 7.13 mg metal oxides in 1.0 g fish gelatin.
Table 3 gives the approximate amino acid composition
for the fish gelatin and mammalian gelatin. The main
difference between the two gelatins is, as pointed out earlier,
the content of Hyp and Pro.
Fig. 1. Molecular weight distributions from SEC-MALLS analysis of FG1
for (a) ultra-centrifuged and standard sample at room temperature, and (b)
standard sample analysed at room temperature and 60 8C.
3.2. Molecular weight characterisation
Fig. 1 gives the molecular weight distribution for FG1.
The weight average molecular weights for the three fish
Table 3
The approximate amino acid composition in Norland Fish gelatin extracted
from cold water fish skins and from calf skins (Norland Prod. Inc.,
1999–2001)
Amino acids
Ala
Arg
Asp
Cys
Glu
Gly
His
Hyl
Hyp
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Try
Tyr
Val
Residues/1000 amino acids
Fish gelatin
Mammalian gelatin
112
49
48
–
72
347
11
5
60
11
21
28
13
13
96
63
24
–
9
18
114
51
45
–
71
313
5
11
86
11
25
34
6
13
135
37
18
–
3
22
gelatins are given in Table 4. The SEC-MALLS experiments revealed that the fish gelatin, as expected, contained
components with different molecular weights. It has
previously been found for mammalian gelatins that the
broad molecular weight distribution is probably due to the
production process, which can give single a-chains, two achains covalently cross-linked to give b-chains, and three
covalently cross-linked a-chains named g-chains (Veis,
1964). These three types of chains have been identified by
electrophoresis and chromatography both for mammalian
and fish gelatins (Gómez-Guillén et al., 2002; Norland,
1990), and the weight average molecular weight of one achain was reported to be between 95 and 100 kDa (Norland,
1990; Piez, 1968; Veis, 1964). The molecular weight of one
a-chain can be predicted from the fact that one aa-chain
consists of approximately 1000 amino acid residues with an
average molecular weight of 110 g/mole, giving a molecular
Table 4
SEC-MALLS and intrinsic viscosity data for the fish gelatins
Sample
Mw (kDa)
Mw =Mn
½h (ml/g)
FG1
FG2
FG3
199 ^ 18
170 ^ 19
140 ^ 8
2.6
3.4
2.1
42
47
42
I.J. Haug et al. / Food Hydrocolloids 18 (2004) 203–213
weight of approximately 110 kDa for one a-chain (Veis,
1964; Ward & Courts, 1977).
The molecular weight distribution curves in Fig. 1(a) and
(b) have two distinctive maxima, at around 100 and
200 kDa. The first maximum at , 100 kDa fits with the
size of one a-chain, whereas the second maximum at
, 200 kDa could be due to b-chains. The second maximum
has a small shoulder and a tail indicating molecules with
higher molecular weights. This could be due to the presence
of g-chains and aggregates, respectively. The molecular
weight distribution indicates that the fish gelatin sample
analysed at room temperature contains mainly a-chains,
approximately 65 (w/w)%, and very small amounts of gchains (1.5 (w/w)%) and high molecular weight aggregates
(0.07 (w/w)%). This small amount of high molecular weight
components will, however, give a considerable contribution
to the weight average molecular weight.
Fig. 1(a) shows the molecular weight distribution for a
standard FG sample and an ultra-centrifuged FG sample
analysed at room temperature. The offset of the curves is
due to differences in injected mass. The amount of a-chains
compared to the total mass of the ultra-centrifuged sample
seems to be slightly higher than for the standard sample, and
the molecular weight is shifted towards lower values. This
could indicate breakage of covalent bonds in some of the
high molecular weight components. The ultra-centrifuged
sample also has a tail after the second maximum, which
probably means that ultra-centrifugation did not totally
remove high molecular weight aggregates.
The fish gelatin samples were analysed at different
temperatures from ambient temperature to 60 8C as shown
in Fig. 1(b). At 60 8C the molecular weight distribution
seems to have a higher and narrower maximum at 100 kDa
indicating a higher fraction of a-chains. Accordingly, the bchain signal decreases and the shoulder on this signal
become less visible. This could be due to splitting of
multiple chains into single a-chains at the elevated
temperature.
207
3.4. Gel properties—effect of temperature
The main differences between fish and mammalian
gelatins are the gel modulus and the gelling and melting
temperature. Gelling and melting temperatures have
previously been defined in different ways in the literature.
In this rheological study the gelling and melting temperature
are taken as the temperatures were the phase angle is at its
transition point, d ¼ 458 (tan d ¼ 1), upon cooling or
heating at a temperature gradient of 0.5 8C/min and a
frequency of 1 Hz. Differences in storage modulus, G0 ; is
illustrated in Fig. 2(a) where a 10 (w/w)% FG1 and 10 (w/
w)% cattle hide gelatin (BG) were investigated with a cone
and plate geometry. Fig. 2(b) displays the changes in phase
angle with varying temperature, and shows that BG gels at
approximately 24 8C, while the corresponding value for FG
is 4.5 8C. The storage modulus, G0 ; is higher for the
mammalian gelatin at both 20 and 4 8C compared to fish
gelatin, and at 4 8C the modulus is as much as approximately 10 times higher for the mammalian gelatin. In
3.3. Hydrodynamic properties
The intrinsic viscosity for fish gelatin, reflecting the
specific hydrodynamic volume of the dissolved gelatin, is
given in Table 4. Pouradier and Venet (1952) plotted the
intrinsic viscosity for two mammalian gelatins, one type A
and one type B, against the number average molecular
weight. The resulting plot gave two straight lines, one for
the type A gelatin and one for the type B gelatin. The fish
gelatin samples in this study are type A gelatins and FG1,
with Mn ¼ 76 kDa and ½h ¼ 42 ml=g; gives a point which
nicely fits onto the published line for the type A mammalian
gelatin. This implies that fish gelatins behave like the
mammalian gelatins with respect to the molecular weight
and hydrodynamic volume.
Fig. 2. Small-strain oscillatory measurements of 10 (w/w)% FG1 and
10 (w/w)% bovine gelatin (BG) on cone and plate geometry showing
difference in G0 at 4 8C (a) and (b) gelling and melting temperature.
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I.J. Haug et al. / Food Hydrocolloids 18 (2004) 203–213
a linear plot it can be seen that both gelatins give rise to
increasing G0 ðtÞ when kept at 4 8C, being typical for nonequilibrium gels. The increase with time is probably due to
reorganisation or growth of junction zones in the gelatin gel
network and cis –trans isomeriation in the a-chains which
slows down the triple helical propagation (Busnel et al.,
1989; Ledward, 1992; Veis, 1964). The fish gelatin and BG
melt at 13 and 30 8C, respectively. The lower G0 and gelling
and melting temperature have previously been explained by
the lower content of the imino acids Pro and Hyp in fish
gelatin (Joly-Duhamel, Hellio, Ajdari, & Djabourov, 2002a;
Norland, 1990; Piez & Gross, 1960).
3.5. Gel properties—effect of gelatin concentration
Since the gelling of gelatin is a non-equilibrium
process, the storage modulus will increase with time at
the annealing temperature. Since some of the experiments
had different time scales, the storage modulus was
extrapolated to infinite time by plotting G0 against
1/time, giving G0infinite ; as shown in Fig. 3, to be able to
compare the experiments. The rheological properties of
FG solutions were tested and Fig. 4 gives G0infinite value at
4 8C for several concentrations of FG1.
In Fig. 4, the experimental G0infinite values for fish gelatin
were fitted to the cascade master functions. The theory for
gelation in point-like covalently cross-linked systems was
developed in the 1940s (Flory, 1941; Stockmayer, 1943),
but has been modified to fit physically cross-linked systems.
The fitting was performed as described by Clark and Farrer
(1995) and Clark and Ross-Murphy (1985). Recently,
Joly-Duhamel et al. (2002a) used a rigid polymer network
model (Jones & Marques, 1990) to compose a master curve
for the elasticity of gelatin gels. This choice of model is
somewhat dubious, and predicts that the elastically active
segments in the gelatin network are constituted by the
helical areas, and not by the random coil segments in
between the helical regions. One of the assumptions in this
network model is that the lengths of the rigid rods are much
Fig. 4. Storage modulus ðG0infinite Þ at 4 8C for different concentration of FG1
fitted to the cascade master function for f ¼ 5; 10 and 20.
larger than the mesh size in the network, which seems rather
questionable.
The generalised front factor a reflects deviations from
ideal rubber elasticity, and when a is close to unity the
system is close to ideal rubber behaviour. The functionality,
f ; is the number of sites available to form cross-links. The
molecular weight of one primary chain was chosen from
SEC-MALLS data to be 100 kDa. Table 5 gives the
calculated, minimum concentration needed to form a gel
at f ¼ 5; 10 and 20. The a-value shows a best fit for f ¼ 5
and 10, and a is closest to unity for f ¼ 10: This has
previously also been found for mammalian gelatin (Clark &
Ross-Murphy, 1985).
For f ¼ 5 and 10, c0 is approximately 2.6 (w/w)% at
4 8C, which is slightly higher than experimentally observed
(, 2%). Previously, c0 for cod gelatin has been found to be
up to 6 (w/w)% (Gilsenan & Ross-Murphy, 1999). For
mammalian gelatins, c0 has been reported to be 0.4 –1.2 (w/
w)% at room temperature (Clark & Ross-Murphy, 1985;
Gilsenan & Ross-Murphy, 2000b). This indicates that
higher concentrations of fish gelatin from cold water fish
species are needed to form a continuous network.
The gel modulus has been found to be proportional to c2
over a wide range of concentrations (Eldridge & Ferry,
1949; Gilsenan and Ross-Murphy, 2000a; Gilsenan and
Ross-Murphy, 2001; te Nijenhuis, 1981). For FG it
was found that G0 / c2 when c . 20%; while the exponent
was greater than 2 at lower concentrations. Clark and
Ross-Murphy (1985) found that the G0 / c2 correlation was
valid when c=c0 . 10 for mammalian gelatin at room
temperature, and this is also the case for the fish gelatin
sample.
Table 5
Values for a and c0 obtained by fitting G0infinite ðcÞ for FG1 to the cascade
master function
Fig. 3. Extrapolation to G0infinite for 10 (w/w)% FG1 at 4 8C measured with
the cone and plate geometry.
f
a
c0 (mg/ml)
5
10
20
1.813
1.1
0.482
25.9
25.7
16.7
I.J. Haug et al. / Food Hydrocolloids 18 (2004) 203–213
3.6. Gel properties—effect of pH
Solutions of 10 (w/v)% FG1 were adjusted to pH values
between 2 and 12, and the storage modulus, loss modulus
and phase angle were recorded on the serrated plate/plate
geometry. As can be seen from Fig. 5, the dynamic storage
modulus, G0 ; after 2 h at 4 8C was nearly constant between
pH 5 and 7, while G0 was slightly increasing between pH 7
and 10 (in a linear plot). It has previously been reported that
the dependence of gel rigidity on pH in the region 4 –10 for
mammalian gelatin is more pronounced at low gelatin
concentrations (Stainsby, 1987). Fig. 5 shows that it is
obvious that G0 for 10 (w/w)% FG1 is also quite dependent
on pH in the area 4 – 10.
Above pH 10, the storage modulus decreases,
probably due to deamination of acid amides and changes
in the charge density to give a high net negatively
charged polymer. The increased charge density can
oppose the ability of chains to make contact and form
junction zones and thus decrease the gel rigidity. The
two amino acids that are most likely to lose ammonia are
Gln and Asn, leading to an increase in the number of
negative charges on the chain. When FG samples with
pH higher than 10 were kept at room temperature over
night in a sealed tube the distinct smell of ammonia
could be noticed when the tube was opened.
The storage modulus at pH values below 5 also
decreased markedly. This is probably due to increased
net positive charge in the chains, which could inhibit
junction zone formation and therefore result in declined
gel rigidity. Fig. 5 also gives the amounts of added acid
and alkali. From the titration curve it can be seen that
FG has a weak buffer capacity between pH 5 and 9. Veis
(1964) gives the changes in gel rigidity on changing pH
for a 2.7 (w/w)% for a type B mammalian gelatin. The
storage modulus in Fig. 5, shows exactly the same
Fig. 5. Changes in G0 as function of pH at 4 8C (average ^ SD). The
amount of added HCl or NaOH to reach the different pH values in
10 (w/w)% FG1 is given as a titration curve.
209
dependence of pH as the type B gelatin described by
Veis (1964).
Solutions of 10% FG1 were adjusted to pH 12 and 3, and
kept over night at 4 8C before pH was adjusted back to the
ambient pH value (5.2). These samples formed weaker gels
compared to the original 10% FG sample, not measurable at
standard conditions in the rheometer. The storage modulus
was, however, probably in the order of a decade lower for
the sample adjusted to pH 12. Solutions adjusted to pH 3
formed gels with only a slightly lower storage modulus than
the original FG solutions. Hence, a partial recovery of
physical properties was observed. The reduction in G0
suggests that the gelatin could be degraded or chemically
changed at extreme pH values.
3.7. Gel properties—effect of ionic strength
The effect of ionic strength on the gel modulus is
presented in Fig. 6. The values of G0 in this figure are taken
after 2 h at 4 8C. When small amounts of salt were added to
the fish gelatin a slightly increase in G0 was observed. This is
probably due to a screening off of long range electrostatic
repulsion allowing tighter association of the gelatin achains into junction zones and promotion of electrostatic
bridging. When higher concentrations of salt were added,
the storage modulus rapidly decreased probably due to
screening off of short range electrostatic interactions and
hence decreasing the ability for a-chains to come into
contact and form electrostatic bridges. The storage modulus
was , 800 Pa when only the intrinsic salts were present.
Fig. 6 shows that addition of 12 mM NaCl gave G0 , 1200
Pa and 250 mM NaCl gave G0 , 600 Pa: Sarabia et al.
(2000) found a decrease in G0 at 0.5 M NaCl, while the gel
rigidity was almost unchanged at 0.1 M NaCl for megrime
skin gelatin. In Fig. 6 there is a decrease in storage modulus
Fig. 6. The storage modulus ðG0 Þ after 2 h at 4 8C for 10 (w/w)% FG1 with
increasing concentrations of NaCl. Measurements were performed on
serrated plate/plate geometry (average ^ SD).
210
I.J. Haug et al. / Food Hydrocolloids 18 (2004) 203–213
at ionic strengths above 0.05 M and this corresponds well
with the results for megrime gelatin. It is well known that
low concentrations of salt only have a small effect on
coacervation of electrostatically stabilised complexes.
Higher ionic strengths, on the other hand, prevent
complexation due to a reduced entropic driving force from
the release of counter-ions (Piculell, Bergfeldt, & Nilsson,
1995; Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998).
It has previously been found for mammalian gelatins that
neutral salts generally give gelatin gels with lower storage
modulus at all concentrations of gelatin (Stainsby, 1987).
The salt dependence of the storage modulus in Fig. 6
indicates that electrostatic interactions can be important in
the stabilisation of the gelatin network.
3.8. Gelling and melting temperatures
The gelling ðtg Þ and melting temperature ðtm Þ; as defined
earlier, were studied as a function of concentration as shown
in Fig. 7(a). The melting temperature seems to be less
dependent upon concentration compared to the gelling
temperature. The gelling temperature increased from 4 8C
for 10% solution to 10 8C for 30% solutions, while the
melting temperature increased from approximately 13–
16 8C. This change in melting temperature is in good
agreement with previous results for cod gelatin when
studied at a cooling rate of 0.5 8C/min and heated at 0.2 8C/
min (Gilsenan & Ross-Murphy, 2000a). An increased
concentration of FG will inevitably lead to shorter distances
between the gelatin a-chains in the solution and formation
of junction zones and gel network will be favoured. The
difference between the gelling and the melting temperature
is most likely caused by some kinetic effects.
The gelling and melting temperature of 10 (w/w)% FG1
solutions were also studied by varying pH and ionic
strength, as given in Fig. 7(b) and (c), respectively. A
10% FG1 solution has an ambient pH-value of 5.2 and gels
at 4.5 8C ðtg;0 Þ and melts at 13 8C ðtm;0 Þ: The change in
gelling and melting temperature at varying pH-values seems
to have the same pH-dependence as the rigidity modulus,
but the decrease at high and low pH is not as pronounced as
for the modulus. As for the storage modulus, the drop in tg
and tm is probably due to increased charge density which
disfavours formation and stabilisation of the gelatin
network.
From Fig. 7(c) it can be seen that the gelling and melting
temperatures are almost unchanged at low ionic strengths.
When the ionic strength is further increased both tg and tm
decline. At high ionic strengths (. 0.5 M) FG does not gel
immediately at 4 8C, but has to be kept at this temperature
for several minutes before the solution solidifies. The time at
4 8C needed before gelation occurred increased with
increasing salt concentration. Addition of 1.0 M NaCl
lowers tm to almost 12 tm;0 : The reduction in tg and tm is
probably due to a reduced electrostatic interaction preventing attractive ionic inter-chain bridging and gelation of FG.
Fig. 7. The gelling ðtg Þ and melting temperature ðtm Þ determined from
small-strain oscillatory measurements as a function of (a) fish gelatin
concentration, (b) pH in 10 (w/w)% solution and (c) ionic strength in
10 (w/w)% solution.
These results are in agreement with earlier work (Sarabia
et al., 2000) on the melting temperature for megrime gelatin.
At 0.1 M NaCl the melting temperature was almost
unchanged, but at 0.5 M NaCl the melting temperature
decreased considerably. Again, this suggests that electrostatic interactions are important for the formation and
stability of the gelatin gel network.
3.9. Chiroptical properties
The optical rotation was followed at several concentrations of FG (data not shown). The optical rotation covers
the transition from a random coil to left-handed helix
conformation. When the temperature was decreased by any
temperature gradient from 25 to 4 8C, the coil – helix
transition temperature seemed to depend on fish gelatin
concentration. This is probably a kinetic effect since it
I.J. Haug et al. / Food Hydrocolloids 18 (2004) 203–213
211
would be expected that the onset of molecular coil – helix
transition should be independent on concentration. Djabourov, Maquet, Theveneau, Leblond and Papon (1985)
studied the kinetics of gelation of aqueous mammalian
gelatin solutions and found that the specific rotation was
dependent on the cooling and heating rate. To avoid this
problem FG was quenched down to 5, 6 and 11 8C and kept
at these temperatures for 5 h to investigate the coil – helix
transition. It was of a certain interest to compare the
fractions of regenerated helices in mammalian gelatin and
FG, and the helix fraction, x; was calculated as described by
Djabourov et al. (1985) and Djabourov, Leblond, and Papon
(1988). The fraction is calculated as amount of helices at
time t (½ameas: ) compared to assumed 100% helix
conformation in collagen ð½a100% helix Þ: In the fish gelatin
solution at 25 8C the molecules are assumed to be in 100%
coil conformation ð½a100% coil Þ: The helix fraction is
calculated according to Eq. (1):
x ¼ ½ameas: 2 ½a100% coil =½a100% helix 2 ½a100% coil
ð1Þ
Fig. 8 shows the fraction of helices in 6 (w/v)% FG3
matured at 5, 6 and 11 8C for 5 h (a) and the helix – coil
transition on increasing temperatures (b). It has previously
been found for mammalian gelatin (4.7 (w/w)%) kept at
20 8C that x ¼ 0:30 – 0:35 after 5 h and that x increases with
time, which is evidence for the non-equilibrium properties
of gelatin. At temperatures above 10 8C the helix fraction
was found to be dependent on gelatin concentration, but the
dependence disappeared below this temperature (Djabourov
et al., 1985, 1988). Since 20 8C is close to the gelation
temperature for the mammalian gelatin, the fraction of
helices at this temperature would be expected to be
approximately the same as for a solution of FG around its
gelling temperature (4 –5 8C). When 6 (w/v)% fish gelatin
was kept at 5 8C for 5 h the fraction of helices was found to
be 0.3, and this is in good agreement with the helix fraction
in mammalian gelatin. It is assumed that the small
difference in concentration between the two gelatins will
not influence the helical content considerably. Comparing
the helix fraction for mammalian and fish gelatin at 5 8C,
where the helical fraction in mammalian gelatin is
independent of concentration, shows a completely different
picture. At 5 8C for mammalian gelatin x ¼ 0:5 – 0:6 after
5 h (Djabourov et al., 1988), which is almost twice as high
as for FG at the same conditions. The difference in
regeneration of ‘collagen structure’ was also found for
different fish species and mammalian gelatin by Joly-Duhamel, Hellio, and Djabourov (2002b) and is found to be
caused by different contents of Hyp and Pro. The
regeneration of helices in different gelatins clearly shows
the correlation between the content of Hyp and Pro, and the
coil –helix transition temperature.
Fig. 8 (a) also shows how the helix fraction changes as
the temperature is increased to higher temperatures (6 and
11 8C). At 6 8C the reformation of helices is only slightly
lower than at 5 8C, while the helix fraction at 11 8C is
Fig. 8. Coil– helix transition in (a) 6 (w/v)% FG3 followed by optical
rotation at 5, 6 and 11 8C and (b) helix–coil transition on heating. x is the
fraction of helices in the gelatin.
considerably lower. This illustrates the thermal effect of
helix formation and the obvious fact that since coil – helix
transition does occur at 11 8C, the coil –helix temperature
must be even higher despite the fact that no macroscopic
gelling occurs. A solution of 2 (w/v)% FG3, which is close
to c0 ; did not form a gel at 4 8C (only increased viscosity),
but optical rotation measurements (data not included)
showed that coil –helix transition still occurs. Hence, it
seems like the amount of helices needs to exceed a critical
value to be sufficient for the formation of a continuous
network. This is in agreement with results from Joly-Duhamel et al. (2002). It is also clear that for fish gelatin from
cold water fish species the amount of regenerated helices is
dependent on concentration at temperatures below 10 8C, in
contrast to what is found for mammalian gelatin by
Djabourov et al. (1985, 1988).
After quenching and annealing the temperature was
raised to 25 8C to follow helix –coil transition (Fig. 8(b))
which seemed to be complete at approximately 20 8C.
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I.J. Haug et al. / Food Hydrocolloids 18 (2004) 203–213
The helix fraction reached zero at the same temperature
independent of the previous maturing temperature, which
was also expected.
4. Conclusions
The main difference between fish and mammalian gelatin
is the content of the imino acids Pro and Hyp, which
stabilises the ordered conformation when gelatin forms a gel
network. The lower content of Hyp and Pro probably gives
fish gelatin its low gel modulus, gelling and melting
temperature. Optical rotation experiments revealed that
the amount of helices in fish gelatin and mammalian gelatin
are approximately identical at their respective gelling
temperatures, and that a critical amount of regenerated
helices is needed to form a gel network. It has been found
that the gel modulus increases at low ionic strength and
decreases with increasing ionic strength. Also the gelling
and melting temperatures are influenced by changes in ionic
strength. This suggests that formation and stability of
junction zone in gelatin could be, directly or indirectly,
influenced by electrostatic interactions. The junction zones
and the gel network may thus be stabilised by both hydrogen
bonds and electrostatic bridging.
Acknowledgements
The authors would like to thank Engineers Ann-Sissel
Ulset for performing the SEC-MALLS analysis. Engineer
Ingrid Aune and diploma student Kirsti Hedalen are thanked
for skilful technical assistance. Thanks are also due to
Professor Bjørn Torger Stokke at the Institute of Physics,
NTNU, Norway, for fitting G0 ðcÞ data to the Cascade Model.
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