Process for extracting gelatin from marine snail (Hexaplex trunculus): Chemical composition and functional properties

Process Biochemistry 47 (2012) 1779–1784

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Process for extracting gelatin from marine snail (Hexaplex trunculus): Chemical composition and functional properties Zied Zarai a , Rafik Balti b,∗ , Hafedh Mejdoub a , Youssef Gargouri a , Adel Sayari a a b

Laboratoire de Biochimie et de Génie Enzymatique des Lipases, Ecole Nationale d’Ingénieurs de Sfax, B.P. “1173”, 3038 Sfax, Tunisia Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d’Ingénieurs de Sfax, B.P. “1173”, 3038 Sfax, Tunisia

a r t i c l e

i n f o

Article history: Received 9 March 2012 Received in revised form 2 June 2012 Accepted 4 June 2012 Available online 18 June 2012 Keywords: Gelatin Marine snail Hexaplex trunculus Functional properties Gel strength

a b s t r a c t Gelatin was extracted, for the first time, from the meat of Tunisian marine snail by the acid extraction process with a yield of 3 g/100 g. Snail meat gelatin (SMG) had high protein (88.62%) but low fat (0.77%) content and contained a high number of imino acids. SMG showed high band intensity for ␣- and ␤components and some degradation peptides. The absorption bands of SMG in Fourier transform infrared spectra were mainly situated in the amide band region (amide I and amide II). The gel strength of the SMG (103 g) was lower than those from other marine species reported in the literature. SMG exhibited a low foam capacity (75.44%) but a high emulsifying stability (38.12 min) and WHC (120%). It can be concluded from the present study that snail meat is a prospective source to produce gelatin in good quality with desirable functional properties. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction Gelatin is an irreversibly thermally denatured and partly hydrolysed form of collagen [1]. In the food industry, gelatin has been widely applied as an ingredient to improve the elasticity, consistency and stability of foods. Depending on the method in which collagens are pre-treated, two different types of gelatin with different characteristics including type-A, acid-treated collagen, and type-B, an alkaline treated counterpart, can be produced [2]. Acid treatment is the most suitable treatment for less fully cross-linked collagens commonly found in pig or fish skins, whereas alkaline treatment is appropriate for the more complex collagens found in bovine hides [1,3]. Bovine and porcine skin and bone have usually been utilized commercially for gelatin production. However, in some countries, the use of gelatin from warm-blooded animals is restricted owing to the transmission of bovine spongiform encephalopathy and religious reasons [4]. To date, however, few alternatives are available, and as a result it has not been possible to eliminate gelatin. Researchers from academia and industry are continually searching for an alternative to gelatin, and to find new sources of gelatin that might be more favourably viewed. Therefore, gelatin from marine resources is a possible alternative for mammalian gelatins [5].

∗ Corresponding author. Tel.: +216 74 274 088; fax: +216 74 275 595. E-mail address: rafi[email protected] (R. Balti).

Gelatin is a product of rapidly growing market. In 2003, the world market for gelatin reached 278,300 tonnes; consisting of 42.4% from pig-skin origin, 29.3% bovine hides, 27.6% bones and 0.7% from other sources [6]. In previous years, Karim and Bhat [7] reported that the annual world output of gelatin increased to 326,000 tonnes with the highest source being pigskin (46%), followed by bovine hides (29.4%), bones (23.1%) and other sources (1.5%). At present, the fish gelatin production is very low, yielding about 1% of the annual world gelatin production of 270,000 metric tonnes [8]. Recently, most of the published data is about gelatins from various fish species such as brownbanded bamboo shark (Chiloscyllium punctatum) and blacktip shark (Carcharhinus limbatus) skins [9], grey triggerfish skin [10], tuna heads [11] and cuttlefish skin [12] have been extracted and characterized. However, few studies have focused and little information regarding the characteristics of gelatins from marine invertebrate species, such as marine snail, has been reported. Tunisia’s marine fisheries produce several thousand metric tonnes of fish and shellfish annually, mainly for exportation. Marine snail (Hexaplex trunculus) is Tunisia’s new leading economically important shellfish species. H. trunculus (also known as Murex trunculus or the banded dye-murex) is a medium-sized species of sea snails, a marine gastropod mollusc in the family Muricidae, the murex shells or rock snails. Therefore, the aims of this investigation were to extract gelatin from the meat of marine snail (H. trunculus) and to study its physicochemical characteristics as well as its functional properties.

1359-5113/$ – see front matter. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.06.007

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2. Materials and methods

2.7. Electrophoretic analysis

2.1. Materials

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli [14] using 70 g/l resolving gel and 40 g/l stacking gel. After electrophoresis, the gel was stained with 1 g/l Coomassie brilliant blue R-250, dissolved in water, methanol and trichloroacetic acid (5:4:1), and de-stained using a solution containing methanol, distilled water, and acetic acid at a ratio of 5:4:1. After electrophoresis, the gel was stained with Coomassie Brilliant blue R-250.

Marine snails (H. trunculus) were collected from the fish market of Sfax city (Tunisia). The samples were placed on ice and transported, immediately, to the laboratory. The outside of H. trunculus was thoroughly cleaned with fresh cold running water. The shell was opened manually. Neither heat nor anaesthetics were used before opening the shells, and it was ensured that no cuts were made or the soft tissues damaged at this stage. The inside was rinsed with fresh cold running water to remove sand or other foreign material. The meat was removed from shell by separating adductor muscles and tissue connecting at hinge and carefully removing the visceral mass. In each case, meat of snails was blended then stored at −20 ◦ C until use. 2.2. Gelatin extraction Snail (H. trunculus) meat was firstly soaked in 0.02 M NaOH with a tissue/solution ratio of 1:6 (w/v) for 60 min at room temperature to remove non-collagenous proteins and pigments. The mixture was stirred continuously and the alkaline solution was changed every 30 min. The pretreatment sturry was centrifuged at 6000 × g for 5 min using a Hermele Z36HK refrigerated centrifuge (Hermle Labortechnik GmbH, Wehingen, Germany). The pelleted tissue was washed by resuspension in deionized water with stirring at room temperature for 10 min, followed by centrifugation at 6000 × g for 5 min. The gelatin was extracted from the washed pelleted tissue with 3% acetic acid at pH 4.0 with a tissue/solution ratio of 1:6 (w/v) for 9 h at 60 ◦ C with gentle stirring. The mixture was then centrifuged at 6000 × g for 10 min to remove insoluble material and the gelatin supernatant was dialysed against 5 volumes of deionized water. Finally, the supernatant was collected and freeze-dried (Bioblock Scientific Christ ALPHA 1-2, IllKrich-Cedex, France). The powder obtained referred to as snail meat gelatin (SMG) was stored at 4 ◦ C until used.

2.8. Fourier transform infrared spectroscopy FT-IR spectra were obtained by a Nicolet Nexus FT-IR spectrometer (Thermo Electron Corporation). The snail gelatin was tested by infrared analysis with the KBr method. 2.9. Determination of emulsifying properties The emulsion activity index (EAI) and the emulsion stability index (ESI) of gelatin were determined according to the method of Pearce and Kinsella [15] with slight modifications. The gelatin solutions were prepared by dissolving dry gelatin in distilled water at 60 ◦ C for 30 min. Thirty millilitres of gelatin solutions 1% (w/v) were homogenized with 10 ml of soybean oil for 1 min at room temperature (22 ± 1 ◦ C) using Moulinex® R62 homogenizer. Aliquots of the emulsion (50 ␮l) were taken from the bottom of the container at 0 and 10 min after homogeneization, and diluted 100-fold with 0.1% SDS solution. The mixtures were mixed thoroughly for 10 s using a vortex mixer. The absorbance of the diluted solutions was measured at 500 nm using a spectrophotometer (T70, UV/VIS Spectrometer, PG Instruments Ltd., China). The absorbances measured immediately (A0 ) and 10 min (A10 ) after emulsion formation were used to calculate the emulsifying activity index (EAI) and the emulsion stability index (ESI) in accordance with as follows Pearce and Kinsella [15] as follows:

EAI (m2 /g) = 2.3. Proximate analysis The moisture, ash and fat contents of the gelatin powder were determined according to the AOAC methods number 927.05, 942.05 and 920.39 B, respectively [13]. The protein content was determined by estimating its total nitrogen content by Kjeldahl method according to the AOAC method number 984.13 [13]. A factor of 5.4 was used to convert the nitrogen value to protein. All measurements were performed in triplicate. The yield of gelatin was calculated based on wet weight of fresh tissue. Yield (%) =

Weight of freeze dried gelatin (g) × 100 Wet weight of fresh tissue (g)

2.4. Determination of gel strength Gel strength of gelatin was determined according to the method of GómezGuillén et al. [3]. Gelatin sample was dissolved in distilled water at 60 ◦ C to obtain the final concentration of 6.67% (w/v). The solution was stirred for 30 min until the gel was solubilized completely and cooled in a refrigerator at 7 ◦ C for 16–18 h. The gel strength of gelatin gel was determined using a Model TA-TX2 texture analyzer with a 5 kN load cell equipped with a 1.27 cm diameter flat-faced cylindrical Teflon plunger. The dimensions of the sample were 3.8 cm in diameter and 2.7 cm in height. Gel strength was expressed as maximum force (in g), required for the plunger to press the gel by 4 mm depression at a rate of 0.5 mm/s. The measurement was performed in triplicate. 2.5. Determination of gelatin colour and gel clarity The colour of dry gelatin sample was determined using a ColorFlex spectrocolorimeter (Hunter Associates Laboratory Inc., Reston, VA, USA) based on three colour co-ordinates, namely L (lightness), a (redness/greenness) and b (yellowness/blueness). The sample was filled in a 64 mm glass sample cup with three readings in the same place and triplicate determinations were taken per sample. The white tile and black glass were used to standardize the equipment. Clarity was determined by measuring transmittance (%T) at 620 nm in spectrophotometer (Thermo spectronic, Cambridge, UK) through 6.67% (w/v) gelatin solution, prepared as described previously. 2.6. Determination of amino acid composition Gelatin samples were oxidized for 17 h with a performic acid/phenol mixture, hydrolysed in 6 M HCl for 24 h at 110 ◦ C (boiled under reflux), pH-adjusted to 2.2, diluted with 0.2 mol/l sodium citrate loading buffer, pH 2.2, and micro-filtered with 0.45 mm Spartan membrane filter prior to analysis on a Beckman 6300 amino acid analyzer (Beckman Instruments Inc., Fullerton, CA, USA).

2 × 2.303 × A0 × N c × ϕ × 10, 000

where N refers to dilution factor, c to the weight of protein per unit volume (g/ml), and ϕ to the oil volumetric fraction (0.25). All determinations are means of at least three measurements. ESI (min) =

A0 × 10 A0 − A10

2.10. Determination of foaming properties Foam expansion (FE) and foam stability (FS) of gelatin solutions were tested according to the method of Shahidi et al. [16], with a slight modification. Twenty millilitres of gelatin solutions (1%, w/v) were homogenized using a Moulinex® R62 homogenizer to incorporate the air for 1 min at room temperature (25 ± 1 ◦ C). The whipped sample was then immediately transferred into a 50 ml graduated cylinder, and the total volume was measured at 0, 30 and 60 min after whipping. Foam capacity was expressed as foam expansion at 0 min, which was calculated according to the following equation: FE (%) =

VT − V0 × 100 V0

Foam stability was calculated as the volume of foam remaining after 30 and 60 min.

FS (%) =

Vt − V0 × 100 V0

where VT is the total volume after whipping (ml); V0 is the volume before whipping; Vt is the total volume after leaving at room temperature for different times (30 and 60 min). All determinations are means of at least three measurements. 2.11. Determination of water holding capacity Water holding capacity (WHC) of gelatin sample was measured by a partially modified method of Lin et al. [17]. Dry gelatin (0.5 g) was placed in a centrifuge tube and weighed. Distilled water (50 ml) was added and held at room temperature for 1 h. The gelatin solution was mixed with vortex mixer for 5 s every 15 min. The mixture was then centrifuged at 450 × g for 20 min. The upper phase was removed and the centrifuge tube was drained for 30 min on a filter paper after tilting to a 45◦ angle. The water holding capacity was calculated as the weight of the contents of the tube after draining divided by the weight of the dried gelatin, and expressed as the weight% of dried gelatin. 2.12. Statistical analysis All data were subjected to Analysis of Variance (ANOVA) and differences between means were evaluated by Duncan’s Multiple Range Test. The SPSS statistic program (Version 10.0) (SPSS, 1.2, 1998) was used for data analysis.

Z. Zarai et al. / Process Biochemistry 47 (2012) 1779–1784 Table 1 Proximate composition of marine snail meat and the extracted gelatin. Values are given as mean ± SD from triplicate determination. Composition (%)

Snail meat

Moisturea Proteina Fata Asha

69.92 15.74 8.99 4.98

± ± ± ±

1.59a 0.40a 0.28a 0.25a

Marine snail gelatin 6.83 88.62 0.77 0.82

± ± ± ±

0.71b 0.45b 0.21b 0.05b

Values in the same row with different letters (a,b) differed significantly (p < 0.05). a Values are given as mean ± SD from triplicate determinations.

3. Results and discussion 3.1. Composition of snail gelatin The proximate composition of snail meat and SMG is shown in Table 1. Fresh snail meat contained moisture as the major component (69.92%), followed by protein (15.74%) and fat (8.99%). SMG had high protein (88.62%) and low ash (0.82%) and fat (0.77%) contents, suggesting the efficient removal of fat and minerals from the meat material. Muyonga et al. [18] reported that protein contents of gelatins derived from skins and bones of young Nile perch were 88.8% and 83.3%, respectively. Gelatins from skins of bigeye snapper and cuttlefish had protein contents of 87.9% and 91.35%, respectively [12,19]. Gelatin as such is a pure protein and nothing else. So, the presence of ash, lipid and other impurity at very low contents are important for the quality of gelatins. SMG had lower ash content than the recommended maximum value of 2.5%. Gelatin was extracted from snail meat with a yield of 3% on the basis of wet weight. This result was similar to those obtained from squid (2.6%) [3], channel catfish (3.9%) [20] and mackerel (3.5%) [21], but lower than other marine species, such as grey triggerfish (5.6%) [10], cuttlefish (7.8%) [12] and tuna (18.1%) [11]. The difference in gelatin recovery from different species could be attributed to the intrinsic characteristics of the matrix used for extraction and collagen molecules, the collagen content, the amount of soluble components in the matrix, the loss of extracted collagen through leaching during the series of washing steps or to an incomplete collagen hydrolysis [22], since these properties vary with the species and the age of the marine species. Moreover, the degree of conversion of collagen into gelatin depends on the processing parameters (temperature, extraction time and pH); the pretreatment conditions, and the properties and the preservation method of the starting raw material [2]. 3.2. Amino acid composition of gelatin The amino acid composition of marine snail gelatin expressed as residues/1000 total amino acid residues is shown in Table 2. The amino acid profile obtained for SMG is comparable to those from gelatins extracted from other marine species and mammals [3,5], being glycine the most predominant residue (321 residues/1000 residues). Other important residues found in SMG were alanine (73), arginine (51), Serine (61), aspartic acid (67), glutamic acid (99) and the imino acids proline (105) and hydroxyproline (98). The content of the acid residues was higher in comparison to those reported previously, and these differences may be due to the acid pretreatment, since during this step in the gelatin extraction, some of the glutamine and asparagine residues might have converted or oxidized into their acidic forms. The high content of proline (Pro) and hydroxyproline (Hyp) in the samples were indicative of collagen and/or its derivatives [23]. The imino acids (Pro + Hyp) content (∼20%) was higher to those previously reported for cuttlefish (Sepia officinalis) and other marine gelatins [3,5,12]. Both residues are known to impart considerable rigidity to the collagen structure [2]. The stability

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Table 2 Amino acid composition of marine snail (H. trunculus) gelatin. Amino acidsa Hydroxyproline (Hyp) Aspartic acid (Asx) Threonine (Thr) Serine (Ser) Glutamic acid (Glx) Proline (Pro) Glycine (Gly) Alanine (Ala) Cysteine (Cys) Valine (Val) Methionine (Met) Isoleucine (Ile) Leucine (Leu) Tyrosine (Tyr) Phenylalanine (Phe) Hydroxylysine (Hyl) Histidine (His) Arginine (Arg) Lysine (Lys) Total Imino acids (Pro + Hyp)

Marine snail gelatin 98 67 28 61 99 105 321 73 0 22 2 12 23 9 10 8 3 51 8 1000 203

Asx = Asp + Asn; Glx = Glu + Gln. a Determinations were performed in triplicate and data correspond to mean values. Standard deviations were in all cases lower than 2%. Proline and lysine hydroxylation are also shown (mean ± SD).

of the triple helical structure in gelatin has been associated with the total content of pyrrolidone amino acids, proline and hydroxyproline [24]. Hydroxyproline plays an important role in the stabilization of the triple helical strands of collagen via its hydrogen bonding ability through its OH group [24]. In addition, the amounts of these imino acids especially that of Hyp depend on the environmental temperature in which the fish lives, this in turn affects the thermal stability of collagens and their derivatives. 3.3. Molecular weight distribution Functional properties of gelatins are influenced by the amino acid composition, the distribution of the molecular weights, structures, and composition of their subunits. Protein patterns of gelatin from snail meat under reducing condition are shown in Fig. 1. ␣1 and ␣2 -chains were found as the major components and similar to that of standard collagen type I. ␤-Component was also observed in the gelatin from snail meat. Gelatin with high proportion of ␣ and ␤ chains has also been extracted from cuttlefish [15], and Alaska pollock [28]. In addition, proteins with molecular weights lower than ␣-chains (∼100 kDa) were also observed in the snail meat gelatin sample. This might be due to the degradation of ␣, ␤- and/or ␥-components during gelatin extraction or preparation. The extraction at higher temperature led to lower molecular weight peptides. Heat-induce breakage of protein components in gelatin occurred during the extraction process of Nile perch skin gelatin and was enhanced with higher temperature of extraction [18]. Furthermore, the degradation of gelatin from bigeye snapper skin caused by some proteinases during extraction process had been reported [19]. The formation of degradation peptides is associated with the low viscosity, low melting point, low setting point, high setting time, as well as decreased bloom strength of gelatin [18,24]. 3.4. Fourier transformed-infrared spectroscopy The frequencies at which major peaks occurred in the SMG FT-IR spectrum are shown in Fig. 2. Four main regions were identified: 3600–2300 cm−1 (amide A), 1656–1644 cm−1 (amide I), 1560–1335 cm−1 (amide II) and 1240–670 cm−1 (amide III). These peaks were similar to those reported by Muyonga et al. [26]. The

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disorder in gelatin, and are associated with the loss of triple helix state, as well as a decrease in molecular order. Thus, the differences detected in the present work might be due to denaturation of collagen into gelatin, which in turn provoke a change in the collagen secondary structure. The extent of these changes seems to be influenced by the gelatin extraction conditions, the number of native crosslinks in the collagen structure, and the amount of collagenous tissues from which gelatin is extracted [26,29].

3.5. Gel strength

Fig. 1. SDS-PAGE patterns of marine snail (H. trunculus) gelatin. 1: 30 ␮g; M: molecular weight markers; ␤: ␤-chain; ␣1 : ␣1 -chain; ␣2 : ␣2 -chain.

amide A peak absorption is due to N H stretching, amide I peak to C O stretching, amide II peak to N H bending and C N stretching vibrations, while the amide III peak is a complex system mainly associated with CH2 residual groups from glycine and proline [27]. The absorption in the amide I region is probably the most useful for infrared spectroscopic analysis of the secondary structure of proteins. Its exact location depends on the hydrogen bonding and conformation of the protein structure. In our study, the amide I peak was observed at 1656 cm−1 , which is in agreement with data reported by Liu et al. [28], who stated that the absorption peak at 1650 cm−1 was characteristic pattern reflecting the amide I of head bone gelatin of channel catfish. Since most proteins have mixed secondary structures, the amide I band often shows several components or shoulders, consequently deconvolution studies can be applied to further investigate conformation changes. In comparison to the FT-IR spectra for other marine gelatins [29,30], SMG showed lower intensity in the amide I, II and III bands. These spectral changes are indicative of greater

Gelatin is highly capable of forming hydrogen bonds with water molecules to form a stable three-dimensional gel. Gel strength is one of the most important functional properties of gelatin, and fish gelatin typically has lower gel strength than mammalian gelatin [4]. Gel strength of gelatin from marine snail (H. trunculus) meat was shown in Table 3. The gel strength obtained in this study (103 g) was similar to that of salmon (108 g) [31] and bigeye snapper (105 g) [19] and lower than that of cuttlefish (181 g) [12], grey triggerfish (168 g) [10], and Nile perch (229 g) [18]. Generally, the gel strength of fish gelatin has been reported in a wide range 124–426 g, compared to 200–300 g for bovine or porcine gelatin. The difference in gel strength may possibly be due to the lower content of imino acids, proline and hydroxyproline found in fish gelatine (16–18%) compared to the mammalian gelatines (24%). Other than the origin of the raw materials, the differences in extraction process used and the intrinsic properties of collagen which varies among fish species could be explain the differences in gel strength. This is supported by an example on gelatin extraction with different concentrations of sodium hydroxide and acetic acid [25].

3.6. Gelatin colour and gel clarity The colour of gelatin extracted fro marine snail (H. trunculus) was expressed in terms of L, a, and b and there were presented in Table 3. In general, satisfactory gelatin should have little colour. Marine snail gelatin showed a high lightness value (L). Gelatin manufacture generally has a good process to clarify the impurities from the gelatin solution, such as chemical clarification and filtration processes. Lower a and b values were also founded in SMG, when compared with other gelatins from marine resources [12]. Both colour and clarity of a gelatin gel are important aesthetic properties, depending on the application for which the gelatin is intended. While marine snail gelatin solution showed the highest transmittance (%T) (Table 3). The turbidity and dark colour of gelatin is commonly caused by inorganic, protein and mucosubstance contaminants, introduced or not removed during its extraction. However, the colour did not affect functional properties of gelatin.

Table 3 Colour, gel strength and gel clarity of marine snail (H. trunculus) gelatin. Properties

Fig. 2. Fourier transform infrared (FTIR) spectra of marine snail (H. trunculus) gelatin.

Gel strength (g)a Colour La aa ba Transmittance (%)a a

Marine snail gelatin 103 ± 1.7 28.62 −1.12 2.85 48.77

± ± ± ±

0.64 0.05 0.32 0.48

Values are given as mean ± SD from triplicate determination.

Z. Zarai et al. / Process Biochemistry 47 (2012) 1779–1784 Table 4 Functional properties of marine snail (H. trunculus). Values are given as mean ± SD from triplicate determination. Properties EAI (m2 /g)a ESI (min)a FE (%)a FS (%)a 30 min 60 min WHC (%)a a

Marine snail gelatin 32.77 ± 1.84 38.12 ± 1.05 75.44 ± 2.66 70.30 ± 0.51 61.86 ± 2.20 120.00 ± 1.65

Values are given as mean ± SD from triplicate determination.

3.7. Emulsifying properties of gelatin Gelatin, and to some extent collagen, are used as a foaming, emulsifying, and wetting agent in food, pharmaceutical, medical, and technical applications due to their surface-active properties. Emulsion activity index (EAI) and emulsion stability index (ESI) of marine snail gelatin was presented in Table 4. At a concentration of 1 g/100 ml, EAI of SMG was 32.77 ± 1.84 m2 /g. This value was higher than those reported for gelatins from cuttlefish (23.67 ± 0.36 m2 /g.) and grey tiggerfish (21.44 ± 0.09 m2 /g) [10,12]. The amphoteric nature with the hydrophobic zones on the peptide chain make gelatin to behave as an emulsifier and it is being used in the manufacture of toffees and water-in-oil emulsions such as low fat margarine, salad dressings, and whipped cream. In addition, high solubility of the protein in the dispersing phase increases the emulsifying efficiency, because the protein molecules should be able to migrate to the surface of the fat droplets rapidly. Emulsions containing gelatin from marine snail was very stable and ESI at a concentration of 1 g/100 ml was 38.12 ± 1.05 min (Table 4). Generally, larger and longer peptides could stabilize the protein film at the interface more effectively. 3.8. Foam capacity and foam stability Foam formation ability is another important property of gelatin for commonly used foods such as marshmallows. Foam expansion (FE) and Foam stability (FS) of marine snail gelatin are shown in Table 4. The foam capacity of marine snail gelatin (75%), at 1 g/100 ml was much lower than that of bovine gelatin (119%) [12]. Foam formation is generally controlled by transportation, penetration and reorganization of protein molecules at the air–water interface. A protein must be capable of migrating rapidly to the air–water interface, unfolding and rearranging at the interface to express good foaming properties [12]. The positive correlation between hydrophobicity of unfolded proteins and foaming characteristics has been reported [32]. At the same concentration, and at 30 and 60 min, FS of marine snail gelatin were 70.30% and 61.86%, respectively. This result indicates that MSG might form a film with stronger and greater elasticity, leading to stable foam. Foam stability of protein solutions is generally positively correlated with molecular weight of peptides. In addition, foam stability depends on the nature of the film and indicates the extent of protein–protein interaction within the matrix. 3.9. Water-holding of gelatin The functional properties of proteins in a food system depend in part on the water holding capacity (WHC) which refers to the ability of protein to imbibe water and retain it against a gravitational force within protein matrix. The dried MSG showed a high value of WHC (120%) (Table 4), which means that it was capable to retain 1.2 g of water per

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gram of gelatin. Since the primary function of hydrocolloids is to retain water, the high value of WHC obtained for MSG suggests the existence of a great number of pores and voids within the gelatin structure. WHC of MSG may be attributed to its microstructure, specifically to its primary chemical structure, hydrophobic/hydrophilic balance and particle size. As shown in Table 2, the amount of hydroxyproline in MSG was 98 residues/1000 residues. The water binding capacity of solubilized gelatin makes it suitable material for reducing drip loss and impairing juiciness in frozen fish or meat products when thawed or cooked, and where denatured protein has suffered a partial loss of its water-holding capacity. 4. Conclusion This work reported the first process for extracting gelatin from the meat of marine snail (H. trunculus). Electrophoretic profile of SMG showed that is contained protein, ␣1 and ␣2 -chains as the major component. Furthermore, the gel strength of SMG was lower than that of mammalian gelatin, but was superior to some gelatins from other marine species previously reported and was less yellowish and reddish in colour. Results from the present study clearly demonstrate that gelatin can be successfully extracted from marine snail with an acceptable quality and having better chemical composition and functional properties. So we can assume that the marine snail is a prospective source to produce gelatin with the desirable characteristics. Acknowledgement This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia. References [1] Foegeding EA, Lanier TC, Hultin HO. Characteristics of edible muscle tissues. In: Fennema OR, editor. Food chem. New York, USA: Marcel Dekker Inc.; 1996. p. 902–6. [2] Johnston-Banks FA. Gelatin. In: Harris P, editor. Food gels. London: Elsevier Science; 1990. p. 233–89. [3] Gómez-Guillén C, Turnay J, Fernández-Díaz M, Ulmo N, Lizarbe M, Montero García P. Structural and physical properties of gelatin extracted from different marine species: a comparative study. Food Hydrocolloid 2002;16:25–34. [4] Gilsenan PM, Ross-Murphy SB. Shear creep of gelatin gels from mammalian and piscine collagens. Int J Biol Macromol 2001;29:53–61. [5] Gómez-Guillén C, Pérez-Mateos M, Gómez-Estaca J, López-Caballero E, Giménez B, Montero P. Fish gelatin: a renewable material for developing active biodegradable films. Trends Food Sci Technol 2009;20:3–16. [6] GEA. Gelatin processing aids. Hudson: GEA Group; 2010. [7] Karim AA, Bhat K. Fish gelatin: properties, challenges and prospects as an alternative to mammalian gelatins. Food Hydrocolloid 2009;23:563–76. [8] Jamilah B, Harvinder KG. Properties of gelatins from skins of fish-black tilapia (Oreochromis mossambicus) and red tilapia (Oreochromis nilotica). J Food Chem 2002;77:81–4. [9] Kittiphattanabawon P, Benjakul S, Visessanguan W, Shahidi F. Effect of extraction temperature on functional properties and antioxidative activities of gelatin from shark skin. Food Bioprocess Technol 2010, http://dx.doi.org/10.1007/s11947-010-0427-0. [10] Jellouli K, Balti R, Bougatef A, Hmidet N, Barkia A, Nasri M. Chemical composition and characteristics of skin gelatin from grey triggerfish (Balistes capriscus). LWT – Food Sci Technol 2011;44:1965–70. [11] Haddar A, Bougatef A, Balti R, Souissi N, Koched W, Nasri M. Physicochemical and functional properties of gelatin from tuna (Thunnus thynnus) head bones. J Food Nutr Res 2011;50:150–9. [12] Balti R, Jridi M, Sila A, Souissi N, Nedjar-Arroume N, Guillochon D, et al. Extraction and functional properties of gelatin from the skin of cuttlefish (Sepia officinalis) using smooth hound crude acid protease-aided process. Food Hydrocolloid 2011;25:943–50. [13] AOAC. Official methods of analysis. 17th ed. Washington, DC: Association of Official Analytical Chemists; 2000. [14] Laemmli UK. Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 1970;227:680–5. [15] Pearce KN, Kinsella JE. Emulsifying properties of proteins: evaluation of a turbidimetric technique. J Agric Food Chem 1978;26:716–23.

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