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Skin gelatin from bigeye snapper and brownstripe red snapper: Chemical compositions and effect of microbial transglutaminase on gel properties
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HYDROCOLLOIDS Food Hydrocolloids 20 (2006) 1216–1222 www.elsevier.com/locate/foodhyd
Skin gelatin from bigeye snapper and brownstripe red snapper: Chemical compositions and effect of microbial transglutaminase on gel properties Akkasit Jongjareonraka, Soottawat Benjakula,, Wonnop Visessanguanb, Munehiko Tanakac a
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Paholyothin Rd., Klong 1, Klong Luang, Pathumthani, 12120, Thailand c Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan 4, Minato, Tokyo, 108-8477, Japan
b
Received 20 April 2005; accepted 13 January 2006
Abstract Fish skin gelatin was extracted from the skin of bigeye snapper (Priacanthus macracanthus) and brownstripe red snapper (Lutjanus vitta) with yields of 6.5% and 9.4% on the basis of wet weight, respectively. Both skin gelatins having high protein but low fat content contained high hydroxyproline content (75.0 and 71.5 mg/g gelatin powder). The bloom strength of gelatin gel from brownstripe red snapper skin gelatin (218.6 g) was greater than that of bigeye snapper skin gelatin (105.7 g) (Po0.05). The addition of microbial transglutaminase (MTGase) at concentrations up to 0.005% and 0.01% (w/v) increased the bloom strength of gelatin gel from bigeye snapper and brownstripe red snapper, respectively (Po0.05). However, the bloom strength of skin gelatin gel from both fish species decreased with further increase in MTGase concentration. SDS-PAGE of gelatin gel added with MTGase showed the decrease in band intensity of protein components, especially, b- and g- components, suggesting the cross-linking of these components induced by MTGase. Microstructure studies revealed that denser and finer structure was observed with the addition of MTGase. r 2006 Elsevier Ltd. All rights reserved. Keywords: Gelatin; Skin; Brownstripe red snapper; Bigeye snapper; Microbial transglutaminase
1. Introduction Gelatin and collagen, its native form, have been widely applied in the food industry as ingredients to improve the elasticity, consistency and stability of foods. It can be used for encapsulation, production of hard and soft capsules, wound dressing, adsorbent pads and edible film formation, making it of interest to the pharmaceutical, biomaterialbased packaging, and photographic industries (Digenis, Gold & Shah, 1994). Gelatin is normally manufactured on a large scale from the waste generated during animal slaughter and processing, such as skin and bone (Patil, Dalev, Mark, Vassileva & Fakirov, 2000). In general, there are two methods to obtain gelatin from skins and bones, an Corresponding author. Tel.: +66 74 286334. fax: +66 74 212889.
E-mail address: [email protected] (S. Benjakul). 0268-005X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2006.01.006
acid process (gelatin A with isoelectric point at pH 6–9) and an alkaline process (gelatin B with isoelectric points at pH 5) (Stainsby, 1987). Chemicals used for pretreatment as well as extraction condition such as temperature and time can influence the length of polypeptide chains and the functional properties of the gelatin (Kolodziejska, Kaczorowski, Piotrowsia & Sadowska, 2004). The gelling properties of gelatin are also influenced by the source of raw material, which vary in proline and hydroxyproline contents. Collagen with different compositions from varying species is associated with the temperature of animals living habitat. The melting temperature of gelatin prepared from the skins of warm-blooded animals and warm-water fish is generally higher than that of gelatin from the skin of fish living in cold-water, owing to the greater imino acid content and increased proline hydroxylation degree (Gilsenan & Ross-Murphy, 2000; Norland,
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1990; Piez & Gross, 1960; Yamaguchi, Lavety, & Love, 1976). To improve the gel property of gelatin, some chemicals such as MgSO4, and glycerol were used (Ferna´ndez-Diaz, Montero, & Go`mez-Guille`n, 2001). Moreover, microbial transglutaminase has been reported to increase the bloom strength of Baltic cod skin gelatin (Kolodziejska et al., 2004). So far, the main sources of gelatin are limited to those of land animal origin such as bovine or porcine skin and bone. However, the outbreak of bovine spongiform encephalopathy (BSE) and the foot-and-mouth disease (FMD) crisis have resulted in anxiety among users of collagen and gelatin products from land-based animals (Helcke, 2000). In addition, gelatin produced from porcine skins or bones cannot be used for some foods due to esthetic and religious objections (Sadowska, Kolodziejska & Niecikowska, 2003). Furthermore, the increases in consumer need for kosher and halal foods have gained a demand for fish gelatin. Therefore, alternative sources, especially fish processing wastes including skin, bone or scale, have been paid increasing attention for gelatin extraction. Fish gelatin has been produced from cod, hake skin (Ferna´ndez-Diaz et al., 2001), megrim skin (Sarabia, Go`mez-Guille`n & Montero, 2000), tilapia (Oreochromis spp.) skin (Grossman & Bergman, 1992; Jamilah & Harvinder, 2002), Nile perch skin and bone (Muyonga, Cole & Duodu, 2004). However, little information regarding the characteristics of gelatin from tropical fish, especially from commercially important species involving those used for surimi production, such as bigeye snapper and brownstripe red snapper, has been reported. Therefore, the aims of this investigation were to prepare and characterize skin gelatins from bigeye snapper and brownstripe red snapper and to study the effect of microbial transglutaminase on their gelling properties. 2. Materials and methods 2.1. Chemicals Microbial transglutaminase (MTGase) powder (ActivaTM TG) was obtained from Ajinomoto Co (Kawasaki, Japan). MTGase powder had 625 units TGase/g powder as assayed by hydroxamate method (Folk, 1970). All chemicals for electrophoresis were purchased from Sigma Chemical Co. (St. Louis, Mo., USA). 2.2. Fish skin preparation Bigeye snapper (Priacanthus macracanthus) and brownstripe red snapper (Lutjanus vitta) with an average total length of 22–25 cm were caught from Songkhla coast along the Gulf of Thailand, stored in ice and off-loaded after 24–36 h of capture. Upon arrival to the dock in Songkhla, fish were stored in ice with a fish/ice ratio of 1:2 (w/w) and transported to the Department of Food Technology, Prince of Songkla University, Hat Yai. Fish were washed
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with a tap water. Skins were then removed, descaled and cut into small pieces (0.5 0.5 cm). Prepared skins were kept on ice prior to gelatin extraction. 2.3. Extraction of fish skin gelatin Gelatin was extracted from fish skin according to the method of Sarabia et al. (2000) with a slight modification. Skins were soaked in 0.2 M NaOH with a skin/solution ratio of 1:10 (w/v) at 4 1C with a gentle stirring. The solution was changed every 30 min for 3 times to remove non-collagenous proteins and pigments. Alkaline-treated skins were then washed with tap water until neutral or faintly basic pHs of wash water were obtained. The skins were then soaked in 0.05 M acetic acid with a skin/solution ratio of 1:10 (w/v) for 3 h at room temperature (25 1C) with a gentle stirring to swell the collagenous material in fish skin matrix. Acid-treated skins were washed as previously described. The swollen fish skins were soaked in distilled water with a skin/water ratio of 1:10 (w/v) at 45 1C for 12 h with a continuous stirring to extract the gelatin from skin matter. The mixture was then filtered using two layers of cheesecloth. The resultant filtrate was freeze-dried and the dry matter was referred to as ‘‘gelatin powder’’. 2.4. Proximate analysis of gelatins The moisture, ash and fat contents of the gelatin powder were determined according to the AOAC methods (AOAC, 1999). Protein content was measured by Kjeldahl method (AOAC, 1999) and a nitrogen conversion factor of 5.4 was used for calculation of protein content (Eastoe & Eastoe, 1952). Hydroxyproline content was determined according to the method of Bergman & Loxley (1963). The conversion factor for calculating the collagen content from hydroxyproline of bigeye snaper and brownstripe red snapper skin were 11.49 and 12.34, respectively (Jongjareonrak, Benjakul, Visessanguan, Nagai & Tanaka, 2005a; Jongjareonrak, Benjakul, Visessanguan & Tanaka, 2005b). 2.5. Determination of amino acid composition Gelatin samples were hydrolyzed under reduced pressure in 4.0 M methanesulfonic acid containing 0.2% (v/v) 3-2(2aminoethyl)indole at 115 1C for 24 h, and the hydrolysates were neutralized with 3.5 M NaOH and diluted with 0.2 M citrate buffer (pH 2.2). An aliquot of 0.4 ml was applied to an amino acid analyzer (MLC-703; Atto Co., Tokyo, Japan). 2.6. Gelatin gel preparation Gelatin gel was prepared according to the British Standard 757:1975 method (BSI (British Standards Institution), 1975) with a slight modification. Gelatin (2.0 g) was mixed with 30 ml of distilled water in a 50 ml-beaker (PYREXs, USA) to obtain a final concentration of 6.67% (w/v). The mixture was stirred thoroughly and left at room temperature for
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30 min to allow the gelatin to absorb water and swell. The mixture was then incubated at 42 1C for 30 min in a temperature-controlled water bath with occasional stirring. The beakers were then kept in a temperature-controlled chamber at 10 1C and allowed to stand for 16–18 h before determination of bloom gel strength. To study the effect of MTGase on gel properties of fish skin gelatin, MTGase at various concentrations (0.001, 0.0025, 0.005, 0.01, and 0.05% (w/v)) was added after the total dissolution. The mixture was then incubated at 40 1C for 30 min, followed by incubating at 10 1C for 16–18 h. The resultant gels were subjected to analysis.
without MTGase having a thickness of 2–3 mm were fixed with 2.5% (v/v) glutaraldehyde in 0.2 M phosphate buffer (pH 7.2). The samples were then rinsed with distilled water for 1 h and dehydrated in ethanol with a serial concentration of 50, 70, 80, 90 and 100% (v/v). Dried samples were mounted on a bronze stub and sputter-coated with gold (Sputter coater SPI-Module, PA, USA). The specimens were observed with a scanning electron microscope (JEOL JSM-5800 LV, Tokyo, Japan) at an acceleration voltage of 10 kV.
2.7. Determination of bloom gel strength
Data were subjected to analysis of variance (ANOVA). Comparison of means was carried out by Duncan’s multiple range test (Steel & Torrie, 1980). Analysis was performed using a SPSS package (SPSS 10.0 for window, SPSS Inc, Chicago, IL).
Bloom gel strength was determined according to the British Standard 757: 1975 method (BSI (British Standards Institution), 1975) with a slight modification. Bloom gel strength at 10 1C was determined by a Model TA-XT2 Texture analyzer (Stable Micro System, Surrey, UK) using a load cell of 5 kN equipped with a 1.27 cm diameter flatfaced cylindrical Teflons plunger. The dimensions of the sample were 3.8 cm in diameter and 2.7 cm in height. The maximum force (in grams) taken, when the penetration distance of 4 mm was obtained, was recorded. The speed of the plunger was 0.5 mm/s. The measurement was performed in triplicate. 2.8. Electrophoretic analysis Protein patterns of gelatin and gelatin gel samples were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970). The samples (1 g) were dissolved in 10 ml of 5% (w/v) SDS solution. The mixture was heated at 85 1C for 1 h in a water bath to dissolve total proteins. Supernatants were collected after centrifuging at 3000g for 3 min. The supernatants were then mixed with sample buffer (0.5 M Tris-HCl, pH 6.8 containing 4% (w/v) SDS, 20% (v/v) glycerol and 10% (v/v) bME at the ratio of 1:1 (v/v). Samples (20 mg protein) were loaded into the polyacrylamide gel made of 7.5% running gel and 4% stacking gel and subjected to electrophoresis at a constant current of 15 mA per gel using a Mini Protean II unit (BioRad Laboratories, Inc., Richmond, CA, USA). After electrophoresis, gel was stained with 0.05% (w/v) Coomassie blue R-250 in 15% (v/v) methanol and 5% (v/v) acetic acid and destained with 30% (v/v) methanol and 10% (v/v) acetic acid. Calf skin acid-soluble type I collagen (Sigma Chemical Co., St. Louis, Mo., USA) was used as a standard collagen. 2.9. Scanning electron microscopy Microstructure of gelatin gels was determined using Scanning Electron Microscopy (SEM). Gelatin gels from bigeye snapper and brownstripe red snapper skin with and
2.10. Statistical analysis
3. Results and discussion 3.1. Composition of fish skin gelatin Gelatins were extracted from bigeye snapper and brownstripe red snapper skin with yields of 6.5% and 9.4%, respectively, on the basis of wet weight. The yield of gelatin extracted from bigeye snapper skin was lower than that of brownstripe red snapper skin. The gelatin yields have been reported to vary among the fish species, mainly due to the differences in collagen content, the compositions of skin as well as the skin matrix. The different yields of skin gelatin have been reported for sole (8.3%), megrim (7.4%), cod (7.2%), hake (6.5%) (Go`mez-Guille`n et al., 2002), red and black tilapia (7.8% and 5.4%, respectively) (Jamilah & Harvinder, 2002), young and adult Nile perch (12.5% and 16%, respectively) (Muyonga et al., 2004). From the results, the lower yield of gelatin from bigeye snapper skin was in accordance with the lower contents of collagen (6.4% ASC and 1.1% PSC), compared with those of brownstripe red snapper skin (9.0% ASC and 4.7% PSC) (Jongjareonrak et al., 2005a, b). Furthermore, the high degree of cross-linking via covalent bonds caused the decrease in solubility of collagen and might lead to the lower content of extractable gelatin (Foegeding, Lanier & Hultin, 1996). The proximate compositions of bigeye snapper and brownstripe red snapper skin gelatins are shown in Table 1. Generally, skin gelatins of both species contained high protein content but low moisture and fat contents, suggesting the efficient removal of water and fat from skin. However, gelatin from bigeye snapper skin was higher in ash content (3.2%), compared with that from brownstripe red snapper (1.9%). Also the ash content obtained was higher than the recommended maximum of 2.6% (Jones, 1997). The different mineral contents between skins of both species might be associated with the varying ash contents obtained. From the results, high hydroxyproline
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Table 1 Proximate composition of gelatins from bigeye snapper and brownstripe red snapper skins
Table 2 Amino acid compositions of gelatins from bigeye snapper and browstripe red snapper skin (residues per 1000 total amino acid residues)
Compositions
Amino acids
Bigeye snapper
Brownstripe red snapper
87.970.8c 8.270.7 3.270.2 0.670.0 75.070.4 862.074.6
88.670.7 7.670.2 1.970.1 0.870.1 71.570.5 882.075.5
a
% wet wt. mg/ 1 g gelatin powder. c Values are given as mean7SD from triplicate determinations. b
and total collagen contents were observed in the extracted gelatins. Collagens constituted approximately 86% and 89% of the dry gelatin from bigeye snapper and brownstripe red snapper skins, respectively, indicating a high purity with negligible contaminating constituents. The amino acid composition of both skin gelatins expressed as residues per 1000 total amino acid residues is shown in Table 2. Slight differences in the amino acid profiles between skin gelatins of two species were observed. In general, both skin gelatins had the amino acid composition similar to that of mother collagens reported by Jongjareonrak et al. (2005a, b). Bigeye snapper and brownstripe red snapper skin gelatins were rich in glycine (19.3% and 20.4%), alanine (10.3% and 10.8%), and proline (13.4% and 14.1%). For hydroxyproline, it accounted about 61 and 56 residues per 1000 residues for bigeye snapper and brownstripe red snapper skin gelatins, respectively. The numbers of imino acids, proline and hydroxyproline, in bigeye snapper and brownstripe red snapper skin gelatins were 195 and 197 residues per 1000 residues, respectively. The imino acid content in skin gelatin from both species were higher than that reported in sole, megrim, cod and hake (174, 175, 156, 173 residues per 1000 residues, respectively) (Go`mez-Guille`n et al., 2002) but lower than that found in porcine skin gelatin (222 residues per 1000 residues) (Cho et al., 2004). The stability of triple helical structure in renatured gelatins was associated with the total content of pyrrolidine imino acids (Ledward, 1986). Hydroxyproline plays an essential role in the stabilization of the triple-helix strands of collagen via its hydrogen bonding ability through its –OH group (Burjandze, 1979; Ledward, 1986). Gelatin with higher content of hydroxyproline is believed to have higher viscoelastic properties and its ability to develop triple helix structures, which are important for stabilizing the gelatin gel structure (Go`mez-Guille`n et al., 2002). 3.2. Effect of MTGase on bloom strength of skin gelatin gels Bloom strength of gelatin gels with and without MTGase was shown in Fig. 1. Bigeye snapper skin gelatin gels
Source of skin gelatin
Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Total
400 Bloom strength (g)
Proteina Moisturea Asha Fata Hydroxyproline contentb Collagen contentb
Source of skin gelatin
Bigeye snapper
Brownstripe red snapper
91 61 32 38 103 134 193 103 21 17 10 27 6 21 38 12 92 1000
84 56 31 39 105 141 204 108 17 15 9 25 5 20 38 9 94 1000
bigeye snapper brownstripe red snapper
a
300 d
e*
f
200 100
b
c a
c*
b
b
b d
0 0
0.001
0.0025 0.005 MTGase (%, w/v)
0.01
0.05
Fig. 1. Bloom strength of gelatin gels from bigeye snapper and browstripe red snapper skins without and with MTGase added at different concentrations. *Values are given as mean7SD from triplicate determinations. Different letters on the bars under the same species indicate the significant differences (Po0.05).
exhibited the lower bloom strength (105.7 g) than that of brownstripe red snapper skin gelatin (218.6 g), suggesting the difference in gel-forming ability of gelatin between two species. The bloom strength of fish skin gelatin gel was reported to be varied with species. Fish skin gelatins with different bloom strength were reported for young and adult Nile perch (217 and 240 g, respectively) (Muyonga et al., 2004), tilapia (263 g) (Grossman & Bergman, 1992), sole, megrim, hake, and cod (350, 340, 100, and 70 g, respectively) (Go`mez-Guille`n et al., 2002). The difference in bloom strength between species was possibly due to the different composition, particularly in terms of amino acid composition and size of protein chains (Muyonga et al., 2004). Protein degradation during gelatin extraction was found in bigeye snapper (Jongjareonrak, Benjakul, Visessanguan, Prodpran & Tanaka, 2006), while less
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degradation was noticeable in brownstripe red snapper skin gelatin. Protein degradation fragments, if present, may reduce the ability of a-chains to anneal correctly by hindering the growth of the existing nucleation sites (Ledward, 1986; Normand, Muller, Ravey & Parker, 2000). From the results, the addition of MTGase generally increased the bloom strength of gelatin gels of both species (Po0.05). The marked increase in bloom strength of gelatin gel from bigeye snapper and brownstripe red snapper skin gelatin was observed with the addition of MTGase up to 0.005% and 0.01%, respectively. The results suggested that MTGase might catalyze the formation of e-(g-glutamyl) lysine linkages between protein chains. However, bloom strength decreased with further increase in MTGase concentration. This result was in agreement with Go`mez-Guille`n, Sarabia, Solas & Montero (2001) who reported that the increases in MTGase concentration from 0.015% to 0.075% lowered the bloom strength of megrim skin gelatin gel. From the result, the addition of MTGase at the appropriate concentration increased bloom strength of skin gelatin of both species effectively. The excessive formation of MTGase-catalyzed covalent bonds was detrimental to gel strength due to formation of intramolecular covalent bonds. As a consequence, intermolecular aggregation might be impeded, leading to reduced gel network formation. Therefore, MTGase at the concentrations of 0.005% and 0.01% (w/v) were appropriate to improve the bloom strength of gelatin gels from bigeye snapper and brownstripe red snapper skins, respectively. 3.3. Effect of MTGase on SDS-PAGE patterns of gelatin gels The protein patterns of gels from bigeye snapper and brownstripe red snapper skin gelatins under reducing condition (in the presence of bME) are shown in Fig. 2. For gelatin gels without MTGase, a1-chains was found as the major component for both samples. However, a2-chains, were not found in gelatin gels from brownstripe red snapper skin. Protein patterns of gelatin gels were generally relevant to those of the starting gelatin powder, especially for brownstripe red snapper gelatin. However, proteins with molecular weights of 71, 65 and 57 kDa were not found in the gel sample from bigeye snapper skin gelatin. No b-component was noticeable in the gel sample, however protein with molecular weight of 143 kDa was found in bigeye snapper gelatin sample. This might be owing to the degradation of these components during gel preparation, particularly during heat treatment. This result was in accordance with Muyonga et al. (2004) who observed that Nile perch skin gelatins contained low-molecular-weight peptides, especially, gelatins extracted with higher temperature. Heatinduced cleavage of protein chains occurred during the extraction process of Nile perch skin gelatin and was
Fig. 2. Protein pattern of gelatin gel from bigeye snapper and brownstripe red snapper skins added with MTGase at different concentrations. HM, C, and G, denote high molecular weight markers, standard collagen type I, and gelatin powder, respectively. Numbers, 0, 1, 2, 3, 4, and 5 indicate MTGase concentrations of 0, 0.001, 0.0025, 0.005, 0.01, and 0.05% (w/v), respectively.
enhanced with higher temperature extraction (Muyonga et al., 2004). From the results, greater amount of peptides with molecular weight lower than a-chains were observed in bigeye snapper skin gelatin than that of brownstripe red snapper skin gelatin. This suggested that collagen of bigeye snapper skin was more degraded during the extraction and some proteinase might be involved in the degradation of gelatin (Jongjareonrak et al., 2006). The formation of degradation fragments is associated with the low viscosity, low melting point, low setting point, high setting time, as well as decreased bloom strength of gelatin (Ledward, 1986; Muyonga et al., 2004; Normand et al., 2000; Tavernier, 1989). From the results, greater bloom strength was found in brownstripe red snapper skin
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gelatin, compared with that from bigeye snapper skin gelatin (Fig. 1). When MTGase was added, band intensity of all components in gelatin gels from both species decreased continuously with increasing MTGase concentration. When MTGase concentration increased, band intensity of b- and g-components as well as a-chains decreased with the concomitant increase in high-molecular-weight aggregate. At the highest concentration used (0.05%), all protein bands almost disappeared. The decrease in protein band intensity was most likely due to the cross-linking via nondisulfide covalent bond formation induced by MTGase (Folk, 1983). The results indicated that all components underwent cross-linking induced by MTGase. This was possibly because most of protein components contained both acyl donor and receptors aligned in the appropriate position for reaction induced by MTGase. Lysine was found in skin gelatin (Table 2) and might act as the acyl receptor of gelatin from both species. Glutamine, reported as glutamic acid, probably functioned as the acyl donor of gelatin. Generally, glutamine undergoes deamidation to glutamic acid under the harsh acid hydrolysis conditions used for analysis (Pickering & Newton, 1990) (Table 2). The decrease in protein band intensity (Fig. 2) was coincidental with the increase in bloom strength (Fig. 1). The aggregation of protein components via the covalent bond directly resulted in the development of a strong gel network, particularly with the appropriate range of MTGase levels. However, excessive cross-linking caused the adverse effect on gel network formation. Thus, the
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alignment or aggregation pattern of gelatin protein components also played an important role in gel formation as well as the strength of gelatin gel obtained. 3.4. Microstructure of gelatin gels The microstructure of gelatin gels from bigeye snapper and brownstripe red snapper skins without and with MTGase was visualized by SEM as shown in Fig. 3. In general, bigeye snapper skin gelatin gel showed the finer and denser strands compared with those of brownstripe red snapper skin gelatin (Fig. 3A and B). This was possibly due to the different characteristics of gelation between species in terms of aggregation and alignment of protein molecules. With addition of MTGase, the denser aggregates with negligible voids in the gel network were observed (Fig. 3B and D). It was suggested that gelatin underwent cross-linking via non-disulfide covalent bonding, resulting in greater intermolecular aggregation between adjacent molecules. The result was coincidental with the lower aand b-components as shown in Fig. 2. These cross-links might be associated with the higher bloom strength of gelatin gel when MTGase was added (Fig. 1). 4. Conclusion Gelatin was extracted from the skin of bigeye snapper and brownstripe red snapper. Both gelatins contained slightly different amino acid composition. The bloom
Fig. 3. Microstructure of gelatin gels (magnification: 10,000 X). A and B: gelatin gels of bigeye snapper skin without and with 0.005% (w/v) MTGase, respectively. C and D: gelatin gels of brownstripe red snapper skin without and with 0.01% (w/v) MTGase, respectively.
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strength of gelatin gel from bigeye snapper skin was lower than that of brownstripe red snapper skin. The addition of MTGase induced the aggregation of proteins, which was associated with the increase in bloom strength of fish skin gelatin from both species. However, bloom strength decreased with the excessive MTGase amount. Acknowledgements The authors thank Professor Akira Shinagawa of the Environment Education Center, Gakushuin Women’s college (Tokyo, Japan), for amino acid analysis. This research was supported by Thailand Research Fund under the Royal Golden Jubilee PhD program for financial support to Akkasit Jongjareonrak (PHD/0060/2544). References AOAC. (1999). Official methods of analysis. 14th ed. Washington, DC: Association of Official Analytical Chemists. Bergman, I., & Loxley, R. (1963). Two improved and simplified methods for the spectrophotometric determination of hydroxyproline. Analytical Chemistry, 35, 1961–1965. BSI (British Standards Institution) (1975). Methods for sampling and testing gelatin (physical and chemical methods). London: BSI. Burjandze, T. V. (1979). Hydroxyproline content and location in relation to collagen thermal stability. Biopolymers, 18, 931–936. Cho, S. M., Kwak, K. S., Park, D. C., Gu, Y. S., Ji, C. I., & Jang, D. H. (2004). Processing optimization and functional properties of gelatin from shark (Isurus oxyrinchus) cartilage. Food Hydrocolloids, 18, 573–579. Digenis, G. A., Gold, T. B., & Shah, V. P. (1994). Cross-linking of gelatin capsules and its relevance to their in vitro–in vivo performance. Journal of Pharmaceutical Science, 83, 915–921. Eastoe, J.E., & Eastoe, B. (1952). A method for the determination of total nitrogen in proteins. In: The British gelatin and glue research association research report, Series B 5 (pp. 1–17). Ferna´ndez-Diaz, M. D., Montero, P., & Go`mez-Guille`n, M. C. (2001). Gel properties of collagens from skins of cod (Gadus morhua) and hake (Merluccius merluccius) and their modification by coenhancers magnesium sulphate, glycerol and transglutaminase. Food Chemistry, 74, 161–167. Foegeding, E., Lanier, T. C., & Hultin, H. O. (1996). Characteristics of edible muscle tissue. In O. R. Fennema (Ed.), Food Chemistry (pp. 879–942). New York: Marcel Dekker. Folk, J. E. (1970). Transglutaminase. Methods in Enzymology, 17, 889–894. Folk, J. E. (1983). Mechanism and basis for specificity of transglutaminase-catalyzed-e(g-glutamyl)lysine bond formation. Advances in Enzymology, 54, 1–57. Gilsenan, P. M., & Ross-Murphy, S. B. (2000). Rheological characterisation of gelatins from mammalian and marine sources. Food Hydrocolloids, 14, 191–196. Go`mez-Guille`n, M. C., Sarabia, A. I., Solas, M. T., & Montero, P. (2001). Effect of microbial transglutaminase on the functional properties of megrim (Lepidorhombus boscii) skin gelatin. Journal of the Science of Food and Agriculture, 81, 665–673. Go`mez-Guille`n, M. C., Turnay, J., Ferna´ndez-Diaz, M. D., Ulmo, N., Lizarbe, M. A., & Montero, P. (2002). Structural and physical properties of gelatin extracted from different marine speices: a comparative study. Food Hydrocolloids, 16, 25–34.
Grossman, S., & Bergman, M. (1992). Process for the production of gelatin from fish skins. US Patent 5,093,474. Helcke, T. (2000). Gelatin, the food technologist’s friend or foe. International Food Ingredients, 1, 6–8. Jamilah, B., & Harvinder, K. G. (2002). Properties of gelatins from skins of fish-black tilapia (Oreochromis mossambicus) and red tilapia (Oreochromis nilotica). Food Chemistry, 77, 81–84. Jones, N. R. (1997). Uses of gelatin in edible products. In A. G. Ward, & A. Courts (Eds.), The science and technology of gelatin (pp. 366–394). London: Academic Press. Jongjareonrak, A., Benjakul, S., Visessanguan, W., Nagai, T., & Tanaka, M. (2005a). Isolation and characterisation of acid and pepsinsolubilised collagen from brownstripe red snapper (Lutjanus vitta) skin. Food Chemistry, 93, 475–484. Jongjareonrak, A., Benjakul, S., Visessanguan, W., & Tanaka, M. (2005b). Isolation and characterization of collagen from bigeye snapper (Priacanthus marcracanthus) skin. Journal of the Science of Food and Agriculture, 85, 1203–1210. Jongjareonrak, A., Benjakul, S., Visessanguan, W., Prodpran, T., & Tanaka, M. (2006). Characterization of edible films from skin gelatin of brownstripe red snapper and bigeye snapper. Food Hydrocolloids, 20, 492–501. Kolodziejska, I., Kaczorowski, K., Piotrowsia, B., & Sadowska, M. (2004). Modification of properties of gelatin from skins of Baltic cod (Gadus morhua) with transglutaminase. Food Chemistry, 86, 203–209. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature, 227, 680–685. Ledward, D. A. (1986). Gelation of gelatin. In J. R. Mitchell, & D. A. Ledward (Eds.), Functional properties of food macromolecules (pp. 233–289). London: Elsevier Applied Science Publishers. 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 M. N. Voight, & J. K. Botta (Eds.), Advances in fisheries technology and biotechnology for increased profitability (pp. 325–333). Lancaster: Technomic Publishing Co. Normand, V., Muller, S., Ravey, J.-C., & Parker, A. (2000). Gelation kinetics of gelatin: A master curve and network modeling. Macromolecules, 33, 1063–1071. Patil, R. D., Dalev, P. G., Mark, J. E., Vassileva, E., & Fakirov, S. (2000). Biodegradation of chemically modified gelatin films in lake and river waters. Journal of Applied Polymer Science, 76, 29–37. Pickering, M. V., & Newton, P. (1990). Amino acid hydrolysis: Old problems, new solutions. LC/GC, 8, 778–781. Piez, K. A., & Gross, J. (1960). The amino acid compostition of some fish collagens: The relation between composition and structure. Journal of Biological Chemistry, 235, 995–999. Sadowska, M., Kolodziejska, I., & Niecikowska, C. (2003). Isolation of collagen from the skin of Baltic cod (Gadus morhua). Food Chemistry, 81, 257–262. Sarabia, A. I., Go¨mez-Guille`n, M. C., & Montero, P. (2000). The effect of added salts on the viscoelastic properties of fish skin gelatin. Food Chemistry, 70, 71–76. Stainsby, G. (1987). Gelatin gels. In A. M. Pearson, T. R. Dutson, & A. J. Bailey (Eds.), Advances in Meat Research (pp. 209–222). New York: Norstrand Reinhold. Steel, R. G. D., & Torrie, J. H. (1980). Principles and procedures of statistics: A biometrical approach. New York: McGraw-Hill. Tavernier, B. H. (1989). Molecular mass distribution of gelatin and physical properties. Photographic Gelatin Proceedings, 1, 217–228. Yamaguchi, K., Lavety, J., & Love, R. M. (1976). The connective tissues of fish. VIII Comparative studies on hake, cod and catfish collagens. Journal of Food Technology, 11, 389–393.
FOOD
HYDROCOLLOIDS Food Hydrocolloids 20 (2006) 1216–1222 www.elsevier.com/locate/foodhyd
Skin gelatin from bigeye snapper and brownstripe red snapper: Chemical compositions and effect of microbial transglutaminase on gel properties Akkasit Jongjareonraka, Soottawat Benjakula,, Wonnop Visessanguanb, Munehiko Tanakac a
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Paholyothin Rd., Klong 1, Klong Luang, Pathumthani, 12120, Thailand c Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan 4, Minato, Tokyo, 108-8477, Japan
b
Received 20 April 2005; accepted 13 January 2006
Abstract Fish skin gelatin was extracted from the skin of bigeye snapper (Priacanthus macracanthus) and brownstripe red snapper (Lutjanus vitta) with yields of 6.5% and 9.4% on the basis of wet weight, respectively. Both skin gelatins having high protein but low fat content contained high hydroxyproline content (75.0 and 71.5 mg/g gelatin powder). The bloom strength of gelatin gel from brownstripe red snapper skin gelatin (218.6 g) was greater than that of bigeye snapper skin gelatin (105.7 g) (Po0.05). The addition of microbial transglutaminase (MTGase) at concentrations up to 0.005% and 0.01% (w/v) increased the bloom strength of gelatin gel from bigeye snapper and brownstripe red snapper, respectively (Po0.05). However, the bloom strength of skin gelatin gel from both fish species decreased with further increase in MTGase concentration. SDS-PAGE of gelatin gel added with MTGase showed the decrease in band intensity of protein components, especially, b- and g- components, suggesting the cross-linking of these components induced by MTGase. Microstructure studies revealed that denser and finer structure was observed with the addition of MTGase. r 2006 Elsevier Ltd. All rights reserved. Keywords: Gelatin; Skin; Brownstripe red snapper; Bigeye snapper; Microbial transglutaminase
1. Introduction Gelatin and collagen, its native form, have been widely applied in the food industry as ingredients to improve the elasticity, consistency and stability of foods. It can be used for encapsulation, production of hard and soft capsules, wound dressing, adsorbent pads and edible film formation, making it of interest to the pharmaceutical, biomaterialbased packaging, and photographic industries (Digenis, Gold & Shah, 1994). Gelatin is normally manufactured on a large scale from the waste generated during animal slaughter and processing, such as skin and bone (Patil, Dalev, Mark, Vassileva & Fakirov, 2000). In general, there are two methods to obtain gelatin from skins and bones, an Corresponding author. Tel.: +66 74 286334. fax: +66 74 212889.
E-mail address: [email protected] (S. Benjakul). 0268-005X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2006.01.006
acid process (gelatin A with isoelectric point at pH 6–9) and an alkaline process (gelatin B with isoelectric points at pH 5) (Stainsby, 1987). Chemicals used for pretreatment as well as extraction condition such as temperature and time can influence the length of polypeptide chains and the functional properties of the gelatin (Kolodziejska, Kaczorowski, Piotrowsia & Sadowska, 2004). The gelling properties of gelatin are also influenced by the source of raw material, which vary in proline and hydroxyproline contents. Collagen with different compositions from varying species is associated with the temperature of animals living habitat. The melting temperature of gelatin prepared from the skins of warm-blooded animals and warm-water fish is generally higher than that of gelatin from the skin of fish living in cold-water, owing to the greater imino acid content and increased proline hydroxylation degree (Gilsenan & Ross-Murphy, 2000; Norland,
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1990; Piez & Gross, 1960; Yamaguchi, Lavety, & Love, 1976). To improve the gel property of gelatin, some chemicals such as MgSO4, and glycerol were used (Ferna´ndez-Diaz, Montero, & Go`mez-Guille`n, 2001). Moreover, microbial transglutaminase has been reported to increase the bloom strength of Baltic cod skin gelatin (Kolodziejska et al., 2004). So far, the main sources of gelatin are limited to those of land animal origin such as bovine or porcine skin and bone. However, the outbreak of bovine spongiform encephalopathy (BSE) and the foot-and-mouth disease (FMD) crisis have resulted in anxiety among users of collagen and gelatin products from land-based animals (Helcke, 2000). In addition, gelatin produced from porcine skins or bones cannot be used for some foods due to esthetic and religious objections (Sadowska, Kolodziejska & Niecikowska, 2003). Furthermore, the increases in consumer need for kosher and halal foods have gained a demand for fish gelatin. Therefore, alternative sources, especially fish processing wastes including skin, bone or scale, have been paid increasing attention for gelatin extraction. Fish gelatin has been produced from cod, hake skin (Ferna´ndez-Diaz et al., 2001), megrim skin (Sarabia, Go`mez-Guille`n & Montero, 2000), tilapia (Oreochromis spp.) skin (Grossman & Bergman, 1992; Jamilah & Harvinder, 2002), Nile perch skin and bone (Muyonga, Cole & Duodu, 2004). However, little information regarding the characteristics of gelatin from tropical fish, especially from commercially important species involving those used for surimi production, such as bigeye snapper and brownstripe red snapper, has been reported. Therefore, the aims of this investigation were to prepare and characterize skin gelatins from bigeye snapper and brownstripe red snapper and to study the effect of microbial transglutaminase on their gelling properties. 2. Materials and methods 2.1. Chemicals Microbial transglutaminase (MTGase) powder (ActivaTM TG) was obtained from Ajinomoto Co (Kawasaki, Japan). MTGase powder had 625 units TGase/g powder as assayed by hydroxamate method (Folk, 1970). All chemicals for electrophoresis were purchased from Sigma Chemical Co. (St. Louis, Mo., USA). 2.2. Fish skin preparation Bigeye snapper (Priacanthus macracanthus) and brownstripe red snapper (Lutjanus vitta) with an average total length of 22–25 cm were caught from Songkhla coast along the Gulf of Thailand, stored in ice and off-loaded after 24–36 h of capture. Upon arrival to the dock in Songkhla, fish were stored in ice with a fish/ice ratio of 1:2 (w/w) and transported to the Department of Food Technology, Prince of Songkla University, Hat Yai. Fish were washed
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with a tap water. Skins were then removed, descaled and cut into small pieces (0.5 0.5 cm). Prepared skins were kept on ice prior to gelatin extraction. 2.3. Extraction of fish skin gelatin Gelatin was extracted from fish skin according to the method of Sarabia et al. (2000) with a slight modification. Skins were soaked in 0.2 M NaOH with a skin/solution ratio of 1:10 (w/v) at 4 1C with a gentle stirring. The solution was changed every 30 min for 3 times to remove non-collagenous proteins and pigments. Alkaline-treated skins were then washed with tap water until neutral or faintly basic pHs of wash water were obtained. The skins were then soaked in 0.05 M acetic acid with a skin/solution ratio of 1:10 (w/v) for 3 h at room temperature (25 1C) with a gentle stirring to swell the collagenous material in fish skin matrix. Acid-treated skins were washed as previously described. The swollen fish skins were soaked in distilled water with a skin/water ratio of 1:10 (w/v) at 45 1C for 12 h with a continuous stirring to extract the gelatin from skin matter. The mixture was then filtered using two layers of cheesecloth. The resultant filtrate was freeze-dried and the dry matter was referred to as ‘‘gelatin powder’’. 2.4. Proximate analysis of gelatins The moisture, ash and fat contents of the gelatin powder were determined according to the AOAC methods (AOAC, 1999). Protein content was measured by Kjeldahl method (AOAC, 1999) and a nitrogen conversion factor of 5.4 was used for calculation of protein content (Eastoe & Eastoe, 1952). Hydroxyproline content was determined according to the method of Bergman & Loxley (1963). The conversion factor for calculating the collagen content from hydroxyproline of bigeye snaper and brownstripe red snapper skin were 11.49 and 12.34, respectively (Jongjareonrak, Benjakul, Visessanguan, Nagai & Tanaka, 2005a; Jongjareonrak, Benjakul, Visessanguan & Tanaka, 2005b). 2.5. Determination of amino acid composition Gelatin samples were hydrolyzed under reduced pressure in 4.0 M methanesulfonic acid containing 0.2% (v/v) 3-2(2aminoethyl)indole at 115 1C for 24 h, and the hydrolysates were neutralized with 3.5 M NaOH and diluted with 0.2 M citrate buffer (pH 2.2). An aliquot of 0.4 ml was applied to an amino acid analyzer (MLC-703; Atto Co., Tokyo, Japan). 2.6. Gelatin gel preparation Gelatin gel was prepared according to the British Standard 757:1975 method (BSI (British Standards Institution), 1975) with a slight modification. Gelatin (2.0 g) was mixed with 30 ml of distilled water in a 50 ml-beaker (PYREXs, USA) to obtain a final concentration of 6.67% (w/v). The mixture was stirred thoroughly and left at room temperature for
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30 min to allow the gelatin to absorb water and swell. The mixture was then incubated at 42 1C for 30 min in a temperature-controlled water bath with occasional stirring. The beakers were then kept in a temperature-controlled chamber at 10 1C and allowed to stand for 16–18 h before determination of bloom gel strength. To study the effect of MTGase on gel properties of fish skin gelatin, MTGase at various concentrations (0.001, 0.0025, 0.005, 0.01, and 0.05% (w/v)) was added after the total dissolution. The mixture was then incubated at 40 1C for 30 min, followed by incubating at 10 1C for 16–18 h. The resultant gels were subjected to analysis.
without MTGase having a thickness of 2–3 mm were fixed with 2.5% (v/v) glutaraldehyde in 0.2 M phosphate buffer (pH 7.2). The samples were then rinsed with distilled water for 1 h and dehydrated in ethanol with a serial concentration of 50, 70, 80, 90 and 100% (v/v). Dried samples were mounted on a bronze stub and sputter-coated with gold (Sputter coater SPI-Module, PA, USA). The specimens were observed with a scanning electron microscope (JEOL JSM-5800 LV, Tokyo, Japan) at an acceleration voltage of 10 kV.
2.7. Determination of bloom gel strength
Data were subjected to analysis of variance (ANOVA). Comparison of means was carried out by Duncan’s multiple range test (Steel & Torrie, 1980). Analysis was performed using a SPSS package (SPSS 10.0 for window, SPSS Inc, Chicago, IL).
Bloom gel strength was determined according to the British Standard 757: 1975 method (BSI (British Standards Institution), 1975) with a slight modification. Bloom gel strength at 10 1C was determined by a Model TA-XT2 Texture analyzer (Stable Micro System, Surrey, UK) using a load cell of 5 kN equipped with a 1.27 cm diameter flatfaced cylindrical Teflons plunger. The dimensions of the sample were 3.8 cm in diameter and 2.7 cm in height. The maximum force (in grams) taken, when the penetration distance of 4 mm was obtained, was recorded. The speed of the plunger was 0.5 mm/s. The measurement was performed in triplicate. 2.8. Electrophoretic analysis Protein patterns of gelatin and gelatin gel samples were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970). The samples (1 g) were dissolved in 10 ml of 5% (w/v) SDS solution. The mixture was heated at 85 1C for 1 h in a water bath to dissolve total proteins. Supernatants were collected after centrifuging at 3000g for 3 min. The supernatants were then mixed with sample buffer (0.5 M Tris-HCl, pH 6.8 containing 4% (w/v) SDS, 20% (v/v) glycerol and 10% (v/v) bME at the ratio of 1:1 (v/v). Samples (20 mg protein) were loaded into the polyacrylamide gel made of 7.5% running gel and 4% stacking gel and subjected to electrophoresis at a constant current of 15 mA per gel using a Mini Protean II unit (BioRad Laboratories, Inc., Richmond, CA, USA). After electrophoresis, gel was stained with 0.05% (w/v) Coomassie blue R-250 in 15% (v/v) methanol and 5% (v/v) acetic acid and destained with 30% (v/v) methanol and 10% (v/v) acetic acid. Calf skin acid-soluble type I collagen (Sigma Chemical Co., St. Louis, Mo., USA) was used as a standard collagen. 2.9. Scanning electron microscopy Microstructure of gelatin gels was determined using Scanning Electron Microscopy (SEM). Gelatin gels from bigeye snapper and brownstripe red snapper skin with and
2.10. Statistical analysis
3. Results and discussion 3.1. Composition of fish skin gelatin Gelatins were extracted from bigeye snapper and brownstripe red snapper skin with yields of 6.5% and 9.4%, respectively, on the basis of wet weight. The yield of gelatin extracted from bigeye snapper skin was lower than that of brownstripe red snapper skin. The gelatin yields have been reported to vary among the fish species, mainly due to the differences in collagen content, the compositions of skin as well as the skin matrix. The different yields of skin gelatin have been reported for sole (8.3%), megrim (7.4%), cod (7.2%), hake (6.5%) (Go`mez-Guille`n et al., 2002), red and black tilapia (7.8% and 5.4%, respectively) (Jamilah & Harvinder, 2002), young and adult Nile perch (12.5% and 16%, respectively) (Muyonga et al., 2004). From the results, the lower yield of gelatin from bigeye snapper skin was in accordance with the lower contents of collagen (6.4% ASC and 1.1% PSC), compared with those of brownstripe red snapper skin (9.0% ASC and 4.7% PSC) (Jongjareonrak et al., 2005a, b). Furthermore, the high degree of cross-linking via covalent bonds caused the decrease in solubility of collagen and might lead to the lower content of extractable gelatin (Foegeding, Lanier & Hultin, 1996). The proximate compositions of bigeye snapper and brownstripe red snapper skin gelatins are shown in Table 1. Generally, skin gelatins of both species contained high protein content but low moisture and fat contents, suggesting the efficient removal of water and fat from skin. However, gelatin from bigeye snapper skin was higher in ash content (3.2%), compared with that from brownstripe red snapper (1.9%). Also the ash content obtained was higher than the recommended maximum of 2.6% (Jones, 1997). The different mineral contents between skins of both species might be associated with the varying ash contents obtained. From the results, high hydroxyproline
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Table 1 Proximate composition of gelatins from bigeye snapper and brownstripe red snapper skins
Table 2 Amino acid compositions of gelatins from bigeye snapper and browstripe red snapper skin (residues per 1000 total amino acid residues)
Compositions
Amino acids
Bigeye snapper
Brownstripe red snapper
87.970.8c 8.270.7 3.270.2 0.670.0 75.070.4 862.074.6
88.670.7 7.670.2 1.970.1 0.870.1 71.570.5 882.075.5
a
% wet wt. mg/ 1 g gelatin powder. c Values are given as mean7SD from triplicate determinations. b
and total collagen contents were observed in the extracted gelatins. Collagens constituted approximately 86% and 89% of the dry gelatin from bigeye snapper and brownstripe red snapper skins, respectively, indicating a high purity with negligible contaminating constituents. The amino acid composition of both skin gelatins expressed as residues per 1000 total amino acid residues is shown in Table 2. Slight differences in the amino acid profiles between skin gelatins of two species were observed. In general, both skin gelatins had the amino acid composition similar to that of mother collagens reported by Jongjareonrak et al. (2005a, b). Bigeye snapper and brownstripe red snapper skin gelatins were rich in glycine (19.3% and 20.4%), alanine (10.3% and 10.8%), and proline (13.4% and 14.1%). For hydroxyproline, it accounted about 61 and 56 residues per 1000 residues for bigeye snapper and brownstripe red snapper skin gelatins, respectively. The numbers of imino acids, proline and hydroxyproline, in bigeye snapper and brownstripe red snapper skin gelatins were 195 and 197 residues per 1000 residues, respectively. The imino acid content in skin gelatin from both species were higher than that reported in sole, megrim, cod and hake (174, 175, 156, 173 residues per 1000 residues, respectively) (Go`mez-Guille`n et al., 2002) but lower than that found in porcine skin gelatin (222 residues per 1000 residues) (Cho et al., 2004). The stability of triple helical structure in renatured gelatins was associated with the total content of pyrrolidine imino acids (Ledward, 1986). Hydroxyproline plays an essential role in the stabilization of the triple-helix strands of collagen via its hydrogen bonding ability through its –OH group (Burjandze, 1979; Ledward, 1986). Gelatin with higher content of hydroxyproline is believed to have higher viscoelastic properties and its ability to develop triple helix structures, which are important for stabilizing the gelatin gel structure (Go`mez-Guille`n et al., 2002). 3.2. Effect of MTGase on bloom strength of skin gelatin gels Bloom strength of gelatin gels with and without MTGase was shown in Fig. 1. Bigeye snapper skin gelatin gels
Source of skin gelatin
Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Total
400 Bloom strength (g)
Proteina Moisturea Asha Fata Hydroxyproline contentb Collagen contentb
Source of skin gelatin
Bigeye snapper
Brownstripe red snapper
91 61 32 38 103 134 193 103 21 17 10 27 6 21 38 12 92 1000
84 56 31 39 105 141 204 108 17 15 9 25 5 20 38 9 94 1000
bigeye snapper brownstripe red snapper
a
300 d
e*
f
200 100
b
c a
c*
b
b
b d
0 0
0.001
0.0025 0.005 MTGase (%, w/v)
0.01
0.05
Fig. 1. Bloom strength of gelatin gels from bigeye snapper and browstripe red snapper skins without and with MTGase added at different concentrations. *Values are given as mean7SD from triplicate determinations. Different letters on the bars under the same species indicate the significant differences (Po0.05).
exhibited the lower bloom strength (105.7 g) than that of brownstripe red snapper skin gelatin (218.6 g), suggesting the difference in gel-forming ability of gelatin between two species. The bloom strength of fish skin gelatin gel was reported to be varied with species. Fish skin gelatins with different bloom strength were reported for young and adult Nile perch (217 and 240 g, respectively) (Muyonga et al., 2004), tilapia (263 g) (Grossman & Bergman, 1992), sole, megrim, hake, and cod (350, 340, 100, and 70 g, respectively) (Go`mez-Guille`n et al., 2002). The difference in bloom strength between species was possibly due to the different composition, particularly in terms of amino acid composition and size of protein chains (Muyonga et al., 2004). Protein degradation during gelatin extraction was found in bigeye snapper (Jongjareonrak, Benjakul, Visessanguan, Prodpran & Tanaka, 2006), while less
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degradation was noticeable in brownstripe red snapper skin gelatin. Protein degradation fragments, if present, may reduce the ability of a-chains to anneal correctly by hindering the growth of the existing nucleation sites (Ledward, 1986; Normand, Muller, Ravey & Parker, 2000). From the results, the addition of MTGase generally increased the bloom strength of gelatin gels of both species (Po0.05). The marked increase in bloom strength of gelatin gel from bigeye snapper and brownstripe red snapper skin gelatin was observed with the addition of MTGase up to 0.005% and 0.01%, respectively. The results suggested that MTGase might catalyze the formation of e-(g-glutamyl) lysine linkages between protein chains. However, bloom strength decreased with further increase in MTGase concentration. This result was in agreement with Go`mez-Guille`n, Sarabia, Solas & Montero (2001) who reported that the increases in MTGase concentration from 0.015% to 0.075% lowered the bloom strength of megrim skin gelatin gel. From the result, the addition of MTGase at the appropriate concentration increased bloom strength of skin gelatin of both species effectively. The excessive formation of MTGase-catalyzed covalent bonds was detrimental to gel strength due to formation of intramolecular covalent bonds. As a consequence, intermolecular aggregation might be impeded, leading to reduced gel network formation. Therefore, MTGase at the concentrations of 0.005% and 0.01% (w/v) were appropriate to improve the bloom strength of gelatin gels from bigeye snapper and brownstripe red snapper skins, respectively. 3.3. Effect of MTGase on SDS-PAGE patterns of gelatin gels The protein patterns of gels from bigeye snapper and brownstripe red snapper skin gelatins under reducing condition (in the presence of bME) are shown in Fig. 2. For gelatin gels without MTGase, a1-chains was found as the major component for both samples. However, a2-chains, were not found in gelatin gels from brownstripe red snapper skin. Protein patterns of gelatin gels were generally relevant to those of the starting gelatin powder, especially for brownstripe red snapper gelatin. However, proteins with molecular weights of 71, 65 and 57 kDa were not found in the gel sample from bigeye snapper skin gelatin. No b-component was noticeable in the gel sample, however protein with molecular weight of 143 kDa was found in bigeye snapper gelatin sample. This might be owing to the degradation of these components during gel preparation, particularly during heat treatment. This result was in accordance with Muyonga et al. (2004) who observed that Nile perch skin gelatins contained low-molecular-weight peptides, especially, gelatins extracted with higher temperature. Heatinduced cleavage of protein chains occurred during the extraction process of Nile perch skin gelatin and was
Fig. 2. Protein pattern of gelatin gel from bigeye snapper and brownstripe red snapper skins added with MTGase at different concentrations. HM, C, and G, denote high molecular weight markers, standard collagen type I, and gelatin powder, respectively. Numbers, 0, 1, 2, 3, 4, and 5 indicate MTGase concentrations of 0, 0.001, 0.0025, 0.005, 0.01, and 0.05% (w/v), respectively.
enhanced with higher temperature extraction (Muyonga et al., 2004). From the results, greater amount of peptides with molecular weight lower than a-chains were observed in bigeye snapper skin gelatin than that of brownstripe red snapper skin gelatin. This suggested that collagen of bigeye snapper skin was more degraded during the extraction and some proteinase might be involved in the degradation of gelatin (Jongjareonrak et al., 2006). The formation of degradation fragments is associated with the low viscosity, low melting point, low setting point, high setting time, as well as decreased bloom strength of gelatin (Ledward, 1986; Muyonga et al., 2004; Normand et al., 2000; Tavernier, 1989). From the results, greater bloom strength was found in brownstripe red snapper skin
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gelatin, compared with that from bigeye snapper skin gelatin (Fig. 1). When MTGase was added, band intensity of all components in gelatin gels from both species decreased continuously with increasing MTGase concentration. When MTGase concentration increased, band intensity of b- and g-components as well as a-chains decreased with the concomitant increase in high-molecular-weight aggregate. At the highest concentration used (0.05%), all protein bands almost disappeared. The decrease in protein band intensity was most likely due to the cross-linking via nondisulfide covalent bond formation induced by MTGase (Folk, 1983). The results indicated that all components underwent cross-linking induced by MTGase. This was possibly because most of protein components contained both acyl donor and receptors aligned in the appropriate position for reaction induced by MTGase. Lysine was found in skin gelatin (Table 2) and might act as the acyl receptor of gelatin from both species. Glutamine, reported as glutamic acid, probably functioned as the acyl donor of gelatin. Generally, glutamine undergoes deamidation to glutamic acid under the harsh acid hydrolysis conditions used for analysis (Pickering & Newton, 1990) (Table 2). The decrease in protein band intensity (Fig. 2) was coincidental with the increase in bloom strength (Fig. 1). The aggregation of protein components via the covalent bond directly resulted in the development of a strong gel network, particularly with the appropriate range of MTGase levels. However, excessive cross-linking caused the adverse effect on gel network formation. Thus, the
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alignment or aggregation pattern of gelatin protein components also played an important role in gel formation as well as the strength of gelatin gel obtained. 3.4. Microstructure of gelatin gels The microstructure of gelatin gels from bigeye snapper and brownstripe red snapper skins without and with MTGase was visualized by SEM as shown in Fig. 3. In general, bigeye snapper skin gelatin gel showed the finer and denser strands compared with those of brownstripe red snapper skin gelatin (Fig. 3A and B). This was possibly due to the different characteristics of gelation between species in terms of aggregation and alignment of protein molecules. With addition of MTGase, the denser aggregates with negligible voids in the gel network were observed (Fig. 3B and D). It was suggested that gelatin underwent cross-linking via non-disulfide covalent bonding, resulting in greater intermolecular aggregation between adjacent molecules. The result was coincidental with the lower aand b-components as shown in Fig. 2. These cross-links might be associated with the higher bloom strength of gelatin gel when MTGase was added (Fig. 1). 4. Conclusion Gelatin was extracted from the skin of bigeye snapper and brownstripe red snapper. Both gelatins contained slightly different amino acid composition. The bloom
Fig. 3. Microstructure of gelatin gels (magnification: 10,000 X). A and B: gelatin gels of bigeye snapper skin without and with 0.005% (w/v) MTGase, respectively. C and D: gelatin gels of brownstripe red snapper skin without and with 0.01% (w/v) MTGase, respectively.
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strength of gelatin gel from bigeye snapper skin was lower than that of brownstripe red snapper skin. The addition of MTGase induced the aggregation of proteins, which was associated with the increase in bloom strength of fish skin gelatin from both species. However, bloom strength decreased with the excessive MTGase amount. Acknowledgements The authors thank Professor Akira Shinagawa of the Environment Education Center, Gakushuin Women’s college (Tokyo, Japan), for amino acid analysis. This research was supported by Thailand Research Fund under the Royal Golden Jubilee PhD program for financial support to Akkasit Jongjareonrak (PHD/0060/2544). References AOAC. (1999). Official methods of analysis. 14th ed. Washington, DC: Association of Official Analytical Chemists. Bergman, I., & Loxley, R. (1963). Two improved and simplified methods for the spectrophotometric determination of hydroxyproline. Analytical Chemistry, 35, 1961–1965. BSI (British Standards Institution) (1975). Methods for sampling and testing gelatin (physical and chemical methods). London: BSI. Burjandze, T. V. (1979). Hydroxyproline content and location in relation to collagen thermal stability. Biopolymers, 18, 931–936. Cho, S. M., Kwak, K. S., Park, D. C., Gu, Y. S., Ji, C. I., & Jang, D. H. (2004). Processing optimization and functional properties of gelatin from shark (Isurus oxyrinchus) cartilage. Food Hydrocolloids, 18, 573–579. Digenis, G. A., Gold, T. B., & Shah, V. P. (1994). Cross-linking of gelatin capsules and its relevance to their in vitro–in vivo performance. Journal of Pharmaceutical Science, 83, 915–921. Eastoe, J.E., & Eastoe, B. (1952). A method for the determination of total nitrogen in proteins. In: The British gelatin and glue research association research report, Series B 5 (pp. 1–17). Ferna´ndez-Diaz, M. D., Montero, P., & Go`mez-Guille`n, M. C. (2001). Gel properties of collagens from skins of cod (Gadus morhua) and hake (Merluccius merluccius) and their modification by coenhancers magnesium sulphate, glycerol and transglutaminase. Food Chemistry, 74, 161–167. Foegeding, E., Lanier, T. C., & Hultin, H. O. (1996). Characteristics of edible muscle tissue. In O. R. Fennema (Ed.), Food Chemistry (pp. 879–942). New York: Marcel Dekker. Folk, J. E. (1970). Transglutaminase. Methods in Enzymology, 17, 889–894. Folk, J. E. (1983). Mechanism and basis for specificity of transglutaminase-catalyzed-e(g-glutamyl)lysine bond formation. Advances in Enzymology, 54, 1–57. Gilsenan, P. M., & Ross-Murphy, S. B. (2000). Rheological characterisation of gelatins from mammalian and marine sources. Food Hydrocolloids, 14, 191–196. Go`mez-Guille`n, M. C., Sarabia, A. I., Solas, M. T., & Montero, P. (2001). Effect of microbial transglutaminase on the functional properties of megrim (Lepidorhombus boscii) skin gelatin. Journal of the Science of Food and Agriculture, 81, 665–673. Go`mez-Guille`n, M. C., Turnay, J., Ferna´ndez-Diaz, M. D., Ulmo, N., Lizarbe, M. A., & Montero, P. (2002). Structural and physical properties of gelatin extracted from different marine speices: a comparative study. Food Hydrocolloids, 16, 25–34.
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