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Physico-chemical and film-forming properties of bovine-hide and tuna-skin gelatin: A comparative study
Journal of Food Engineering 90 (2009) 480–486
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Physico-chemical and film-forming properties of bovine-hide and tuna-skin gelatin: A comparative study J. Gómez-Estaca, P. Montero, F. Fernández-Martín, M.C. Gómez-Guillén * Instituto del Frío (CSIC), Meat and Fish Science and Technology, José Antonio Novais, 10, 28040 Madrid, Spain
a r t i c l e
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Article history: Received 9 May 2008 Received in revised form 16 July 2008 Accepted 19 July 2008 Available online 31 July 2008 Keywords: Bovine-hide gelatin Tuna-skin gelatin Biodegradable films Physico-chemical properties
a b s t r a c t A bovine-hide gelatin and a tuna-skin gelatin, both characterized on the basis of their amino acid composition and molecular weight distribution, were used to prepare edible films by casting with glycerol and sorbitol added as plasticizers. The molecular weight distribution of the tuna-skin gelatin exhibited appreciably higher quantities of b-components (covalently linked a-chain dimers), whereas bovine-hide gelatin showed a certain degradation of a1-chains being indicative of a greater proteolysis. Intrinsic differences in the gelatin attributes affected in diverse manner some of the physical properties of the films. Thus, water vapour permeability was higher in the bovine-hide gelatin film, whereas deformability was considerably higher (10 times higher) in the tuna-skin gelatin film. In contrast, breaking force and water solubility were basically unaffected by gelatin origin. Analysis of the thermal properties revealed both films to be wholly amorphous with similar glass transition temperature values thanks to the plasticizing effects of the glycerol and sorbitol and the low moisture contents. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Gelatin is a protein with a wide range of industrial applications employed worldwide. It enhances the functional properties of food products by improving their elasticity, consistency, and stability, and it may also be used as an outer film to protect food against drying, light, and oxygen, especially in those cases where oxidative and microbiological deterioration occurs (Arvanitoyannis, 2002). Gelatin is obtained by hydrolyzing the collagen present in the bones and skin generated as waste during animal slaughtering and processing. Bovine and porcine wastes are the most frequent sources to obtain gelatin of good quality. Other sources of gelatin are becoming increasingly relevant, such as fish bones and skins. The waste produced by fish filleting can account for as much as 75% of the total weight of catches (Shahidi, 1994), and further processing to yield gelatin can help offset harmful environmental effects. The quality of a fish gelatin is determined mainly by its bloom strength and heat stability (melting and gelling temperatures). Certain uses, however, do not require these physical attributes to be as high as those of mammalian gelatins, e.g., encapsulation. For a long time, marine gelatins have been shown to present inferior rheological properties as compared to mammalian gelatins, which is especially true in the case of gelatins from cold-water fish species (Leuenberger, 1991; Gudmundsson and Hafsteinsson, 1997; Haug et al., 2004). Nevertheless, recent studies showed that certain fish * Corresponding author. Tel.: +34 91 5445607; fax: +34 91 5493627. E-mail address: [email protected] (M.C. Gómez-Guillén). 0260-8774/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2008.07.022
gelatins might have not superior but similar quality characteristics compared to mammalian gelatin, depending on the species gelatin extracted from and the processing conditions (Choi and Regenstein, 2000; Cho et al., 2005; Zhou et al., 2006; Yang et al., 2007). The lower values for the physical properties of gelatins from cold-water fish species have largely been related to the considerably lower number of proline and hydroxyproline-rich regions in the collagen molecule (Ledward, 1986). The different physical properties of gelatins are related not only to the amino acid composition but also to the relative a-chain, b- or c-component, and higher-molecular-weight aggregate contents and to the presence of lower-molecular-weight protein fragments (Johnston-Banks, 1990). For this reason, the extraction procedure greatly influences the properties of the resulting gelatin. More severe treatment conditions are widely agreed to be detrimental to a gelatin’s physical properties. Nevertheless, high heat is commonly used to increase yields of commercial mammalian gelatins. In the case of fish gelatins, the normally lower degree of crosslinking in the native collagen (Montero et al., 1990; Bateman et al., 1996) allows milder acid and heat treatment conditions, thus yielding gelatin preparations of reasonably high quality (Gómez-Guillén et al., 2002). In view of the growing interest in biodegradable films, any consideration of the quality of a gelatin should take into account not only its gel-forming properties but also its film-forming ability, along with the physical properties of the resulting films. Both the intrinsic differences between mammalian and fish gelatins and the different extraction conditions employed may influence the properties and the potential applications of films made from a given gelatin.
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There has been a review on gelatin films (Arvanitoyannis, 2002), and a considerable body of recently published work on the use of gelatin to obtain edible films is available in the literature (Menegalli et al., 1999; Sobral et al., 2001; Simon-Lukasik and Ludescher, 2004; Bertan et al., 2005). However, the bulk of this information concerns commercial mammalian gelatins. Although researchers are now increasingly turning their attention to fish gelatin films (Muyonga et al., 2004; Jongjareonrak et al., 2006a,b; Gómez-Guillén et al., 2007; Carvalho et al., 2008), the list of literature references dealing with these latter films is considerably shorter. The present literature seems to bear out that there are some differences in the physical properties of films obtained from mammalian and fish gelatins, the former being stronger and more permeable to water vapour and the latter more elastic (Sobral et al., 2001; Thomazine et al., 2005; Avena-Bustillos et al., 2006; Gómez-Guillén et al., 2007) although it remains somewhat unclear. Comparability of the data is limited because of the wide range of different experimental conditions employed for film producing, i.e., plasticizer type and concentration, dehydration temperature, film thickness and conditioning, etc. In addition, the recent knowledge about extraction and characterization of gelatin from many fish species, may difficult generalizing about fish gelatin properties. The purposes of this study were: (i) to characterize the physicochemical attributes of two different origin gelatins (one from tunaskin, the other from bovine-hide) on the basis of the more distinct parameters: aminoacid composition and molecular weight distribution; as well as thermal and rheological properties in the presence of plasticizers and (ii) to determine how these attributes affected the properties of the resulting films, produced in identical conditions to provide a proper comparison. 2. Materials and methods 2.1. Characterization of the gelatins The tuna-skin gelatin was prepared in our laboratory according to the method described by Gómez-Guillén and Montero (2001). The bovine-hide gelatin (Bloom 200/220) was commercially obtained from Sancho de Borja S.L. (Saragossa, Spain). 2.2. Amino acid composition An amount of 1 mg/ml of dry gelatin was dissolved in distilled water, and samples (50 ll) were dried and hydrolyzed in vacuumsealed glass tubes at 108 °C for 18 h in the presence of continuously boiling 5.7 N HCl containing 0.1% phenol with norleucine as internal standard. After hydrolysis, samples were vacuum-dried, dissolved in application buffer, and injected onto a Biochrom 20 amino acid analyser (Pharmacia, Barcelona, Spain). DL-5-Hydroxylysine hydrochloride and cis-4-hydroxy-D-proline (Sigma, St. Louis, MO, USA) were also used as standards to determine the amount of hydroxylysine and hydroxyproline, respectively.
2.3. Molecular weight profile The molecular weight distribution of the bovine-hide and tunaskin gelatins was determined by SDS–polyacrylamide gel electrophoresis. Gelatin (5 mg/ml) solutions were mixed with loading buffer (2% SDS, 5% mercaptoethanol, and 0.002% bromophenol blue) in a proportion of 1:4. Samples were heat-denatured at 90 °C for 5 min and analysed according to Laemmli (1970) in a Mini Protean II unit (Bio-Rad Laboratories, Hercules, CA, USA) using 4% stacking gels and 6% resolving gels at 25 mA/gel. Loading volume was 15 ll in all lanes. Protein bands were stained with Coomassie brilliant blue R-250. Type I collagen from foetal calf was used as a
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marker for a-chain and b-component mobilities. Also a molecular weight standard composed of myosin (212 kDa), a2-macroglobulin (170 kDa), b-galactosidase (116 kDa), transferrin (76 kDa) and glutamic dehydrogenase (53 kDa) (Amersham Pharmacia biotech, Buckinghamshire, UK) was employed. 2.4. Film-forming solution and film preparation The film-forming solutions were prepared using gelatin at a concentration of 4 g/100 ml of distilled water. Sorbitol (0.15 g/g gelatin) and glycerol (0.15 g/g gelatin) were employed as plasticizers. The mixtures were warmed and stirred at 40 °C for 15 min to obtain a good blend, and the films were made by casting an amount of 40 ml in square dishes (12 12 cm) and drying in a convection oven at 45 °C for 15 h. Prior to determinations the films were conditioned over a saturated solution of NaBr (58% relative humidity) at 22 °C in desiccators for 2 d. The moisture content of films, determined by AOAC method 24003 (AOAC, 1984) after the conditioning period, was 10.23% (±0.30) for the bovine-hide gelatin films and 13.46% (±0.34) for the tuna gelatin films. Film thickness was measured using a digital micrometer (Mitutoyo, model MDC-25M, Kanagawa, Japan), averaging nine different locations. 2.5. Characterization of the film-forming solutions 2.5.1. Dynamic viscoelastic properties Dynamic viscoelastic analysis of the film-forming solutions was carried out as described in Gómez-Guillén et al. (2007), on a Bohlin CSR-10 rheometer rotary viscometer (Bohlin Instruments Ltd., Gloucestershire, UK) using a cone-plate geometry (cone angle = 4°, gap = 0.15 mm). Cooling and heating from 40 to 6 °C and back to 40 °C took place at a scan rate of 1 °C/min, frequency of 1 Hz, and a target strain of 0.2 mm. The elastic modulus (G0 ; Pa), viscous modulus (G00 ; Pa) and phase angle (°) were determined as functions of temperature. Several determinations were performed for each sample, with an experimental error of less than 6% in all cases. 2.5.2. Gel strength The film-forming solutions were poured into glasses 2.3 cm in diameter and 3.6 cm in height and left to mature in a refrigerator at 2 °C for 16–18 h. Gel strength at 8–9 °C was determined, as described in Gómez-Guillén et al. (2002), on an Instron model 4501 Universal Testing Machine (Instron Co., Canton, MA, USA) with a 100-N load cell, a cross-head speed of 60 mm/min, and a flat-faced cylindrical plunger 1.27 cm in diameter. The maximum force (g) was determined when the plunger had penetrated 4 mm into the gelatin gels. 2.6. Characterization of the films 2.6.1. Mechanical properties A puncture test was performed to determine the breaking force and deformation of films at the breaking point (Gómez-Guillén et al. (2007). Films were placed in a cell 5.6 cm in diameter and perforated to the breaking point using an Instron model 4501 Universal Testing Machine (Instron Co., Canton, MA, USA) with a round-ended stainless-steel plunger (3 mm in diameter) at a cross-head speed of 60 mm/min and a 100-N load-cell. Breaking force was expressed in N and breaking deformation in %, according to Sobral et al. (2001). All determinations are the means of at least five measurements. 2.6.2. Thermal properties Calorimetric analysis was performed with a model TA-Q1000 differential scanning calorimeter (DSC) (TA Instruments, New Castle, DE, USA) previously calibrated by running high purity indium
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(melting point 156.4 °C; enthalpy of melting 28.44 W/g). Samples of approximately 10 mg (±0.002 mg) were weighed out using a model ME235S electronic balance (Sartorious, Goettingen, Germany) and were tightly encapsulated in aluminium pans and scanned under dry nitrogen purge (50 ml/min). Freshly conditioned films were rapidly cooled to 0 °C and scanned at between 0 and 90 °C at a heating rate of 10 °C/min. Glass transition temperatures, Tg (°C), were determined only on the first heating scans, values obtained on the second scans being deemed not relevant because of the practical impossibility of reproducing the original film conditioning. The glass transition temperature was estimated as the midpoint of the line drawn between the temperature at the intersection of the initial tangent with the tangent through the inflection point of the trace and the temperature of the intersection of the tangent through the inflection point with the final tangent. Tg data have been reported as the mean values of at least duplicate samples for each film, usually within ±1 °C. 2.6.3. Water solubility Film portions measuring 4 cm2 were placed in aluminium capsules with 15 ml of distilled water and shaken gently at 22 °C for 15 h. The solution was then filtered through Whatman no. 1 filter paper to recover the remaining undissolved film, which was desiccated at 105 °C for 24 h. Film solubility was calculated by the equation FS (%) = ((W0 Wf)/W0) 100, where W0 was the initial weight of the film expressed as dry matter and Wf was the weight of the undissolved desiccated film residue. All tests were carried out in triplicate. 2.6.4. Water vapour permeability Water vapour permeability was determined following the method described by Sobral et al. (2001). Films were attached over the openings of cells (permeation area = 15.9 cm2) containing silica gel, and the cells were placed in desiccators with distilled water at 22 °C. The cells were weighed daily for 7 d. Water vapour permeability was calculated using the equation WVP = w x t1 A1 DP1, where w was weight gain (g), x film thickness (mm), t elapsed time for the weight gain (h), and DP the partial vapour pressure difference between the dry atmosphere and pure water (2642 Pa at 22 °C). Results have been expressed as g mm h1 cm2 Pa1. All tests were carried out in duplicate. 2.7. Statistical analysis Statistical tests were performed using the SPSSÒ computer program (SPSS Statistical Software Inc., Chicago, IL, USA). One-way analysis of variance was carried out. Differences between pairs of means were assessed on the basis of confidence intervals using the Tukey-b test. The level of significance was p 6 0.05.
3. Results and discussion 3.1. Characterization of the gelatins Table 1 presents the amino acid composition of the gelatins. Both the bovine-hide and the tuna-skin gelatins exhibited typical type I collagen Gly content, representing approximately 1/3 of the total amino acids. As described by Asghar and Henrickson (1982), 50–60% of a-chains consist of tripeptides having the general formula Gly-X-Y, where X is generally proline and Y is mainly hydroxyproline. The proline plus hydroxyproline (imino acids) content was higher in the bovine-hide gelatin than in the tuna-skin one (210 vs. 185 residues/1000, respectively). Gelatins made from warm-blooded animal tissues have been reported to have a higher imino acid content, hydroxyproline in particular (Norland, 1990),
Table 1 Amino acid composition of the bovine-hide and the tuna-skin gelatines Number of residues/1000
Hyp Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg Hyl
Bovine-hide
Tuna-skin
83 46 33 39 74 127 342 113 19 4 11 24 4 12 4 25 47 5
78 44 21 48 71 107 336 119 28 16 7 21 3 13 7 25 52 6
and this promotes triple helix formation and stabilization of the gelatin at low temperatures (Burjandze, 1979) thanks to the hydrogen bonding ability of the –OH group on the hydroxyproline. In the present work the Hyp content of the tuna-skin gelatin was relatively high (78 residues/1000) although lower than the one for bovine-hide gelatin (83 residues/1000), due to the fact that this fish species do not come from very cold waters. Similarly, a relatively high content of hydroxyproline (74 residues/1000) has been recently reported in gelatin extracted from tuna fin (Aewsiri et al., 2008). Gelatins from warm-water fishes have a higher imino acid content than gelatins from cold-water fish species, closer to mammalian ones (Gilseman and Ross-Murphy, 2000a; Avena-Bustillos et al., 2006). With respect to the hydrophobic nature of our gelatins, both of them presented a similar content of hydrophobic residues of around 310 per 1000 residues. This is again attributed to the relatively warm water in which tuna lives. Cold-water fish gelatins are typically more hydrophobic. The electrophoretic profile of the bovine-hide gelatin was considerably more heterogeneous than that of the tuna-skin gelatin, thereby causing bands on the SDS gel to be more diffuse (Fig. 1). This is indicative of a broad range of molecular weights, probably attributable to the normal use of more severe extraction conditions than for the fish gelatin, for which extraction was carried out at temperatures below 45 °C. Both types of gelatin contained achains weighing around 100 kDa and b-components weighing around 200 kDa, typical of type I collagen. Nevertheless, there were appreciable differences in a-chain yields during extraction. The fish gelatin exhibited a a1/ a2 ratio of around 2, indicating that the native structure is maintained. In contrast, intensities for the bands for the a1 and a2 chains in the mammalian gelatin were similar, indicating possible degradation of the a1-chains during the extraction process. This is an interesting factor to bear in mind, since, as reported by Sims et al. (1997), a1-chains have greater gelling ability than a2-chains. The amount of b-components was also considerably higher in the fish gelatin, suggesting that the milder extraction conditions were more conducive to extracting intact, covalently linked a-chain dimers. Both gelatins had appreciable amounts of higher-molecularweight (>200 kDa) fractions of several crosslinked a-chains, but the bands were more distinct in the case of the fish gelatin, indicating less dispersal of molecular weights in the gelatin. Very highmolecular-weight polymers resulting from residual heat-stable crosslinks were also present in both types of gelatin, as revealed by the high-intensity bands at the tops of the polyacrylamide gels.
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The <100 kDa polypeptide fraction appeared to be more abundant in the bovine-hide gelatin, again largely attributable to more extensive cleavage of the -chain peptide bonds during gelatin extraction. Moreover, the presence of a high-intensity band at the bottom of the resolving gel in the mammalian gelatin indicated the presence of extensively hydrolyzed fractions that did not appear in the fish gelatin.
HMW-a
β
3.2. Characterization of the film-forming solutions
α1 α2
Tuna-skin
Bovine-hide
Tuna-skin
G' (Pa)
Fig. 1. Electrophoretic profile of the bovine-hide and tuna-skin gelatins. HMW-a, high-molecular-weight aggregates.
Fig. 2 shows the changes in the viscoelastic properties of the film-forming solutions during the cooling and subsequent heating ramps. The bovine-hide gelatin showed higher elastic modulus (G0 ) values at low temperatures, indicative of enhanced ability to refold into a triple helix (Ledward, 1986; Gómez-Guillén et al., 2002). The phase angle exhibited higher thermal transition points in the bovine-hide gelatin on both, the cooling and heating ramps, an indication that it was more heat stable. The average gelling temperature and melting temperature for the bovine-hide gelatin were, respectively, 20 °C and 30 °C, whereas for the tuna-skin gelatin were 15 °C and 22.5 °C. These higher rheological properties and thermostability are typical of mammalian gelatins (Leuenberger, 1991; Gilseman and Ross-Murphy, 2000b) and are mainly related to imino acid composition, hydroxyproline playing a
a
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Temperature (ºC) Bovine-hide
10
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Temperature (ºC)
Tuna-skin
Fig. 2. Changes in the viscoelastic properties (G0 , modulus of elasticity; G00 , modulus of viscosity; °, phase angle) of the film-forming solutions of bovine-hide and tuna-skin gelatin during the cooling (a) and subsequent heating (b) ramps.
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singular role in stabilizing the triple-stranded helix thanks to the hydrogen bonding ability of its –OH group (Burjandze, 1979). As mentioned above, the total imino acid content was higher in the bovine-hide gelatin. As expected, gel strength was significantly (p 6 0.05) lower in the tuna-skin gelatin (167 ± 4 g) than in the bovine-hide gelatin (211 ± 6 g). However, the difference was less pronounced than the difference observed in the elastic modulus. Triple helix formation takes place by association of the different a-chains during cold maturation of the gel. This gives rise to differences according to gelatin characteristics suggesting that the nucleation points initially formed in the tuna-skin gelatin are capable of considerable growth during maturation under refrigeration. A possible explanation for this is the molecular weight distribution of the tuna-skin gelatin employed, with higher amount of native structures -monomers and dimmers-. The tuna-skin gelatin had considerably higher amounts of a1-chains and b components than the bovine-hide gelatin did, and according to Sims et al. (1997) this could be responsible for its relatively high gel strength. This molecular weight distribution, in which the two a-chains comprising the b-component are already crosslinked, enables the chains to form triple helices upon cooling and contributes to helix growth during gel maturation. The presence of glycerol and sorbitol in the film-forming solutions, which were added as plasticizers in a relatively high concentration (30 g/100 g gelatin) should be also taken into consideration. Glycerol has been reported to exert a considerable gel strength-enhancing effect in fish gelatin gels, largely attributed to hydrogen bonding stabilization (Fernández-Díaz et al., 2001); however, it is worth noting that in the study reported by those authors, the glycerol concentration was approximately 7.5-fold higher than in the present one. 3.3. Characterization of the films Both the bovine-hide and the tuna-skin gelatin-based filmforming solutions yielded flexible and transparent films, similar (p 6 0.05) in thickness (109 ± 18 lm for the bovine-hide films and 98 ± 15 for the tuna-skin films). The data from the physical properties analysis are set out in Table 2. Both films presented a similar (p 6 0.05) maximum load value at the breaking point (10.5 N and 8.5 N for bovine-hide and tuna-skin, respectively). On the contrary, the tuna-skin gelatin film had breaking deformation values around 10 times higher than the values for the bovinehide gelatin film. Data from bibliography are not easily comparable because of the differences in film obtaining, type and concentration of plasticizers, gelatin type, measurement methodology, etc. Having this in mind there seems to be a trend to obtain stronger films from mammalian gelatins whereas fish gelatins render more deformable films (Sobral et al., 2001; Thomazine et al., 2005; Avena-Bustillos et al., 2006; Gómez-Guillén et al., 2007; Pérez-Mateos et al., 2009). In the present work both films were elaborated with the same type and concentration of plasticizers, so the differences in the Table 2 Physical properties (puncture force and deformation at the break point, water vapour permeability (WVP) and water solubility) of the bovine-hide and the tuna-skin gelatine films
Puncture force(N) Puncture deformation(%) WVP(108 g mm h1 cm2 Pa1) Water solubility(%) Tg (°C)
Bovine-hide
Tuna-skin
10.7 ± 2.2a 14.1 ± 5a 2.20 ± 0.11a 34.3 ± 0.6a 41.5 ± 1a
8.5 ± 1.6a 154 ± 36b 1.65 ± 0.39b 39.9 ± 1.3a 40.7 ± 1a
Results are expressed as mean value ± standard deviation. Different letters (a, b) indicate significant differences.
mechanical properties should be linked to the physico-chemical characteristics of the gelatin, especially regarding the aminoacid composition, which is greatly a species-specific characteristic, and the molecular weight distribution, which is mostly determined by processing conditions. Regarding the amino acid composition, mammal gelatins are well known to be richer in imino acids (Norland, 1990). As discussed above, the higher imino acid content (Pro + Hyp) strongly determine the superior rheological properties and thermostability of bovine-hide gelatin gels. This is largely attributable to the formation of junction zones by hydrogen bonding, which in turn yield a multitude of intra- and inter-chain interactions important to cold stabilization of the gel network. These interactions are promoted by the availability of more -OH groups to form hydrogen bonds with bridging water molecules inside the triple helices and also between the chains of neighbouring triple helices (Asghar and Henrickson, 1982). Thus, water plays an important role in augmenting the stability of the triple helical structure. According to Djabourov et al. (1985), the structural water fraction increases during cold gelation and is proportional to helix formation. Water molecules are taken into the structure of the triple helix and are hydrogen bonded to the -CO or -NH groups. However, presumably the involvement of hydrogen bonds is lower in dried films, in which most of the water has been removed and the storage temperature is around 20 °C, which may act to cause the differences in imino acid content between the two gelatins to be less appreciable. In this connection, the mechanical properties of gelatin films have been related to the triple helix content of the gelatin employed (Bigi et al., 2004); however, it is known to decrease upon isothermal dehydration of films (Wetzel et al., 1987). Regarding the molecular weight profile, it could be assumed that the presumably more severe treatment conditions needed for the commercial bovine-hide gelatin extraction led to a higher proportion of protein fractions of low molecular weight (<100 kDa). According to the works by Muyonga et al. (2004) and Carvalho et al. (2008) this fact results in a reduction of film strength and an increase in film extensibility. The role of gelatin molecular weight distribution in the rheological properties of the films may be associated to the presence and concentration of plasticizers in the formulation, which in the present study was relatively high (30 g/ 100 g gelatin). Thus, for the same mass concentration of plasticizer, films made using a lower-molecular-weight biopolymer could be more plasticized, because the molar plasticizer/biopolymer ratio is higher (Thomazine et al., 2005). However, in our work both gelatins presented a considerable amount of high-molecular-weight aggregates (Fig. 1) which would improve the film strength in both cases. Moreover, the predominance of b-components in the fish gelatin could also act to noticeably strengthen the corresponding films, despite the lower iminoacid content. Regarding the differences in film deformation, which was 10fold higher for the tuna-skin films, data from literature show us that fish gelatins produce more deformable films than mammal ones (Sobral et al., 2001; Thomazine et al., 2005; Gómez-Guillén et al., 2007; Pérez-Mateos et al., 2009). In the present work, this finding is not possible to be explained in terms of a higher plasticizing effect associated to the gelatin molecular weight distribution, since bovine-hide gelatin appeared to have a higher amount of hydrolyzed peptide fractions which are prompt to interact with plasticizer molecules. Thus, one possible explanation lies in the different imino acid content in both gelatins. The higher Pro + Hyp content in the bovine-hide gelatin may impose conformational constraints by imparting a higher degree of molecular rigidity to the corresponding films, which would obviously lead to a lower deformability. Fig. 3 plots the DSC traces. Despite being encapsulated in nonhermetically sealed pans, samples lost only 1–2% of their weight
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after scanning, which indicated that the films had good water retention capacity. This resulted in a certain bending of the trace due to water vapourization, which somehow distorted the DSC profiles at the glass transition point. On the other hand, glass transition seemed to overlap with an enthalpy relaxation phenomenon. Both water vapourization and enthalpy relaxation made precise measurement of the Tg values somewhat difficult. The films were wholly amorphous, since they did not exhibit any melting event in the temperature region that would otherwise be expected based on the heat stability characteristics of the gelatins. The plasticizing effects of the glycerol plus sorbitol (Sobral et al., 2001) and water may inhibit gelatin crystallization. Additionally, moisture reduction up to the films typical levels (10–14% depending on the gelatin) caused the complex systems to move from regions of gel–sol– gel transitions to vitreous domains. Tg values were similar in both films, somewhat lower in the tuna-skin gelatin film, though the difference was not significant (Table 2). Cold-water fish gelatins have been reported (Gilseman and Ross-Murphy, 2000a) to be less heat stable than mammalian gelatins, mostly due to the lower imino acid content. In contrast, the properties of warm-water fish gelatins may be quite similar to those of mammalian gelatins (Gilseman and Ross-Murphy, 2000b). Although tuna cannot be considered a warm-water fish species it is not located at very cold-water seas as for example cod or hake are. Table 2 also lists the water vapour permeability (WVP) of the films. The tuna-skin gelatin film was less permeable than the bovine-hide gelatin film (p 6 0.05). One more time the explanation could lie in the differences in the amino acid composition of the two gelatins. Avena-Bustillos et al. (2006) observed that the hydrophobic nature of the amino acids of a gelatin highly determine the water vapour permeability of the corresponding films, being the fresh water fish species gelatin films the less permeable, followed by the warm-water fish ones and finally the mammal gelatin films. However in our work the differences recorded in water vapour permeability cannot be absolutely ascribed to amino acid composition because the total amount of hydrophobic amino acids was quite similar for both gelatins (310 for bovine-hide gelatin vs. 311 for tuna-skin one). However Hyp, which is a hydrophilic amino acid, was higher for the bovine-hide gelatin film, and this could have contributed in part to its higher water vapour permeability. The molecular weight distribution has also been thought to exert an effect. The bovine-hide gelatin presented a considerably higher amount of protein fractions up to 100 kDa which could be more susceptible to the plasticizing effects of glycerol and sorbitol than
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the bigger fragments. Both glycerol and sorbitol are well known to cause an increase in the water vapour permeability of gelatin films due to its hydrophilic –OH groups and this effect is directly related to plasticizer concentration (Sobral et al., 2001; Jongjareonrak et al., 2006a). Contrary to expectation, the water solubility of the tuna-skin gelatin film was not significantly different from that of the bovine-hide gelatin film (Table 2). The bovine-hide gelatin may presumably present lower water solubility due to the straightening effects of the hydrogen bonds promoted by imino acids. However, as discussed for the mechanical properties, the lower water content seems to play a singular role reducing the straightening effects of hydrogen bonding in the films. It seems to be more determinant the nature and extent of the very high-molecular-weight fraction, in which residual heat-stable crosslinks predominate. The electrophoretic analysis did not reveal any substantial differences in this fraction in the two types of gelatin. The low extraction temperature together with intrinsic attributes of the tuna-skin collagen used in this study may help retain some heat-stable crosslinked fractions in the resulting gelatin, leading to relatively low water solubility of the film. Carvalho et al. (2008) elaborated films with two differently processed Atlantic halibut skin gelatins, which did not present any fraction of very high-molecular-weight, and found them to be totally soluble in water. Other study dealing with cod-skin gelatin film has yielded water solubility values as high as 90–100% (Pérez-Mateos et al., 2009), compared with values of around 30% for bovine gelatin film (Bertan et al., 2005). Unfortunately, these works do report any data about neither the molecular weight distribution nor the amino acid profile of the gelatins.
4. Conclusions As expected, the different origin and extraction process of the bovine-hide and the tuna-skin gelatins influenced the physical properties of the corresponding filmogenic solutions. However this was not so evident in the films, presumably due to the lower involvement of hydrogen bonding as a consequence of the reduction of the water content. The breaking force and the water solubility were scarcely affected by the gelatin origin (bovine-hide or tuna-skin) and presented similar values in both films. On the contrary, the puncture deformation and the water vapour permeability were quite different, being attributed to the amino acid profile and the molecular weight distribution of the gelatins. Acknowledgements
a Heat Flow (Endo Down)
This research was financed by the ‘‘Ministerio de Educación y Ciencia”, under Project AGL2005-02380.
b
Tg
References
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Temperature (ºC) Fig. 3. DSC traces and Tg values for the bovine-hide (a) and tuna-skin (b) gelatin films.
Aewsiri, T., Benjakul, S., Visessanguan, W., Tanaka, M., 2008. Chemical compositions and functional properties of gelatin from pre-cooked tuna fin. International Journal of Food Science and Technology 43 (4), 685–693. AOAC (Association of Official Analytical Chemists), 1984. Official Methods of Analysis, 12th ed. Elsevier, Washington, DC. Arvanitoyannis, I.S., 2002. Formation and properties of collagen and gelatin films and coatings. In: Gennadios, A. (Ed.), Protein-based Films and Coatings. CRC Press, Boca Ratón, Florida, pp. 275–304. Asghar, A., Henrickson, R.L., 1982. Chemical, biochemical, functional, and nutritional characteristics of collagen in food systems. Advances in Food Research, vol. 28. Academic Press, London, pp. 232–372. Avena-Bustillos, R.J., Olsen, C.W., Olson, D.A., Chiou, B., Yee, E., Bechtel, P.J., McHugh, T.H., 2006. Water vapor permeability of mammalian and fish gelatin films. Journal of Food Science 71, 202–207. Bateman, J.F., Lamandé, S.R., Ramshaw, J.A.M., 1996. Collagen superfamily. In: Comper, W.D. (Ed.), Extracellular Matrix. Molecular Components and Interactions, vol. 2. Harwood Academic, UK, pp. 22–27.
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Bertan, L.C., Tanada-Palmu, P.S., Siani, A.C., Grosso, C.R.F., 2005. Effect of fatty acids and ‘Brazilian elemi’ on composite films based on gelatin. Food Hydrocolloids 19, 73–82. Bigi, A., Panzavolta, S., Rubini, K., 2004. Relationship between triple-helix content and mechanical properties of gelatin films. Biomaterials 25, 5575–5680. Burjandze, T.V., 1979. Hydroxy-proline content and location in relation to collagen thermal stability. Biopolymers 18, 931–936. Carvalho, R.A., Sobral, P.J.A., Thomazine, M., Habitante, A.M.Q.B., Giménez, B., Gómez-Guillén, M.C., Montero, P., 2008. Development of edible films based on differently processed Atlantic halibut (Hippoglossus hippoglossus) skin gelatin. Food Hydrocolloids 22, 1117–1123. Cho, S.M., Gu, Y.S., Kim, S.B., 2005. Extracting optimization and physical properties of yellowfin tuna (Thunnus albacares) skin gelatin compared to mammalian gelatins. Food Hydrocolloids 19 (2), 221–229. Choi, S., Regenstein, J.M., 2000. Physicochemical and sensory characteristics of fish gelatin. Journal of Food Science 65 (2), 194–199. Djabourov, M., Maquet, J., Theveneau, H., Leblond, J., Papon, P., 1985. Kinetics of gelation of aqueous gelatin solutions. British Polymer Journal 17, 169–174. Fernández-Díaz, M.D., Montero, P., Gómez-Guillén, M.C., 2001. Gel properties of collagens from skins of cod (Gadus morhua) and hake (Merluccius merluccius) and their modification by the coenhancers magnesium sulphate, glycerol and transglutaminase. Food Chemistry 74, 161–167. Gilseman, P.M., Ross-Murphy, S.B., 2000a. Viscoelasticity of thermoreversible gelatin gels from mammalian and piscine collagens. Journal of Rheology 44, 871–883. Gilseman, P.M., Ross-Murphy, S.B., 2000b. Rheological characterisation of gelatins from mammalian and marine sources. Food Hydrocolloids 14, 191–195. Gómez-Guillén, M.C., Montero, P., 2001. Method for the Production of Gelatin of Marine Origin and Product thus obtained. International Patent PCT/S01/00275. Gómez-Guillén, M.C., Ihl, M., Bifani, V., Silva, A., Montero, P., 2007. Edible films made from tuna-fish gelatin with antioxidant extracts of two different murta ecotypes leaves (Ugni molinae Turcz). Food Hydrocolloids 21, 1133– 1143. Gómez-Guillén, M.C., Turnay, J., Fernández-Díaz, M.D., Ulmo, N., Lizarbe, M.A., Montero, P., 2002. Structural and physical properties of gelatin extracted from different marine species: a comparative study. Food Hydrocolloids 16, 25–34. Gudmundsson, M., Hafsteinsson, H., 1997. Gelatin from cod skins as affected by chemical treatments. Journal of Food Science 62 (1), 37–39 +47. Haug, I.J., Draget, K.I., Smidsrød, O., 2004. Physical and rheological properties of fish gelatin compared to mammalian gelatin. Food Hydrocolloids 18 (2), 203– 213. Johnston-Banks, F.A., 1990. Gelatin. In: Harris, P. (Ed.), Food Gels. Elsevier, London, pp. 233–289. Jongjareonrak, A., Benjakul, S., Visessanguan, W., Tanaka, M., 2006a. Effects of plasticizers on the properties of edible films from skin gelatin of bigeye snapper and brownstripe red snapper. European Food Research and Technology 222, 229–235.
Jongjareonrak, A., Benjakul, S., Visessanguan, W., Tanaka, M., 2006b. Characterization of edible films from skin gelatin of brownstripe red snapper and bigeye snapper. Food Hydrocolloids 20, 492–501. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 227, 860–865. Ledward, D.A., 1986. Gelation of gelatin. In: Mitchell, J.R., Ledward, D.A. (Eds.), Functional Properties of Food Macromolecules. Elsevier, London, pp. 171–201. Leuenberger, B.H., 1991. Investigation of viscosity and gelation properties of different mammalian and fish gelatins. Food Hydrocolloids 5, 353–361. Menegalli, F.C., Sobral, P.J.A., Roques, M.A., Laurent, S., 1999. Characteristics of gelatin biofilms in relation to drying process conditions near melting. Drying Technology 17, 1697–1706. Montero, P., Borderías, J., Turnay, J., Leyzarbe, M.A., 1990. Characterization of hake (Merluccius merluccius L.) and trout (Salmo irideus gibb) collagen. Journal of Agricultural and Food Chemistry 38, 604–609. Muyonga, J.H., Cole, C.G.B., Duodu, K.G., 2004. Extraction and physico-chemical characterisation of Nile perch (Lates niloticus) skin and bone gelatin. Food Hydrocolloids 18, 581–592. Norland, R.E., 1990. Fish gelatin. In: Voight, M.N., Botta, J.K. (Eds.), Advances in Fisheries Technology and Biotechnology for Increased Profitability. Technomic Publishing Co., Lancaster, PA, pp. 325–333. Pérez-Mateos, M., Montero, P., Gómez-Guillén, M.C., 2009. Formulation and stability of biodegradable films made from cod gelatin and sunflower oil blends. Food Hydrocolloids 23, 53–61. Shahidi, F., 1994. Seafood processing by-products. In: Shahidi, F., Botta, J.R. (Eds.), Seafoods Chemistry, Processing, Technology and Quality. Blackie Academic and Professional, Glasgow, pp. 320–334. Simon-Lukasik, K.V., Ludescher, R.D., 2004. Erythrosin b phosphorescence as a probe of oxygen diffusion in amorphous gelatin films. Food Hydrocolloids 18, 621– 630. Sims, T.J., Bailey, A.J., Field, D.S., 1997. The chemical basis of molecular weight differences in gelatins. The Imaging Science Journal 45, 171–177. Sobral, P.J.A., Menegalli, F.C., Hubinger, M.D., Roques, M.A., 2001. Mechanical, water vapor barrier and thermal properties of gelatin based edible films. Food Hydrocolloids 15, 423–432. Thomazine, M., Carvalho, R.A., Sobral, P.I.A., 2005. Physical properties of gelatin films plasticized by blends of glycerol and sorbitol. Journal of Food Science 70, 172–176. Wetzel, R., Buder, E., Hermel, H., Hüttner, A., 1987. Conformations of different gelatins in solutions and in films an analysis of circular dichroism (CD) measurements. Colloid and Polymer Science 265 (12), 1036–1045. Yang, H., Wang, Y., Jiang, M., Oh, J., Herring, J., Zhou, P., 2007. 2-Step optimization of the extraction and subsequent physical properties of channel catfish (Ictalurus punctatus) skin gelatin. Journal of Food Science 72 (4), 188–195. Zhou, P., Mulvaney, S.J., Regenstein, J.M., 2006. Properties of Alaska pollock skin gelatin: a comparison with tilapia and pork skin gelatins. Journal of Food Science 71 (6), 313–321.
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Physico-chemical and film-forming properties of bovine-hide and tuna-skin gelatin: A comparative study J. Gómez-Estaca, P. Montero, F. Fernández-Martín, M.C. Gómez-Guillén * Instituto del Frío (CSIC), Meat and Fish Science and Technology, José Antonio Novais, 10, 28040 Madrid, Spain
a r t i c l e
i n f o
Article history: Received 9 May 2008 Received in revised form 16 July 2008 Accepted 19 July 2008 Available online 31 July 2008 Keywords: Bovine-hide gelatin Tuna-skin gelatin Biodegradable films Physico-chemical properties
a b s t r a c t A bovine-hide gelatin and a tuna-skin gelatin, both characterized on the basis of their amino acid composition and molecular weight distribution, were used to prepare edible films by casting with glycerol and sorbitol added as plasticizers. The molecular weight distribution of the tuna-skin gelatin exhibited appreciably higher quantities of b-components (covalently linked a-chain dimers), whereas bovine-hide gelatin showed a certain degradation of a1-chains being indicative of a greater proteolysis. Intrinsic differences in the gelatin attributes affected in diverse manner some of the physical properties of the films. Thus, water vapour permeability was higher in the bovine-hide gelatin film, whereas deformability was considerably higher (10 times higher) in the tuna-skin gelatin film. In contrast, breaking force and water solubility were basically unaffected by gelatin origin. Analysis of the thermal properties revealed both films to be wholly amorphous with similar glass transition temperature values thanks to the plasticizing effects of the glycerol and sorbitol and the low moisture contents. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Gelatin is a protein with a wide range of industrial applications employed worldwide. It enhances the functional properties of food products by improving their elasticity, consistency, and stability, and it may also be used as an outer film to protect food against drying, light, and oxygen, especially in those cases where oxidative and microbiological deterioration occurs (Arvanitoyannis, 2002). Gelatin is obtained by hydrolyzing the collagen present in the bones and skin generated as waste during animal slaughtering and processing. Bovine and porcine wastes are the most frequent sources to obtain gelatin of good quality. Other sources of gelatin are becoming increasingly relevant, such as fish bones and skins. The waste produced by fish filleting can account for as much as 75% of the total weight of catches (Shahidi, 1994), and further processing to yield gelatin can help offset harmful environmental effects. The quality of a fish gelatin is determined mainly by its bloom strength and heat stability (melting and gelling temperatures). Certain uses, however, do not require these physical attributes to be as high as those of mammalian gelatins, e.g., encapsulation. For a long time, marine gelatins have been shown to present inferior rheological properties as compared to mammalian gelatins, which is especially true in the case of gelatins from cold-water fish species (Leuenberger, 1991; Gudmundsson and Hafsteinsson, 1997; Haug et al., 2004). Nevertheless, recent studies showed that certain fish * Corresponding author. Tel.: +34 91 5445607; fax: +34 91 5493627. E-mail address: [email protected] (M.C. Gómez-Guillén). 0260-8774/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2008.07.022
gelatins might have not superior but similar quality characteristics compared to mammalian gelatin, depending on the species gelatin extracted from and the processing conditions (Choi and Regenstein, 2000; Cho et al., 2005; Zhou et al., 2006; Yang et al., 2007). The lower values for the physical properties of gelatins from cold-water fish species have largely been related to the considerably lower number of proline and hydroxyproline-rich regions in the collagen molecule (Ledward, 1986). The different physical properties of gelatins are related not only to the amino acid composition but also to the relative a-chain, b- or c-component, and higher-molecular-weight aggregate contents and to the presence of lower-molecular-weight protein fragments (Johnston-Banks, 1990). For this reason, the extraction procedure greatly influences the properties of the resulting gelatin. More severe treatment conditions are widely agreed to be detrimental to a gelatin’s physical properties. Nevertheless, high heat is commonly used to increase yields of commercial mammalian gelatins. In the case of fish gelatins, the normally lower degree of crosslinking in the native collagen (Montero et al., 1990; Bateman et al., 1996) allows milder acid and heat treatment conditions, thus yielding gelatin preparations of reasonably high quality (Gómez-Guillén et al., 2002). In view of the growing interest in biodegradable films, any consideration of the quality of a gelatin should take into account not only its gel-forming properties but also its film-forming ability, along with the physical properties of the resulting films. Both the intrinsic differences between mammalian and fish gelatins and the different extraction conditions employed may influence the properties and the potential applications of films made from a given gelatin.
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There has been a review on gelatin films (Arvanitoyannis, 2002), and a considerable body of recently published work on the use of gelatin to obtain edible films is available in the literature (Menegalli et al., 1999; Sobral et al., 2001; Simon-Lukasik and Ludescher, 2004; Bertan et al., 2005). However, the bulk of this information concerns commercial mammalian gelatins. Although researchers are now increasingly turning their attention to fish gelatin films (Muyonga et al., 2004; Jongjareonrak et al., 2006a,b; Gómez-Guillén et al., 2007; Carvalho et al., 2008), the list of literature references dealing with these latter films is considerably shorter. The present literature seems to bear out that there are some differences in the physical properties of films obtained from mammalian and fish gelatins, the former being stronger and more permeable to water vapour and the latter more elastic (Sobral et al., 2001; Thomazine et al., 2005; Avena-Bustillos et al., 2006; Gómez-Guillén et al., 2007) although it remains somewhat unclear. Comparability of the data is limited because of the wide range of different experimental conditions employed for film producing, i.e., plasticizer type and concentration, dehydration temperature, film thickness and conditioning, etc. In addition, the recent knowledge about extraction and characterization of gelatin from many fish species, may difficult generalizing about fish gelatin properties. The purposes of this study were: (i) to characterize the physicochemical attributes of two different origin gelatins (one from tunaskin, the other from bovine-hide) on the basis of the more distinct parameters: aminoacid composition and molecular weight distribution; as well as thermal and rheological properties in the presence of plasticizers and (ii) to determine how these attributes affected the properties of the resulting films, produced in identical conditions to provide a proper comparison. 2. Materials and methods 2.1. Characterization of the gelatins The tuna-skin gelatin was prepared in our laboratory according to the method described by Gómez-Guillén and Montero (2001). The bovine-hide gelatin (Bloom 200/220) was commercially obtained from Sancho de Borja S.L. (Saragossa, Spain). 2.2. Amino acid composition An amount of 1 mg/ml of dry gelatin was dissolved in distilled water, and samples (50 ll) were dried and hydrolyzed in vacuumsealed glass tubes at 108 °C for 18 h in the presence of continuously boiling 5.7 N HCl containing 0.1% phenol with norleucine as internal standard. After hydrolysis, samples were vacuum-dried, dissolved in application buffer, and injected onto a Biochrom 20 amino acid analyser (Pharmacia, Barcelona, Spain). DL-5-Hydroxylysine hydrochloride and cis-4-hydroxy-D-proline (Sigma, St. Louis, MO, USA) were also used as standards to determine the amount of hydroxylysine and hydroxyproline, respectively.
2.3. Molecular weight profile The molecular weight distribution of the bovine-hide and tunaskin gelatins was determined by SDS–polyacrylamide gel electrophoresis. Gelatin (5 mg/ml) solutions were mixed with loading buffer (2% SDS, 5% mercaptoethanol, and 0.002% bromophenol blue) in a proportion of 1:4. Samples were heat-denatured at 90 °C for 5 min and analysed according to Laemmli (1970) in a Mini Protean II unit (Bio-Rad Laboratories, Hercules, CA, USA) using 4% stacking gels and 6% resolving gels at 25 mA/gel. Loading volume was 15 ll in all lanes. Protein bands were stained with Coomassie brilliant blue R-250. Type I collagen from foetal calf was used as a
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marker for a-chain and b-component mobilities. Also a molecular weight standard composed of myosin (212 kDa), a2-macroglobulin (170 kDa), b-galactosidase (116 kDa), transferrin (76 kDa) and glutamic dehydrogenase (53 kDa) (Amersham Pharmacia biotech, Buckinghamshire, UK) was employed. 2.4. Film-forming solution and film preparation The film-forming solutions were prepared using gelatin at a concentration of 4 g/100 ml of distilled water. Sorbitol (0.15 g/g gelatin) and glycerol (0.15 g/g gelatin) were employed as plasticizers. The mixtures were warmed and stirred at 40 °C for 15 min to obtain a good blend, and the films were made by casting an amount of 40 ml in square dishes (12 12 cm) and drying in a convection oven at 45 °C for 15 h. Prior to determinations the films were conditioned over a saturated solution of NaBr (58% relative humidity) at 22 °C in desiccators for 2 d. The moisture content of films, determined by AOAC method 24003 (AOAC, 1984) after the conditioning period, was 10.23% (±0.30) for the bovine-hide gelatin films and 13.46% (±0.34) for the tuna gelatin films. Film thickness was measured using a digital micrometer (Mitutoyo, model MDC-25M, Kanagawa, Japan), averaging nine different locations. 2.5. Characterization of the film-forming solutions 2.5.1. Dynamic viscoelastic properties Dynamic viscoelastic analysis of the film-forming solutions was carried out as described in Gómez-Guillén et al. (2007), on a Bohlin CSR-10 rheometer rotary viscometer (Bohlin Instruments Ltd., Gloucestershire, UK) using a cone-plate geometry (cone angle = 4°, gap = 0.15 mm). Cooling and heating from 40 to 6 °C and back to 40 °C took place at a scan rate of 1 °C/min, frequency of 1 Hz, and a target strain of 0.2 mm. The elastic modulus (G0 ; Pa), viscous modulus (G00 ; Pa) and phase angle (°) were determined as functions of temperature. Several determinations were performed for each sample, with an experimental error of less than 6% in all cases. 2.5.2. Gel strength The film-forming solutions were poured into glasses 2.3 cm in diameter and 3.6 cm in height and left to mature in a refrigerator at 2 °C for 16–18 h. Gel strength at 8–9 °C was determined, as described in Gómez-Guillén et al. (2002), on an Instron model 4501 Universal Testing Machine (Instron Co., Canton, MA, USA) with a 100-N load cell, a cross-head speed of 60 mm/min, and a flat-faced cylindrical plunger 1.27 cm in diameter. The maximum force (g) was determined when the plunger had penetrated 4 mm into the gelatin gels. 2.6. Characterization of the films 2.6.1. Mechanical properties A puncture test was performed to determine the breaking force and deformation of films at the breaking point (Gómez-Guillén et al. (2007). Films were placed in a cell 5.6 cm in diameter and perforated to the breaking point using an Instron model 4501 Universal Testing Machine (Instron Co., Canton, MA, USA) with a round-ended stainless-steel plunger (3 mm in diameter) at a cross-head speed of 60 mm/min and a 100-N load-cell. Breaking force was expressed in N and breaking deformation in %, according to Sobral et al. (2001). All determinations are the means of at least five measurements. 2.6.2. Thermal properties Calorimetric analysis was performed with a model TA-Q1000 differential scanning calorimeter (DSC) (TA Instruments, New Castle, DE, USA) previously calibrated by running high purity indium
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(melting point 156.4 °C; enthalpy of melting 28.44 W/g). Samples of approximately 10 mg (±0.002 mg) were weighed out using a model ME235S electronic balance (Sartorious, Goettingen, Germany) and were tightly encapsulated in aluminium pans and scanned under dry nitrogen purge (50 ml/min). Freshly conditioned films were rapidly cooled to 0 °C and scanned at between 0 and 90 °C at a heating rate of 10 °C/min. Glass transition temperatures, Tg (°C), were determined only on the first heating scans, values obtained on the second scans being deemed not relevant because of the practical impossibility of reproducing the original film conditioning. The glass transition temperature was estimated as the midpoint of the line drawn between the temperature at the intersection of the initial tangent with the tangent through the inflection point of the trace and the temperature of the intersection of the tangent through the inflection point with the final tangent. Tg data have been reported as the mean values of at least duplicate samples for each film, usually within ±1 °C. 2.6.3. Water solubility Film portions measuring 4 cm2 were placed in aluminium capsules with 15 ml of distilled water and shaken gently at 22 °C for 15 h. The solution was then filtered through Whatman no. 1 filter paper to recover the remaining undissolved film, which was desiccated at 105 °C for 24 h. Film solubility was calculated by the equation FS (%) = ((W0 Wf)/W0) 100, where W0 was the initial weight of the film expressed as dry matter and Wf was the weight of the undissolved desiccated film residue. All tests were carried out in triplicate. 2.6.4. Water vapour permeability Water vapour permeability was determined following the method described by Sobral et al. (2001). Films were attached over the openings of cells (permeation area = 15.9 cm2) containing silica gel, and the cells were placed in desiccators with distilled water at 22 °C. The cells were weighed daily for 7 d. Water vapour permeability was calculated using the equation WVP = w x t1 A1 DP1, where w was weight gain (g), x film thickness (mm), t elapsed time for the weight gain (h), and DP the partial vapour pressure difference between the dry atmosphere and pure water (2642 Pa at 22 °C). Results have been expressed as g mm h1 cm2 Pa1. All tests were carried out in duplicate. 2.7. Statistical analysis Statistical tests were performed using the SPSSÒ computer program (SPSS Statistical Software Inc., Chicago, IL, USA). One-way analysis of variance was carried out. Differences between pairs of means were assessed on the basis of confidence intervals using the Tukey-b test. The level of significance was p 6 0.05.
3. Results and discussion 3.1. Characterization of the gelatins Table 1 presents the amino acid composition of the gelatins. Both the bovine-hide and the tuna-skin gelatins exhibited typical type I collagen Gly content, representing approximately 1/3 of the total amino acids. As described by Asghar and Henrickson (1982), 50–60% of a-chains consist of tripeptides having the general formula Gly-X-Y, where X is generally proline and Y is mainly hydroxyproline. The proline plus hydroxyproline (imino acids) content was higher in the bovine-hide gelatin than in the tuna-skin one (210 vs. 185 residues/1000, respectively). Gelatins made from warm-blooded animal tissues have been reported to have a higher imino acid content, hydroxyproline in particular (Norland, 1990),
Table 1 Amino acid composition of the bovine-hide and the tuna-skin gelatines Number of residues/1000
Hyp Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg Hyl
Bovine-hide
Tuna-skin
83 46 33 39 74 127 342 113 19 4 11 24 4 12 4 25 47 5
78 44 21 48 71 107 336 119 28 16 7 21 3 13 7 25 52 6
and this promotes triple helix formation and stabilization of the gelatin at low temperatures (Burjandze, 1979) thanks to the hydrogen bonding ability of the –OH group on the hydroxyproline. In the present work the Hyp content of the tuna-skin gelatin was relatively high (78 residues/1000) although lower than the one for bovine-hide gelatin (83 residues/1000), due to the fact that this fish species do not come from very cold waters. Similarly, a relatively high content of hydroxyproline (74 residues/1000) has been recently reported in gelatin extracted from tuna fin (Aewsiri et al., 2008). Gelatins from warm-water fishes have a higher imino acid content than gelatins from cold-water fish species, closer to mammalian ones (Gilseman and Ross-Murphy, 2000a; Avena-Bustillos et al., 2006). With respect to the hydrophobic nature of our gelatins, both of them presented a similar content of hydrophobic residues of around 310 per 1000 residues. This is again attributed to the relatively warm water in which tuna lives. Cold-water fish gelatins are typically more hydrophobic. The electrophoretic profile of the bovine-hide gelatin was considerably more heterogeneous than that of the tuna-skin gelatin, thereby causing bands on the SDS gel to be more diffuse (Fig. 1). This is indicative of a broad range of molecular weights, probably attributable to the normal use of more severe extraction conditions than for the fish gelatin, for which extraction was carried out at temperatures below 45 °C. Both types of gelatin contained achains weighing around 100 kDa and b-components weighing around 200 kDa, typical of type I collagen. Nevertheless, there were appreciable differences in a-chain yields during extraction. The fish gelatin exhibited a a1/ a2 ratio of around 2, indicating that the native structure is maintained. In contrast, intensities for the bands for the a1 and a2 chains in the mammalian gelatin were similar, indicating possible degradation of the a1-chains during the extraction process. This is an interesting factor to bear in mind, since, as reported by Sims et al. (1997), a1-chains have greater gelling ability than a2-chains. The amount of b-components was also considerably higher in the fish gelatin, suggesting that the milder extraction conditions were more conducive to extracting intact, covalently linked a-chain dimers. Both gelatins had appreciable amounts of higher-molecularweight (>200 kDa) fractions of several crosslinked a-chains, but the bands were more distinct in the case of the fish gelatin, indicating less dispersal of molecular weights in the gelatin. Very highmolecular-weight polymers resulting from residual heat-stable crosslinks were also present in both types of gelatin, as revealed by the high-intensity bands at the tops of the polyacrylamide gels.
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The <100 kDa polypeptide fraction appeared to be more abundant in the bovine-hide gelatin, again largely attributable to more extensive cleavage of the -chain peptide bonds during gelatin extraction. Moreover, the presence of a high-intensity band at the bottom of the resolving gel in the mammalian gelatin indicated the presence of extensively hydrolyzed fractions that did not appear in the fish gelatin.
HMW-a
β
3.2. Characterization of the film-forming solutions
α1 α2
Tuna-skin
Bovine-hide
Tuna-skin
G' (Pa)
Fig. 1. Electrophoretic profile of the bovine-hide and tuna-skin gelatins. HMW-a, high-molecular-weight aggregates.
Fig. 2 shows the changes in the viscoelastic properties of the film-forming solutions during the cooling and subsequent heating ramps. The bovine-hide gelatin showed higher elastic modulus (G0 ) values at low temperatures, indicative of enhanced ability to refold into a triple helix (Ledward, 1986; Gómez-Guillén et al., 2002). The phase angle exhibited higher thermal transition points in the bovine-hide gelatin on both, the cooling and heating ramps, an indication that it was more heat stable. The average gelling temperature and melting temperature for the bovine-hide gelatin were, respectively, 20 °C and 30 °C, whereas for the tuna-skin gelatin were 15 °C and 22.5 °C. These higher rheological properties and thermostability are typical of mammalian gelatins (Leuenberger, 1991; Gilseman and Ross-Murphy, 2000b) and are mainly related to imino acid composition, hydroxyproline playing a
a
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Temperature (ºC) Bovine-hide
10
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Tuna-skin
Fig. 2. Changes in the viscoelastic properties (G0 , modulus of elasticity; G00 , modulus of viscosity; °, phase angle) of the film-forming solutions of bovine-hide and tuna-skin gelatin during the cooling (a) and subsequent heating (b) ramps.
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singular role in stabilizing the triple-stranded helix thanks to the hydrogen bonding ability of its –OH group (Burjandze, 1979). As mentioned above, the total imino acid content was higher in the bovine-hide gelatin. As expected, gel strength was significantly (p 6 0.05) lower in the tuna-skin gelatin (167 ± 4 g) than in the bovine-hide gelatin (211 ± 6 g). However, the difference was less pronounced than the difference observed in the elastic modulus. Triple helix formation takes place by association of the different a-chains during cold maturation of the gel. This gives rise to differences according to gelatin characteristics suggesting that the nucleation points initially formed in the tuna-skin gelatin are capable of considerable growth during maturation under refrigeration. A possible explanation for this is the molecular weight distribution of the tuna-skin gelatin employed, with higher amount of native structures -monomers and dimmers-. The tuna-skin gelatin had considerably higher amounts of a1-chains and b components than the bovine-hide gelatin did, and according to Sims et al. (1997) this could be responsible for its relatively high gel strength. This molecular weight distribution, in which the two a-chains comprising the b-component are already crosslinked, enables the chains to form triple helices upon cooling and contributes to helix growth during gel maturation. The presence of glycerol and sorbitol in the film-forming solutions, which were added as plasticizers in a relatively high concentration (30 g/100 g gelatin) should be also taken into consideration. Glycerol has been reported to exert a considerable gel strength-enhancing effect in fish gelatin gels, largely attributed to hydrogen bonding stabilization (Fernández-Díaz et al., 2001); however, it is worth noting that in the study reported by those authors, the glycerol concentration was approximately 7.5-fold higher than in the present one. 3.3. Characterization of the films Both the bovine-hide and the tuna-skin gelatin-based filmforming solutions yielded flexible and transparent films, similar (p 6 0.05) in thickness (109 ± 18 lm for the bovine-hide films and 98 ± 15 for the tuna-skin films). The data from the physical properties analysis are set out in Table 2. Both films presented a similar (p 6 0.05) maximum load value at the breaking point (10.5 N and 8.5 N for bovine-hide and tuna-skin, respectively). On the contrary, the tuna-skin gelatin film had breaking deformation values around 10 times higher than the values for the bovinehide gelatin film. Data from bibliography are not easily comparable because of the differences in film obtaining, type and concentration of plasticizers, gelatin type, measurement methodology, etc. Having this in mind there seems to be a trend to obtain stronger films from mammalian gelatins whereas fish gelatins render more deformable films (Sobral et al., 2001; Thomazine et al., 2005; Avena-Bustillos et al., 2006; Gómez-Guillén et al., 2007; Pérez-Mateos et al., 2009). In the present work both films were elaborated with the same type and concentration of plasticizers, so the differences in the Table 2 Physical properties (puncture force and deformation at the break point, water vapour permeability (WVP) and water solubility) of the bovine-hide and the tuna-skin gelatine films
Puncture force(N) Puncture deformation(%) WVP(108 g mm h1 cm2 Pa1) Water solubility(%) Tg (°C)
Bovine-hide
Tuna-skin
10.7 ± 2.2a 14.1 ± 5a 2.20 ± 0.11a 34.3 ± 0.6a 41.5 ± 1a
8.5 ± 1.6a 154 ± 36b 1.65 ± 0.39b 39.9 ± 1.3a 40.7 ± 1a
Results are expressed as mean value ± standard deviation. Different letters (a, b) indicate significant differences.
mechanical properties should be linked to the physico-chemical characteristics of the gelatin, especially regarding the aminoacid composition, which is greatly a species-specific characteristic, and the molecular weight distribution, which is mostly determined by processing conditions. Regarding the amino acid composition, mammal gelatins are well known to be richer in imino acids (Norland, 1990). As discussed above, the higher imino acid content (Pro + Hyp) strongly determine the superior rheological properties and thermostability of bovine-hide gelatin gels. This is largely attributable to the formation of junction zones by hydrogen bonding, which in turn yield a multitude of intra- and inter-chain interactions important to cold stabilization of the gel network. These interactions are promoted by the availability of more -OH groups to form hydrogen bonds with bridging water molecules inside the triple helices and also between the chains of neighbouring triple helices (Asghar and Henrickson, 1982). Thus, water plays an important role in augmenting the stability of the triple helical structure. According to Djabourov et al. (1985), the structural water fraction increases during cold gelation and is proportional to helix formation. Water molecules are taken into the structure of the triple helix and are hydrogen bonded to the -CO or -NH groups. However, presumably the involvement of hydrogen bonds is lower in dried films, in which most of the water has been removed and the storage temperature is around 20 °C, which may act to cause the differences in imino acid content between the two gelatins to be less appreciable. In this connection, the mechanical properties of gelatin films have been related to the triple helix content of the gelatin employed (Bigi et al., 2004); however, it is known to decrease upon isothermal dehydration of films (Wetzel et al., 1987). Regarding the molecular weight profile, it could be assumed that the presumably more severe treatment conditions needed for the commercial bovine-hide gelatin extraction led to a higher proportion of protein fractions of low molecular weight (<100 kDa). According to the works by Muyonga et al. (2004) and Carvalho et al. (2008) this fact results in a reduction of film strength and an increase in film extensibility. The role of gelatin molecular weight distribution in the rheological properties of the films may be associated to the presence and concentration of plasticizers in the formulation, which in the present study was relatively high (30 g/ 100 g gelatin). Thus, for the same mass concentration of plasticizer, films made using a lower-molecular-weight biopolymer could be more plasticized, because the molar plasticizer/biopolymer ratio is higher (Thomazine et al., 2005). However, in our work both gelatins presented a considerable amount of high-molecular-weight aggregates (Fig. 1) which would improve the film strength in both cases. Moreover, the predominance of b-components in the fish gelatin could also act to noticeably strengthen the corresponding films, despite the lower iminoacid content. Regarding the differences in film deformation, which was 10fold higher for the tuna-skin films, data from literature show us that fish gelatins produce more deformable films than mammal ones (Sobral et al., 2001; Thomazine et al., 2005; Gómez-Guillén et al., 2007; Pérez-Mateos et al., 2009). In the present work, this finding is not possible to be explained in terms of a higher plasticizing effect associated to the gelatin molecular weight distribution, since bovine-hide gelatin appeared to have a higher amount of hydrolyzed peptide fractions which are prompt to interact with plasticizer molecules. Thus, one possible explanation lies in the different imino acid content in both gelatins. The higher Pro + Hyp content in the bovine-hide gelatin may impose conformational constraints by imparting a higher degree of molecular rigidity to the corresponding films, which would obviously lead to a lower deformability. Fig. 3 plots the DSC traces. Despite being encapsulated in nonhermetically sealed pans, samples lost only 1–2% of their weight
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after scanning, which indicated that the films had good water retention capacity. This resulted in a certain bending of the trace due to water vapourization, which somehow distorted the DSC profiles at the glass transition point. On the other hand, glass transition seemed to overlap with an enthalpy relaxation phenomenon. Both water vapourization and enthalpy relaxation made precise measurement of the Tg values somewhat difficult. The films were wholly amorphous, since they did not exhibit any melting event in the temperature region that would otherwise be expected based on the heat stability characteristics of the gelatins. The plasticizing effects of the glycerol plus sorbitol (Sobral et al., 2001) and water may inhibit gelatin crystallization. Additionally, moisture reduction up to the films typical levels (10–14% depending on the gelatin) caused the complex systems to move from regions of gel–sol– gel transitions to vitreous domains. Tg values were similar in both films, somewhat lower in the tuna-skin gelatin film, though the difference was not significant (Table 2). Cold-water fish gelatins have been reported (Gilseman and Ross-Murphy, 2000a) to be less heat stable than mammalian gelatins, mostly due to the lower imino acid content. In contrast, the properties of warm-water fish gelatins may be quite similar to those of mammalian gelatins (Gilseman and Ross-Murphy, 2000b). Although tuna cannot be considered a warm-water fish species it is not located at very cold-water seas as for example cod or hake are. Table 2 also lists the water vapour permeability (WVP) of the films. The tuna-skin gelatin film was less permeable than the bovine-hide gelatin film (p 6 0.05). One more time the explanation could lie in the differences in the amino acid composition of the two gelatins. Avena-Bustillos et al. (2006) observed that the hydrophobic nature of the amino acids of a gelatin highly determine the water vapour permeability of the corresponding films, being the fresh water fish species gelatin films the less permeable, followed by the warm-water fish ones and finally the mammal gelatin films. However in our work the differences recorded in water vapour permeability cannot be absolutely ascribed to amino acid composition because the total amount of hydrophobic amino acids was quite similar for both gelatins (310 for bovine-hide gelatin vs. 311 for tuna-skin one). However Hyp, which is a hydrophilic amino acid, was higher for the bovine-hide gelatin film, and this could have contributed in part to its higher water vapour permeability. The molecular weight distribution has also been thought to exert an effect. The bovine-hide gelatin presented a considerably higher amount of protein fractions up to 100 kDa which could be more susceptible to the plasticizing effects of glycerol and sorbitol than
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the bigger fragments. Both glycerol and sorbitol are well known to cause an increase in the water vapour permeability of gelatin films due to its hydrophilic –OH groups and this effect is directly related to plasticizer concentration (Sobral et al., 2001; Jongjareonrak et al., 2006a). Contrary to expectation, the water solubility of the tuna-skin gelatin film was not significantly different from that of the bovine-hide gelatin film (Table 2). The bovine-hide gelatin may presumably present lower water solubility due to the straightening effects of the hydrogen bonds promoted by imino acids. However, as discussed for the mechanical properties, the lower water content seems to play a singular role reducing the straightening effects of hydrogen bonding in the films. It seems to be more determinant the nature and extent of the very high-molecular-weight fraction, in which residual heat-stable crosslinks predominate. The electrophoretic analysis did not reveal any substantial differences in this fraction in the two types of gelatin. The low extraction temperature together with intrinsic attributes of the tuna-skin collagen used in this study may help retain some heat-stable crosslinked fractions in the resulting gelatin, leading to relatively low water solubility of the film. Carvalho et al. (2008) elaborated films with two differently processed Atlantic halibut skin gelatins, which did not present any fraction of very high-molecular-weight, and found them to be totally soluble in water. Other study dealing with cod-skin gelatin film has yielded water solubility values as high as 90–100% (Pérez-Mateos et al., 2009), compared with values of around 30% for bovine gelatin film (Bertan et al., 2005). Unfortunately, these works do report any data about neither the molecular weight distribution nor the amino acid profile of the gelatins.
4. Conclusions As expected, the different origin and extraction process of the bovine-hide and the tuna-skin gelatins influenced the physical properties of the corresponding filmogenic solutions. However this was not so evident in the films, presumably due to the lower involvement of hydrogen bonding as a consequence of the reduction of the water content. The breaking force and the water solubility were scarcely affected by the gelatin origin (bovine-hide or tuna-skin) and presented similar values in both films. On the contrary, the puncture deformation and the water vapour permeability were quite different, being attributed to the amino acid profile and the molecular weight distribution of the gelatins. Acknowledgements
a Heat Flow (Endo Down)
This research was financed by the ‘‘Ministerio de Educación y Ciencia”, under Project AGL2005-02380.
b
Tg
References
0.1 W/g
10
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30
40
50
60
70
80
Temperature (ºC) Fig. 3. DSC traces and Tg values for the bovine-hide (a) and tuna-skin (b) gelatin films.
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