- Email: [email protected]
Effect of pH on the properties of protein-based film from bigeye snapper (Priacanthus tayenus) surimi
Bioresource Technology 98 (2007) 221–225
Short Communication
EVect of pH on the properties of protein-based Wlm from bigeye snapper (Priacanthus tayenus) surimi Krittabhart Chinabhark a, Soottawat Benjakul
a,¤
, Thummanoon Prodpran
b
a
b
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Department of Material Product Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Received 14 March 2005; received in revised form 11 November 2005; accepted 13 November 2005 Available online 27 December 2005
Abstract The eVects of the pH and protein content on the properties and compositional changes of protein-based Wlms from bigeye snapper (Priacanthus tayenus) surimi were investigated. Tensile strength of bigeye snapper surimi Wlms prepared at the acidic (pH 3) and alkaline (pH 11) conditions were not signiWcantly diVerent (P > 0.05). Nevertheless, elongation at break of Wlms prepared at pH 3 was higher than that at pH 11 (P < 0.05). The acidic and alkaline conditions had no eVect on water vapor permeability of the Wlms obtained. However, the Wlm prepared at acidic condition was more yellowish than that prepared at alkaline pH. Protein content inXuenced the mechanical properties and color of Wlms. © 2005 Elsevier Ltd. All rights reserved. Keywords: Surimi; Film; Bigeye snapper; Protein; Fish muscle
1. Introduction Natural biopolymers have been paid increasing attention for manufacturing edible or biodegradable materials owing to their biocompatibility. Protein-based Wlms have been used commercially for packaging or coating. For instance, collagen Wlm has been used as casing for meat product (Krochta, 2002). A variety of proteins from both plant and animal can be used as Wlm-forming agents. Surimi is the concentrated myoWbrillar proteins prepared from Wsh mince that is washed with water and blended with cryoprotectants (Park and Morrissey, 2000). Surimi has been known to exhibit the gelling property, which makes it useful as a food base in seafood analogue. Furthermore, surimi or Wsh proteins were used for Wlm preparation (Shiku et al., 2003; Paschoalick et al., 2003). Proteins are thermoplastic heteropolymers containing both polar and non-polar amino acids, which are able to form numerous *
Corresponding author. Tel.: +66 74 286334; fax: +66 74 212889. E-mail address: [email protected] (S. Benjakul).
0960-8524/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.11.012
intermolecular linkages. Generally, globular proteins must be denatured by heat, acid, base and/or solvent to form more extended structures that are required for Wlm formation (Krochta, 2002). Solubilizing process directly aVected the property of Wlm. Fish myoWbrillar protein Wlm showed the diVerent properties, depending on the pH of Wlm-forming solution (Shiku et al., 2003). Thailand is one of the largest surimi producers in Southeast Asia. Bigeye snapper (Priacanthus spp.) is one of Wsh species commonly used for surimi production owing to its excellent gel-forming ability (Benjakul et al., 2001). Apart from gelation, the appropriate development of protein Wlm from bigeye snapper surimi should be an alternative promising means to obtain the nutritional and biodegradable Wlm. So far, the information concerning the eVect of pH on the properties of Wlm produced from frozen surimi containing cryoprotectants is scarce. The objective of this investigation was to study the eVect of pH on the properties of protein-based Wlm from bigeye snapper surimi and to monitor the compositional changes of Wlm-forming solution as inXuenced by diVerent pHs.
222
K. Chinabhark et al. / Bioresource Technology 98 (2007) 221–225
2. Methods 2.1. Frozen surimi Frozen surimi (grade A), produced from bigeye snapper (Priacanthus tayenus), was purchased locally. Moisture, protein, ash, fat and carbohydrate contents of surimi were measured (AOAC, 1999). Protein patterns were determined by SDS–PAGE using 4% stacking gel and 10% running gel according to the method of Laemmli (1970). 2.2. Preparation of Wlm-forming solution Frozen surimi was thawed using a running water (26– 27 °C) until the core temperature reached 0 °C. The Wlmforming solution was prepared as described by Shiku et al. (2003) with a slight modiWcation. Thawed surimi was added with the distilled water to obtain the Wnal protein concentrations of 1% and 2% (w/v). The mixture was homogenized at 13,000g for 1 min using a homogenizer (IKA Labortechnik, Selangor, Malaysia). Glycerol was then added at 50% (w/w) of protein content. The mixture was stirred gently for 30 min at room temperature. The pH of the mixture was adjusted to 3 or 11 using 1 N HCl and 1 N NaOH, respectively, to solubilize the protein by electrostatic repulsion. The Wlm-forming solution obtained was subjected to centrifugation at 3000g for 10 min at room temperature to remove air bubbles and aggregated proteins. The supernatant was transferred carefully using a transfer pipette. The solution was used for Wlm casting. 2.3. Film casting and drying The Wlm-forming solution (4 g) was cast onto a rimmed silicone resin plate (50 £ 50 mm) and air blown for 12 h at room temperature prior to further drying in a ventilated oven at 25 °C and 50% relative humidity (RH) for 24 h in an environmental chamber (WTB Binder, Tuttlingen, Germany). The resulting Wlms were manually peeled oV and used for analyses. 2.4. Determination of Wlm properties 2.4.1. Film thickness and mechanical properties The thickness of Wlm was measured using a micrometer (Gotech, Model GT-313-A, Gotech testing machines Inc., Taiwan). Five random positions of each Wlm of Wve Wlms were used for thickness determination. Prior to the measurement of mechanical properties, the Wlms were conditioned for 48 h at 25 °C and 50% RH prior to testing. Tensile strength (TS) and elongation at break (EAB) were determined as described by Iwata et al. (2000) with a slight modiWcation using the Universal Testing Machine (Lloyd Instruments, Hampshire, UK). Eight samples (2 £ 5 cm) with initial grip length of 3 cm were used for testing. Crosshead speed was 0.5 mm/s.
2.4.2. Water vapor permeability (WVP) WVP of Wlms was determined using a modiWed ASTM method as described by Shiku et al. (2003). Five Wlms were used for WVP testing and the measurement was run in duplicate. 2.4.3. Color and transparency Color of the Wlm was determined as L¤, a¤ and b¤ using CIE colorimeter (Hunter associates laboratory, Inc., VA, USA). The Wlms were subjected to the measurement at 600 nm using the UV-1601 spectrophotometer (Shimadzu, Kyoto, Japan) as described by Han and Floros (1997). The transparency of the Wlms was calculated as follows: Transparency D ¡logT600/x, where T600 is the transmittance at 600 nm and x is the Wlm thickness (mm). 2.5. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) The protein patterns of surimi sample and the Wlms were analyzed by SDS–PAGE according to the method of Laemmli (1970) using a 10% running gel and 4% stacking gel. To prepare the surimi sample for analysis, surimi (3 g) was solubilized in 27 ml of 1% SDS (85 °C) as described by Benjakul et al. (2001). To solubilize the Wlms, the samples were mixed with a solubilizing solution containing 1% SDS and 8 M urea, homogenized at 13,000 rpm for 1 min using an IKA homogenizer and stirred continuously for 24 h at room temperature. The supernatants obtained after centrifugation at 3000g for 5 min were subjected to SDS–PAGE analysis. 2.6. EVects of pH on the compositional changes of Wlm-forming solution The surimi was solubilized using acid and alkaline at pH 3 and 11, respectively, as described previously. The solution was allowed to stand at room temperature and taken for analysis at 0, 2, 5, 7 and 10 h. At the time designated, the solution was neutralized using either 1 N NaOH or 1 N HCl. Then, the neutralized solution was mixed with 5% SDS at a ratio of 1:1 (v/v). The mixture was incubated at 85 °C for 15 min and centrifugation at 3500g for 5 min. The supernatants were subjected to SDS–PAGE analysis and reducing sugar determination. 2.7. Determination of reducing sugar The reducing sugar was determined according to the method of Nelson–Somogyi as modiWed by Chaplin and Kennedy (1994). 2.8. Statistical analysis Analysis of variance (ANOVA) was performed and mean comparisons were carried out by Duncan’s multiple range test (Steel and Torrie, 1980). Analysis was performed
K. Chinabhark et al. / Bioresource Technology 98 (2007) 221–225
using the SPSS package (SPSS 11.0 for Windows, SPSS Inc., Chicago, IL). 3. Results and discussion 3.1. Chemical compositions of surimi Bigeye snapper surimi showed 78.78 § 0.03% moisture, 14.06 § 0.16% protein and 5.98 § 0.29% carbohydrate contents. Cryoprotectants, including sucrose and/or sorbitol, were the major sources of carbohydrate. Fat (0.04 § 0.00%) and ash (0.60 § 0.16%) contents were negligible. Electrophoretic study of bigeye snapper surimi revealed that myosin heavy chain constituted the major protein, followed by the actin (Fig. 1). 3.2. EVect of pH on the properties of Wlm from bigeye snapper surimi At the same protein content used, tensile strength (TS) of surimi Wlms prepared at the acidic (pH 3) and alkaline (pH 11) conditions was not diVerent (P > 0.05) (Table 1). Nevertheless, elongation at break (EAB) of Wlms prepared at pH 3 was much higher than that prepared at pH 11 (P < 0.05). The unfolded proteins obtained from either acid or alkaline solubilizing process underwent the aggregation through hydrogen, ionic, hydrophobic and covalent bonding, particularly when the water was removed. At the extreme acidic or alkaline pH values, strong electrostatic
Fig. 1. Protein patterns of surimi Wlm from bigeye snapper. M: highmolecular-weight protein marker; MHC: myosin heavy chain; S; surimi; A1: 1% protein content, pH 3; B1: 1% protein content, pH 11; A2: 2% protein content, pH 3; B2: 2% protein content, pH 11.
223
repulsion of ionized groups occurred, leading to solublization of proteins, which is prerequisite for Wlm formation. The degree of chain extension and the nature of sequence of amino acid residues might aVect the mechanical properties of protein Wlm (Krochta, 1997). The main associative forces involved in surimi Wlms were hydrogen bonds, hydrophobic and ionic interactions (Shiku et al., 2004). At the same pH used, Wlm with greater protein content prepared at pH 3 exhibited higher TS (P < 0.05) (Table 1). Proteins at the higher amount might aggregate intermolecularly to a greater extent, compared with the lower amount, resulting in the stronger interaction as evidenced by the increased TS. Nevertheless, no diVerences in TS between Wlms with diVerent protein contents were observed, when Wlms were prepared at pH 11. Similar EAB was found between Wlms with 1% and 2% protein content at the same pH used. Generally, the greater protein content resulted in non-signiWcant higher EAB than the lower one. No diVerences in WVP were found between Wlms prepared at pH 3 and 11 (P > 0.05), when the same protein content was used (Table 1). WVP of surimi Wlms prepared at both pHs increased with increasing protein contents (P < 0.05). A higher amount of protein was probably associated with a higher amount of polar groups in surimi Wlm, which could absorb more water from the surrounding atmosphere. Since the surimi Wlm with higher protein content had the higher thickness, WVP of Wlms might be inXuenced by thickness. Increased transmission of water vapor through protein-based Wlm was also caused by the presence of glycerol, a hydrophilic plasticizer (Cuq et al., 1995). Sucrose and sorbitol used as cryoprotectants also provided the polar groups in the surimi Wlms, where hydrogen bonding could be formed with water. L¤, a¤ and b¤-values of bigeye snapper surimi Wlms prepared at diVerent pHs, and protein contents are shown in Table 2. At the same protein content used, Wlm prepared at pH 3 had the greater b¤-value but lower L¤-value (P < 0.05). The result suggested that the surimi Wlms prepared at acidic pH was more yellowish than those prepared at alkaline pH as evidenced by greater b¤-value. Film prepared at pH 3 had the lower lightness than that prepared at pH 11. For a¤value, no marked diVerences were observed between Wlms prepared at pH 3 and 11 (P > 0.05). From the result, it indicated that acidic condition might induce the formation of yellowish pigment, especially via Maillard reaction. At the same pH used, protein content aVected the color diVerently.
Table 1 Physical and mechanical properties of bigeye snapper surimi Wlm Surimi Wlms
TSA (MPa)
EABA (%)
WVPB (£ 10¡10 g m¡1 s¡1 Pa¡1)
ThicknessB (mm)
1% pH 3 1% pH 11 2% pH 3 2% pH 11
3.31 § 0.11bc 3.21 § 0.30b 3.89 § 0.40a 3.59 § 0.30ab
69.19 § 23.92a 22.69 § 13.17b 79.96 § 17.73a 29.27 § 6.67b
0.90 § 0.04b 0.84 § 0.03b 1.17 § 0.08a 1.11 § 0.12a
0.018 § 0.003b 0.020 § 0.004b 0.031 § 0.002a 0.032 § 0.003a
The diVerent superscripts in the same column indicate the signiWcant diVerences (P < 0.05). A Mean § SD from eight determinations. B Mean § SD from Wve determinations.
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Table 2 L¤, a¤ and b¤-values of bigeye snapper surimi Wlm Surimi Wlms 1% pH 3 1% pH 11 2% pH 3 2% pH 11
L¤A
a¤A c
90.41 § 0.07 91.17 § 0.10a 90.23 § 0.04d 90.93 § 0.02b
TransparencyA
b¤A a
¡1.23 § 0.10 ¡1.29 § 0.16a ¡1.70 § 0.07b ¡1.55 § 0.13b
b
1.44 § 0.04 1.09 § 0.05c 3.76 § 0.56a 1.45 § 0.38b
4.33 § 0.12b 4.29 § 0.08b 5.93 § 0.19a 5.84 § 0.25a
The diVerent superscripts in the same column indicate the signiWcant diVerences (P < 0.05). A Mean § SD from three determinations.
The greater b¤-value with lower L¤- and a¤-values were noticeable with Wlms having 2% protein content, compared with those containing the lower protein content (1%) (P < 0.05). Thus, the Wlms with 2% protein content had more yellowness but lower lightness than those having 1% protein content. The Wlm was more transparent when the lower protein content was used (P < 0.05) (Table 2). No diVerences in transparency were observed between Wlms prepared at diVerent pHs, when the same protein content was used (P > 0.05). Thus the thickness of Wlm possibly inXuenced the color and transparency. No MHC band was found in both Wlms (Fig. 1). The surimi Wlms also contained the lower band intensity of actin, compared with those of surimi. Those changes were observed with the concomitant appearance of proteins with the lower molecular weight. This suggested that the degradation of proteins, especially MHC, occurred in the Wlms. From the result, diVerent protein patterns were found between Wlms prepared at pH 3 and 11. The degraded proteins with molecular weight of 140–150 kDa were found in the Wlm with acid solubilizing process, while the proteins with the molecular weight ranging from 60 to 70 kDa were obtained in the Wlm with alkaline solubilizing process. Therefore, the cleavage of myosin might be taken place at diVerent sites, leading to the diVerences in the molecular distribution. Actin was also degraded into diVerent degradation products by two diVerent protein solubilizing processes. Therefore, the acid or alkaline solubilizing process might induce the degradation of muscle protein in surimi. This possibly contributed to the diVerent characteristics, both mechanical properties (Table 1) and color (Table 2) of surimi Wlms produced by both processes.
by either acids or enzymes (BeBiller and Whistler, 1996). Since the reducing sugar content in acidic Wlm-forming solutions was higher than alkaline Wlm-forming solutions, browning of the surimi Wlms prepared by the former solution was greater than the latter as shown by the higher b¤value in the Wlms prepared at acidic condition. The protein patterns of acidic and alkaline Wlm-forming solutions are shown in Fig. 2. At 0 h, the protein pattern of Wlm-forming solution was very similar to that of surimi. For alkaline solution, MHC was more degraded as the exposure time increased up to 10 h. Actin was also hydrolyzed with increasing exposure time under alkaline condition. At acidic condition, negligible degradation was observed throughout 10 h (Fig. 2A). The result suggested that autolysis caused by endogenous proteases was greater at alkaline pH. Bigeye snapper muscle was reported to contain the heat-stable alkaline proteinase, which could degrade MHC eVectively (Benjakul et al., 2003). From the result, the degradation took place to a higher extent at alkaline pH, leading to the decreased peptide chain length. This
3.3. Compositional changes of surimi Wlm-forming solution with acidic and alkaline pHs The reducing sugar content of Wlm-forming solutions with pH 3 and 11 increased as the exposure time increased up to 10 h (P < 0.05) (data not shown). However, the rate of increase was much greater in the acidic solution, particularly with increasing exposure time. Acidic condition might induce the hydrolysis of sucrose used as the cryoprotectant in surimi, resulting in the increased reducing sugar released, both glucose and fructose (data not shown). Acid eVectively hydrolyzes disaccharide to monosaccharide. Generally, hydrolysis of glycosidic bonds joining monosaccharide (glycosyl) units in di-and polysaccharides can be catalyzed
Fig. 2. Protein patterns of acidic (A) and alkaline (B) Wlm-forming solutions from bigeye snapper surimi with diVerent exposure times. MHC: myosin heavy chain. The numbers designate the exposure time (h).
K. Chinabhark et al. / Bioresource Technology 98 (2007) 221–225
might be associated with the lower EAB in surimi Wlm prepared at alkaline pH (Table 2). 4. Conclusions Mechanical properties and color of the protein-based Wlms from bigeye snapper surimi were aVected by pHs and protein content. Acidic condition rendered the Wlms with the greater EAB with the higher yellowness, while alkaline condition favored the protein degradation. pH also aVected the hydrolysis of sugar, which accordingly inXuenced the color formation in the Wlm via Maillard reaction. Acknowledgement We thank Prince of Songkla University for the Wnancial support. References American Society for Testing and Materials, 1989. In: Annual Book of ASTM Standards. ASTM, Philadelphia, p. 1989. AOAC, 1999. OYcial Method of Analysis, 14th ed. Association of Agricultural Chemists, Washington, DC. BeBiller, J.N., Whistler, R.L., 1996. Carbohydrate. In: Fennema, O.R. (Ed.), Food Chemistry. Marcel Dekkers, Inc., New York, pp. 157–223. Benjakul, S., Visessanguan, W., Srivilai, C., 2001. Gel properties of bigeye snapper (Priacanthus tayenus) surimi as aVected by setting and porcine plasma proteins. J. Food Quality 24, 453–471. Benjakul, S., Visessanguan, W., Leepongwattana, K., 2003. Comparative study on proteolysis of two species of bigeye snapper, Priacanthus
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macracanthus and Priacanthus tayenus. J. Sci. Food Agric. 83, 871– 879. Chaplin, M.F, Kennedy, J.F., 1994. Monosaccharides. Oxford University Press, New York. p . 110. Cuq, B., Aymad, C., Cuq, J., Quilbert, S., 1995. Edible packaging Wlms based on Wsh myoWbrillar proteins: formulation and functional properties. J. Food Sci. 60, 1369–1373. Han, J.H., Floros, J.D., 1997. Casting antimicrobial packaging Wlms and measuring their physical properties and antimicrobial activity. Plastic Film Sheet J. 13, 287–298. Iwata, K., Ishizaki, S., Handa, A., Tanaka, M., 2000. Preparation and characterization of edible Wlms from Wsh water-soluble proteins. Fish. Sci. 66, 372–378. Krochta, J.M., 1997. Edible protein Wlms and coatings. In: Damodaran, S., Paraf, A. (Eds.), Food Proteins and Their Applications. Marcel Dekker, Inc., New York, pp. 529–549. Krochta, J.M., 2002. Protein as raw materials for Wlms and coatings deWnitions, current status, and opportunities. In: Gennadios, A. (Ed.), Protein Based-Films and Coating. CRC Press, New York, pp. 1–39. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 227, 680–685. Park, J.W., Morrissey, M.T., 2000. Manufacturing of surimi from light muscle Wsh. In: Park, J.W. (Ed.), Surimi and Surimi Seafood. Marcel Dekker, Inc., New York, pp. 23–58. Paschoalick, T.M., Garcia, F.T., Sobral, P.J.A., Habitante, A.M.Q.B., 2003. Characterization of some functional properties of edible Wlms based on muscle proteins of Nile tilapia. Food Hydrocolloids 17, 419–427. Shiku, Y., Hamaguchi, P.Y., Benjakul, S., Visessanguan, W., Tanaka, M., 2004. EVect of surimi quality on properties of edible Wlms based on Alaska pollack. Food Chem. 86, 493–499. Shiku, Y., Hamaguchi, P.Y., Tanaka, M., 2003. EVect of pH on the preparation of edible Wlms based on Wsh myoWbrillar proteins. Fish. Sci. 69, 1026–1032. Steel, R.G.D., Torrie, J.H., 1980. Principles and Procedure of Statistics; A Biometrical Approach. McGraw-Hill, New York.
Short Communication
EVect of pH on the properties of protein-based Wlm from bigeye snapper (Priacanthus tayenus) surimi Krittabhart Chinabhark a, Soottawat Benjakul
a,¤
, Thummanoon Prodpran
b
a
b
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Department of Material Product Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Received 14 March 2005; received in revised form 11 November 2005; accepted 13 November 2005 Available online 27 December 2005
Abstract The eVects of the pH and protein content on the properties and compositional changes of protein-based Wlms from bigeye snapper (Priacanthus tayenus) surimi were investigated. Tensile strength of bigeye snapper surimi Wlms prepared at the acidic (pH 3) and alkaline (pH 11) conditions were not signiWcantly diVerent (P > 0.05). Nevertheless, elongation at break of Wlms prepared at pH 3 was higher than that at pH 11 (P < 0.05). The acidic and alkaline conditions had no eVect on water vapor permeability of the Wlms obtained. However, the Wlm prepared at acidic condition was more yellowish than that prepared at alkaline pH. Protein content inXuenced the mechanical properties and color of Wlms. © 2005 Elsevier Ltd. All rights reserved. Keywords: Surimi; Film; Bigeye snapper; Protein; Fish muscle
1. Introduction Natural biopolymers have been paid increasing attention for manufacturing edible or biodegradable materials owing to their biocompatibility. Protein-based Wlms have been used commercially for packaging or coating. For instance, collagen Wlm has been used as casing for meat product (Krochta, 2002). A variety of proteins from both plant and animal can be used as Wlm-forming agents. Surimi is the concentrated myoWbrillar proteins prepared from Wsh mince that is washed with water and blended with cryoprotectants (Park and Morrissey, 2000). Surimi has been known to exhibit the gelling property, which makes it useful as a food base in seafood analogue. Furthermore, surimi or Wsh proteins were used for Wlm preparation (Shiku et al., 2003; Paschoalick et al., 2003). Proteins are thermoplastic heteropolymers containing both polar and non-polar amino acids, which are able to form numerous *
Corresponding author. Tel.: +66 74 286334; fax: +66 74 212889. E-mail address: [email protected] (S. Benjakul).
0960-8524/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.11.012
intermolecular linkages. Generally, globular proteins must be denatured by heat, acid, base and/or solvent to form more extended structures that are required for Wlm formation (Krochta, 2002). Solubilizing process directly aVected the property of Wlm. Fish myoWbrillar protein Wlm showed the diVerent properties, depending on the pH of Wlm-forming solution (Shiku et al., 2003). Thailand is one of the largest surimi producers in Southeast Asia. Bigeye snapper (Priacanthus spp.) is one of Wsh species commonly used for surimi production owing to its excellent gel-forming ability (Benjakul et al., 2001). Apart from gelation, the appropriate development of protein Wlm from bigeye snapper surimi should be an alternative promising means to obtain the nutritional and biodegradable Wlm. So far, the information concerning the eVect of pH on the properties of Wlm produced from frozen surimi containing cryoprotectants is scarce. The objective of this investigation was to study the eVect of pH on the properties of protein-based Wlm from bigeye snapper surimi and to monitor the compositional changes of Wlm-forming solution as inXuenced by diVerent pHs.
222
K. Chinabhark et al. / Bioresource Technology 98 (2007) 221–225
2. Methods 2.1. Frozen surimi Frozen surimi (grade A), produced from bigeye snapper (Priacanthus tayenus), was purchased locally. Moisture, protein, ash, fat and carbohydrate contents of surimi were measured (AOAC, 1999). Protein patterns were determined by SDS–PAGE using 4% stacking gel and 10% running gel according to the method of Laemmli (1970). 2.2. Preparation of Wlm-forming solution Frozen surimi was thawed using a running water (26– 27 °C) until the core temperature reached 0 °C. The Wlmforming solution was prepared as described by Shiku et al. (2003) with a slight modiWcation. Thawed surimi was added with the distilled water to obtain the Wnal protein concentrations of 1% and 2% (w/v). The mixture was homogenized at 13,000g for 1 min using a homogenizer (IKA Labortechnik, Selangor, Malaysia). Glycerol was then added at 50% (w/w) of protein content. The mixture was stirred gently for 30 min at room temperature. The pH of the mixture was adjusted to 3 or 11 using 1 N HCl and 1 N NaOH, respectively, to solubilize the protein by electrostatic repulsion. The Wlm-forming solution obtained was subjected to centrifugation at 3000g for 10 min at room temperature to remove air bubbles and aggregated proteins. The supernatant was transferred carefully using a transfer pipette. The solution was used for Wlm casting. 2.3. Film casting and drying The Wlm-forming solution (4 g) was cast onto a rimmed silicone resin plate (50 £ 50 mm) and air blown for 12 h at room temperature prior to further drying in a ventilated oven at 25 °C and 50% relative humidity (RH) for 24 h in an environmental chamber (WTB Binder, Tuttlingen, Germany). The resulting Wlms were manually peeled oV and used for analyses. 2.4. Determination of Wlm properties 2.4.1. Film thickness and mechanical properties The thickness of Wlm was measured using a micrometer (Gotech, Model GT-313-A, Gotech testing machines Inc., Taiwan). Five random positions of each Wlm of Wve Wlms were used for thickness determination. Prior to the measurement of mechanical properties, the Wlms were conditioned for 48 h at 25 °C and 50% RH prior to testing. Tensile strength (TS) and elongation at break (EAB) were determined as described by Iwata et al. (2000) with a slight modiWcation using the Universal Testing Machine (Lloyd Instruments, Hampshire, UK). Eight samples (2 £ 5 cm) with initial grip length of 3 cm were used for testing. Crosshead speed was 0.5 mm/s.
2.4.2. Water vapor permeability (WVP) WVP of Wlms was determined using a modiWed ASTM method as described by Shiku et al. (2003). Five Wlms were used for WVP testing and the measurement was run in duplicate. 2.4.3. Color and transparency Color of the Wlm was determined as L¤, a¤ and b¤ using CIE colorimeter (Hunter associates laboratory, Inc., VA, USA). The Wlms were subjected to the measurement at 600 nm using the UV-1601 spectrophotometer (Shimadzu, Kyoto, Japan) as described by Han and Floros (1997). The transparency of the Wlms was calculated as follows: Transparency D ¡logT600/x, where T600 is the transmittance at 600 nm and x is the Wlm thickness (mm). 2.5. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) The protein patterns of surimi sample and the Wlms were analyzed by SDS–PAGE according to the method of Laemmli (1970) using a 10% running gel and 4% stacking gel. To prepare the surimi sample for analysis, surimi (3 g) was solubilized in 27 ml of 1% SDS (85 °C) as described by Benjakul et al. (2001). To solubilize the Wlms, the samples were mixed with a solubilizing solution containing 1% SDS and 8 M urea, homogenized at 13,000 rpm for 1 min using an IKA homogenizer and stirred continuously for 24 h at room temperature. The supernatants obtained after centrifugation at 3000g for 5 min were subjected to SDS–PAGE analysis. 2.6. EVects of pH on the compositional changes of Wlm-forming solution The surimi was solubilized using acid and alkaline at pH 3 and 11, respectively, as described previously. The solution was allowed to stand at room temperature and taken for analysis at 0, 2, 5, 7 and 10 h. At the time designated, the solution was neutralized using either 1 N NaOH or 1 N HCl. Then, the neutralized solution was mixed with 5% SDS at a ratio of 1:1 (v/v). The mixture was incubated at 85 °C for 15 min and centrifugation at 3500g for 5 min. The supernatants were subjected to SDS–PAGE analysis and reducing sugar determination. 2.7. Determination of reducing sugar The reducing sugar was determined according to the method of Nelson–Somogyi as modiWed by Chaplin and Kennedy (1994). 2.8. Statistical analysis Analysis of variance (ANOVA) was performed and mean comparisons were carried out by Duncan’s multiple range test (Steel and Torrie, 1980). Analysis was performed
K. Chinabhark et al. / Bioresource Technology 98 (2007) 221–225
using the SPSS package (SPSS 11.0 for Windows, SPSS Inc., Chicago, IL). 3. Results and discussion 3.1. Chemical compositions of surimi Bigeye snapper surimi showed 78.78 § 0.03% moisture, 14.06 § 0.16% protein and 5.98 § 0.29% carbohydrate contents. Cryoprotectants, including sucrose and/or sorbitol, were the major sources of carbohydrate. Fat (0.04 § 0.00%) and ash (0.60 § 0.16%) contents were negligible. Electrophoretic study of bigeye snapper surimi revealed that myosin heavy chain constituted the major protein, followed by the actin (Fig. 1). 3.2. EVect of pH on the properties of Wlm from bigeye snapper surimi At the same protein content used, tensile strength (TS) of surimi Wlms prepared at the acidic (pH 3) and alkaline (pH 11) conditions was not diVerent (P > 0.05) (Table 1). Nevertheless, elongation at break (EAB) of Wlms prepared at pH 3 was much higher than that prepared at pH 11 (P < 0.05). The unfolded proteins obtained from either acid or alkaline solubilizing process underwent the aggregation through hydrogen, ionic, hydrophobic and covalent bonding, particularly when the water was removed. At the extreme acidic or alkaline pH values, strong electrostatic
Fig. 1. Protein patterns of surimi Wlm from bigeye snapper. M: highmolecular-weight protein marker; MHC: myosin heavy chain; S; surimi; A1: 1% protein content, pH 3; B1: 1% protein content, pH 11; A2: 2% protein content, pH 3; B2: 2% protein content, pH 11.
223
repulsion of ionized groups occurred, leading to solublization of proteins, which is prerequisite for Wlm formation. The degree of chain extension and the nature of sequence of amino acid residues might aVect the mechanical properties of protein Wlm (Krochta, 1997). The main associative forces involved in surimi Wlms were hydrogen bonds, hydrophobic and ionic interactions (Shiku et al., 2004). At the same pH used, Wlm with greater protein content prepared at pH 3 exhibited higher TS (P < 0.05) (Table 1). Proteins at the higher amount might aggregate intermolecularly to a greater extent, compared with the lower amount, resulting in the stronger interaction as evidenced by the increased TS. Nevertheless, no diVerences in TS between Wlms with diVerent protein contents were observed, when Wlms were prepared at pH 11. Similar EAB was found between Wlms with 1% and 2% protein content at the same pH used. Generally, the greater protein content resulted in non-signiWcant higher EAB than the lower one. No diVerences in WVP were found between Wlms prepared at pH 3 and 11 (P > 0.05), when the same protein content was used (Table 1). WVP of surimi Wlms prepared at both pHs increased with increasing protein contents (P < 0.05). A higher amount of protein was probably associated with a higher amount of polar groups in surimi Wlm, which could absorb more water from the surrounding atmosphere. Since the surimi Wlm with higher protein content had the higher thickness, WVP of Wlms might be inXuenced by thickness. Increased transmission of water vapor through protein-based Wlm was also caused by the presence of glycerol, a hydrophilic plasticizer (Cuq et al., 1995). Sucrose and sorbitol used as cryoprotectants also provided the polar groups in the surimi Wlms, where hydrogen bonding could be formed with water. L¤, a¤ and b¤-values of bigeye snapper surimi Wlms prepared at diVerent pHs, and protein contents are shown in Table 2. At the same protein content used, Wlm prepared at pH 3 had the greater b¤-value but lower L¤-value (P < 0.05). The result suggested that the surimi Wlms prepared at acidic pH was more yellowish than those prepared at alkaline pH as evidenced by greater b¤-value. Film prepared at pH 3 had the lower lightness than that prepared at pH 11. For a¤value, no marked diVerences were observed between Wlms prepared at pH 3 and 11 (P > 0.05). From the result, it indicated that acidic condition might induce the formation of yellowish pigment, especially via Maillard reaction. At the same pH used, protein content aVected the color diVerently.
Table 1 Physical and mechanical properties of bigeye snapper surimi Wlm Surimi Wlms
TSA (MPa)
EABA (%)
WVPB (£ 10¡10 g m¡1 s¡1 Pa¡1)
ThicknessB (mm)
1% pH 3 1% pH 11 2% pH 3 2% pH 11
3.31 § 0.11bc 3.21 § 0.30b 3.89 § 0.40a 3.59 § 0.30ab
69.19 § 23.92a 22.69 § 13.17b 79.96 § 17.73a 29.27 § 6.67b
0.90 § 0.04b 0.84 § 0.03b 1.17 § 0.08a 1.11 § 0.12a
0.018 § 0.003b 0.020 § 0.004b 0.031 § 0.002a 0.032 § 0.003a
The diVerent superscripts in the same column indicate the signiWcant diVerences (P < 0.05). A Mean § SD from eight determinations. B Mean § SD from Wve determinations.
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Table 2 L¤, a¤ and b¤-values of bigeye snapper surimi Wlm Surimi Wlms 1% pH 3 1% pH 11 2% pH 3 2% pH 11
L¤A
a¤A c
90.41 § 0.07 91.17 § 0.10a 90.23 § 0.04d 90.93 § 0.02b
TransparencyA
b¤A a
¡1.23 § 0.10 ¡1.29 § 0.16a ¡1.70 § 0.07b ¡1.55 § 0.13b
b
1.44 § 0.04 1.09 § 0.05c 3.76 § 0.56a 1.45 § 0.38b
4.33 § 0.12b 4.29 § 0.08b 5.93 § 0.19a 5.84 § 0.25a
The diVerent superscripts in the same column indicate the signiWcant diVerences (P < 0.05). A Mean § SD from three determinations.
The greater b¤-value with lower L¤- and a¤-values were noticeable with Wlms having 2% protein content, compared with those containing the lower protein content (1%) (P < 0.05). Thus, the Wlms with 2% protein content had more yellowness but lower lightness than those having 1% protein content. The Wlm was more transparent when the lower protein content was used (P < 0.05) (Table 2). No diVerences in transparency were observed between Wlms prepared at diVerent pHs, when the same protein content was used (P > 0.05). Thus the thickness of Wlm possibly inXuenced the color and transparency. No MHC band was found in both Wlms (Fig. 1). The surimi Wlms also contained the lower band intensity of actin, compared with those of surimi. Those changes were observed with the concomitant appearance of proteins with the lower molecular weight. This suggested that the degradation of proteins, especially MHC, occurred in the Wlms. From the result, diVerent protein patterns were found between Wlms prepared at pH 3 and 11. The degraded proteins with molecular weight of 140–150 kDa were found in the Wlm with acid solubilizing process, while the proteins with the molecular weight ranging from 60 to 70 kDa were obtained in the Wlm with alkaline solubilizing process. Therefore, the cleavage of myosin might be taken place at diVerent sites, leading to the diVerences in the molecular distribution. Actin was also degraded into diVerent degradation products by two diVerent protein solubilizing processes. Therefore, the acid or alkaline solubilizing process might induce the degradation of muscle protein in surimi. This possibly contributed to the diVerent characteristics, both mechanical properties (Table 1) and color (Table 2) of surimi Wlms produced by both processes.
by either acids or enzymes (BeBiller and Whistler, 1996). Since the reducing sugar content in acidic Wlm-forming solutions was higher than alkaline Wlm-forming solutions, browning of the surimi Wlms prepared by the former solution was greater than the latter as shown by the higher b¤value in the Wlms prepared at acidic condition. The protein patterns of acidic and alkaline Wlm-forming solutions are shown in Fig. 2. At 0 h, the protein pattern of Wlm-forming solution was very similar to that of surimi. For alkaline solution, MHC was more degraded as the exposure time increased up to 10 h. Actin was also hydrolyzed with increasing exposure time under alkaline condition. At acidic condition, negligible degradation was observed throughout 10 h (Fig. 2A). The result suggested that autolysis caused by endogenous proteases was greater at alkaline pH. Bigeye snapper muscle was reported to contain the heat-stable alkaline proteinase, which could degrade MHC eVectively (Benjakul et al., 2003). From the result, the degradation took place to a higher extent at alkaline pH, leading to the decreased peptide chain length. This
3.3. Compositional changes of surimi Wlm-forming solution with acidic and alkaline pHs The reducing sugar content of Wlm-forming solutions with pH 3 and 11 increased as the exposure time increased up to 10 h (P < 0.05) (data not shown). However, the rate of increase was much greater in the acidic solution, particularly with increasing exposure time. Acidic condition might induce the hydrolysis of sucrose used as the cryoprotectant in surimi, resulting in the increased reducing sugar released, both glucose and fructose (data not shown). Acid eVectively hydrolyzes disaccharide to monosaccharide. Generally, hydrolysis of glycosidic bonds joining monosaccharide (glycosyl) units in di-and polysaccharides can be catalyzed
Fig. 2. Protein patterns of acidic (A) and alkaline (B) Wlm-forming solutions from bigeye snapper surimi with diVerent exposure times. MHC: myosin heavy chain. The numbers designate the exposure time (h).
K. Chinabhark et al. / Bioresource Technology 98 (2007) 221–225
might be associated with the lower EAB in surimi Wlm prepared at alkaline pH (Table 2). 4. Conclusions Mechanical properties and color of the protein-based Wlms from bigeye snapper surimi were aVected by pHs and protein content. Acidic condition rendered the Wlms with the greater EAB with the higher yellowness, while alkaline condition favored the protein degradation. pH also aVected the hydrolysis of sugar, which accordingly inXuenced the color formation in the Wlm via Maillard reaction. Acknowledgement We thank Prince of Songkla University for the Wnancial support. References American Society for Testing and Materials, 1989. In: Annual Book of ASTM Standards. ASTM, Philadelphia, p. 1989. AOAC, 1999. OYcial Method of Analysis, 14th ed. Association of Agricultural Chemists, Washington, DC. BeBiller, J.N., Whistler, R.L., 1996. Carbohydrate. In: Fennema, O.R. (Ed.), Food Chemistry. Marcel Dekkers, Inc., New York, pp. 157–223. Benjakul, S., Visessanguan, W., Srivilai, C., 2001. Gel properties of bigeye snapper (Priacanthus tayenus) surimi as aVected by setting and porcine plasma proteins. J. Food Quality 24, 453–471. Benjakul, S., Visessanguan, W., Leepongwattana, K., 2003. Comparative study on proteolysis of two species of bigeye snapper, Priacanthus
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