Physical and rheological properties of fish gelatin compared to mammalian gelatin

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: [email protected] (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 (Go´mez-Guille´n & 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, Go´mez-Guille´n, & 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

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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-Gera¨te 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 (Go´mez-Guille´n 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

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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.

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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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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

G0infinite

Fig. 3. Extrapolation to the cone and plate geometry.

for 10 (w/w)% FG1 at 4 8C measured with

f

a

c0 (mg/ml)

5 10 20

1.813 1.1 0.482

25.9 25.7 16.7

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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.

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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).

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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

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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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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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