Combination effects of chicken plasma protein and setting phenomenon on gel properties and cross-linking of bigeye snapper muscle proteins

ARTICLE IN PRESS

LWT 38 (2005) 353–362 www.elsevier.com/locate/lwt

Combination effects of chicken plasma protein and setting phenomenon on gel properties and cross-linking of bigeye snapper muscle proteins Saroat Rawdkuena, Soottawat Benjakula,, Wonnop Visessanguanb, Tyre C. Lanierc a

Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, 90112, Thailand National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Phaholyothin Rd., Klong1, Klong Luang, Pathumthani, 12120, Thailand c Department of Food Science, North Calorina State University, Raleigh, NC 27695-7624, USA

b

Received 26 January 2004; accepted 8 June 2004

Abstract Effects of chicken plasma protein (CPP) in combination with setting on surimi gel properties and protein cross-linking were investigated. Addition of CPP (0.5 g/100 g), CaCl2 (10 mmole/kg) and 200 units of thrombin/g CPP in combination with setting at 40 1C for 30 min prior to heating at 90 1C for 20 min resulted in the highest breaking force and deformation (Po0.05). Regardless of CPP addition, myosin heavy chain (MHC) in surimi proteins and natural actomyosin (NAM) underwent polymerization to some extent in the presence of CaCl2 and thrombin. The cross-linking of MHC in surimi proteins were markedly suppressed by the addition of EGTA and NH4Cl, transglutaminase (TGase) inhibitors. No cross-linking of myosin was observed when CPP, CaCl2 and thrombin were added, even with the prolonged incubation time. The result revealed that CPP showed no cross-linking activity and gel strengthening effect of CPP was attributed to its filler effect and proteolytic inhibitory activity. r 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Myosin heavy chain; Surimi; Setting; Cross-linking; Chicken plasma

1. Introduction Surimi possesses the functionality including binding, gelling and emulsifying properties and can be used as a functional protein ingredient in several products (Lanier, 1986). The thermal aggregation of myosin molecule is a crucial process for developing the elastic gels (Sano, Noguchi, Tsuchiya, & Matsumoto, 1988). The crosslinking and aggregation of protein molecules into threedimensional solid-like networks results in elastic gel with high water holding capacity (Sano et al., 1988). To increase the gel strength, setting has been widely implicated in surimi gel preparation. Setting is a phenomenon describing increased gel strength after Corresponding author. Tel.: +66-7428-6334; fax: +66-7421-2889.

E-mail address: [email protected] (S. Benjakul).

pre-incubation of surimi paste at a certain temperature between 5 and 40 1C for a specific period of time (Lanier, 2000). Benjakul and Visessanguan (2003) reported that the optimum condition for setting of surimi sol from bigeye snapper (Priacanthus tayenus and P. macracanthus) were 40 and 25 1C for 2 and 3 h, respectively. Setting at 25 1C for an appropriate time also improves gelling properties of surimi produced from other tropical fish (Benjakul, Chantarasuwan, & Visessanguan, 2003). An improvement of textural properties is attributed to an endogenous transglutaminase (TGase) that catalyses the cross-linking reaction of muscle proteins, especially myosin (Kimura, Sugimoto, Toyoda, Seki, Arai, & Fujita, 1991; Kishi, Nozawa, & Seki, 1991). Setting of surimi is enhanced by sufficient calcium content since endogenous TGase is a Ca2+-dependent

0023-6438/$30.00 r 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2004.06.016

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enzyme (Kimura et al., 1991; Lee & Park, 1998). e(gglutamyl) lysine dipeptide cross-links formed during setting generally correlates with the increase in gel strength (Imai, Tsukamasa, Sugiyama, Minegishi, & Shimizu, 1996). The formation of nondisulfide covalent cross-links catalysed by an endogenous TGase resulted in the polymerization of myosin (Seki et al., 1990; Kimura et al., 1991; Benjakul & Visessanguan, 2003). Plasma proteins are the promising sources of soluble proteins, which have been recognized as a useful ingredient in cooked meat products due to its excellent gelling properties (Caldironi & Ockerman, 1982). Additionally, it has been reported to exhibit proteinase inhibitory activity and gel strengthening ability during heat-induced gelation of surimi (Seymour, Peter, Morrissey, & An, 1997; Benjakul & Visessanguan, 2000; Visessanguan, Benjakul, & An, 2000) From our previous study, chicken plasma protein (CPP) was able to enhance the gel strength by acting as filler in surimi gel matrix and also as protease inhibitor (Rawdkuen, Benjakul, Visessanguan, & Lanier, 2004a,b). To improve the gel strength of surimi, the appropriate addition of CPP in combination with proper setting induced by either endogenous or plasma TGase could be a promising approach. However, no information regarding cross-linking activity of chicken plasma has been reported. Therefore, the objective of this study was to determine the cross-linking activity of chicken plasma towards bigeye snapper muscle and surimi proteins and to study the combination effect of CPP and setting on surimi gel properties.

2. Materials and methods 2.1. Chemicals Trisodium citrate and sodium chloride were purchased from Merck (Darmstadt, Germany). Adenosine 50 -triphosphate (ATP), b-mercaptoethanol (bME), ethylene glycol-bis (b-aminoethyl ether) N, N, N0 , N0 -tetra acetic acid (EGTA), thrombin from bovine plasma and L-tyrosine were obtained from Sigma Chemical Co. (St Louis, MO, USA). Sodium dodecyl sulfate (SDS), N, N, N0 , N0 -tetramethyl ethylene diamine (TEMED) and Coomassie Blue R-250 were obtained from Bio-Rad Laboratories (Hercules, CA, USA). 2.2. CPP preparation Chicken blood was obtained from a slaughterhouse in Hat Yai, Songkhla, Thailand. During collection, onetenth volume of trisodium citrate (3.8 g/100 ml) was added to prevent coagulation. The blood was centrifuged twice at 1500  g for 15 min at 4 1C to remove red blood cells using a Sorvall Model RC-B Plus centrifuge

(Newtown, CT, USA). The supernatant was then freezedried and kept at 18 1C until used. 2.3. Natural actomyosin (NAM) preparation NAM was prepared from bigeye snapper muscle according to the method of Nowsad, Kanoh, and Niwa (1994). Briefly, fish muscle (100 g) was homogenized with 500 ml of cold NaH2PO4 (3.38 mmole/l), Na2HPO4 (15.5 mmole/l) (pH 7.5) using an IKA homogenizer (Selangor, Malaysia) at a speed of 11,000 rpm for 2 min.The homogenate was centrifuged at 6000  g for 10 min, and washed for totally six times. NAM pellet was solubilized in a 500 ml of KCl (0.8 mole/l), KH2PO4 (3.38 mmole/l), Na2HPO4 (20.5 mmole/l) (pH 8.0) with continuous stirring. NAM was precipitated with four volumes of water. The mixture was centrifuged at 15,000  g for 30 min and NAM pellet was collected. All the procedures were done in a cold room at 4 1C. 2.4. Myosin preparation Myosin was extracted according to the method described by Martone, Busconi, Folco, Trucco, and Sanchez (1986) with a slight modification. Bigeye snapper fillets was finely chopped and added with ten volumes of buffer A (0.10 mole/l KCl, 0.02 g/100 ml NaN3 and 20 mmole/l Tris–HCl, pH 7.5). After incubation on ice for 10 min with occasional stirring, washed muscle was recovered by centrifugation at 1000  g for 10 min.The pellet was suspended in five volumes of buffer B (0.45 mole/l KCl, 5 mmole/l bME, 0.2 mole/l Mg(CH3COO)2, 1 mmole/l EGTA and 20 mmole/l Tris–maleate, pH 6.8), and ATP was added to a final concentration of 10 mmole/l. The mixture was kept on ice for 1 h and centrifuged at 10,000  g for 15 min. Supernatant was recovered, and 25 volumes of NaHCO3 (1 mmole/l) were slowly added. The mixture was incubated for 15 min on ice. Precipitated myosin was collected by centrifugation at 12,000  g and resuspended gently with five volumes of buffer C (0.5 mole/l KCl, 5 mmole/l bME and 20 mmole/l Tris–HCl, pH 7.5), followed by the addition of three volumes of NaHCO3. MgCl2 (1 mmole/l) was added to obtain the final concentration of 10 mmole/l. The mixture was incubated overnight and centrifuged at 22,000  g for 15 min. Myosin was recovered as the pellet. The purity of extracted myosin was estimated by SDS-PAGE. 2.5. Study on the effect of CPP in combination with CaCl2 and thrombin on surimi gel properties Frozen surimi from bigeye snapper grade SA (breaking force of 600–800 g; deformation of 12–14 mm)

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obtained from Man A Frozen Food Co, Ltd. (Songkhla, Thailand) was used. Frozen surimi was partially thawed at 4 1C for 2–3 h, cut into small pieces and chopped by a Moulinex Masterchef 350 mixer (Paris, France). Surimi paste was added with CPP (0.5 g/100 g) and CaCl2 (10 mmole/kg) in presence of various levels of thrombin (0, 100, 150 and 200 units/g CPP). Surimi paste added with CPP (0.5 g/100 g) or with CaCl2 (10 mmole/kg) was used as the controls. The moisture of the mixture was adjusted to 80 g/100 g with iced water and NaCl (2.5 g/100 g) was added. The mixture was chopped for 4 min.The surimi sol was stuffed into polyvinylidine casing with a diameter of 2.5 cm and both ends were sealed tightly. The sol was incubated at 40 1C for 30 min, followed by heating at 90 1C for 20 min in a temperature-controlled water bath (Memmert, Schwabach, Germany). These gels were referred to as ‘‘kamaboko gel’’. For directly heated gel, surimi sol was subjected to heating at 90 1C for 20 min. After heating, all gels were cooled in iced water for 30 min and stored at 4 1C overnight prior to analysis. 2.6. Determination of surimi gel compositions and properties 2.6.1. Texture analysis Texture analysis of surimi gels was carried out using a Model TA-XT2 texture analyser (Stable Micro System, Surrey, UK). Gels were equilibrated at room temperature (28–30 1C) before analysis. Five cylindrical samples (2.5 cm in length) were prepared and tested. Breaking force (strength) and deformation (cohesiveness/elasticity) were measured by the texture analyser equipped with a cylindrical plunger (5 mm diameter, 60 mm/min depression speed). 2.6.2. Determination of autolysis in surimi gel To 2 g of finely chopped gel samples, 18 ml of TCA (5 g/100 ml) were added and homogenized for 2 min using an IKA homogenizer at a speed of 11,000 rpm.The homogenate was incubated at 4 1C for 1 h and centrifuged at 8000  g for 5 min.TCA-soluble peptides in the supernatant were measured according to the Lowry method (Lowry, Rosenbrough, Farr, & Randall, 1951) and expressed as mmole tyrosine/g sample (Morrissey, Wu, Lin, & An, 1993). 2.6.3. Protein solubility Solubility of surimi gel proteins was determined as described by Benjakul, Visessanguan, and Srivilai (2001). Finely chopped gel sample (1 g) was solubilized with 20 ml of Tris–HCl, (20 mmole/l) pH 8.0 containing SDS (1 g/100 ml), b-mercaptoethanol (2 g/100 ml) and urea (8 mole/l). The mixture was homogenized for 1 min, boiled for 2 min and stirred for 4 h at room temperature

355

(28–30 1C) using a magnetic stirrer (IKA-Werke, Staufen, Germany). The mixture was centrifuged at 10,000  g for 30 min. A 2 ml of cold trichloroacetic acid (TCA) (50 g/100 ml) was added to 10 ml of supernatant. The mixture was kept at 4 1C for 18 h prior to centrifugation at 10,000  g for 20 min.The precipitate was washed with TCA (10 g/100 ml), and solubilized in NaOH (0.5 mole/l). Protein concentration was determined by the Biuret method (Robinson & Hodgen, 1940). Solubility of protein in surimi samples was expressed as the percentage of total protein in surimi gels solubilized directly in NaOH (0.5 mole/l). 2.6.4. SDS-PAGE SDS-PAGE analysis was performed according to the method of Laemmli (1970). To 2 g of sample, 18 ml of SDS solution (5 g/100 ml) was added. The mixture was then homogenized using an IKA Labortechnik homogenizer at a speed of 11,000 rpm for 1 min.The homogenates was incubated at 85 1C in a temperaturecontrolled water bath for 1 h to dissolve total proteins. The sample was centrifuged at 10,000  g for 5 min to remove undissolved debris. Solubilized samples were mixed at 1:1 (v/v) ratio with the sample buffer (0.5 mole/ l Tris–HCl, pH 6.8 containing SDS (4 g/100 ml), glycerol (20 g/100 ml) and bME (10 g/100 ml)) and boiled for 3 min.The samples (20 mg protein) were loaded into the polyacrylamide gel made of 10% 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 electrophoresis system (Bio-Rad Laboratories, Inc, Richmond, CA, USA). After separation, the proteins were stained with Coomassie Brilliant Blue R250 (0.02 g/100 ml) in methanol (50 ml/100 ml) and acetic acid (7.5 ml/100 ml) and destained with methanol (50 ml/100 ml) and acetic acid (7.5 ml/100 ml), followed by methanol (5 ml/100 ml) and acetic acid (7.5 ml/ 100 ml). 2.6.5. Scanning Electron Microscopy (SEM) Microstructure of directly heated gel and kamaboko gel without and with 10 mmole CaCl2/kg or 0.5 g CPP/ 100 g in combination with 10 mmole CaCl2/kg and 200 units of thrombin were determined. Samples with a thickness of 2–3 mm were fixed with glutaraldehyde (2.5 ml/100 ml) in phosphate buffer (0.2 mole/l) (pH 7.2). The samples were then rinsed for 1 h in distilled water before being dehydrated in ethanol with a serial concentration of 50, 70, 80, 90 and 100 ml/100 ml. Dried samples were mounted on a bronze stub and sputtercoated with gold (Sputter coater SPI-Module, PA, USA). The specimens were observed with a SEM (JEOL JSM-5800 LV, Tokyo, Japan) at an acceleration voltage of 10 kV.

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3. Results and discussion 3.1. Effects of CPP in combination with thrombin and/or CaCl2 on textural properties of surimi gels The highest breaking force and deformation of kamaboko gel from bigeye snapper surimi were obtained when CPP (0.5 g/100 g) was added in combination with CaCl2 (10 mmole/kg) and 150–200 units of thrombin/g CPP (Fig. 1). Addition of 0.5 g CPP/100 g or 10 mmole CaCl2/kg resulted in 33.7% and 62.3% increase in breaking force, respectively, compared with the control kamaboko gel (without additives). Breaking force of surimi gel increased by 90.6% and 103.8% with the addition of 0.5g CPP/100 g and 10 mmole CaCl2/kg or the combination of 0.5g CPP/100g, 10 mmole CaCl2/ kg and 200 units of thrombin/g CPP, respectively, 1800 1600 1400

f

c

800

b

600

a

400

10mM CaCl2

CPP+ CaCl2

T100

T150

c

d

d

d

10mM CaCl2

CPP+ CaCl2

T100

T150

T200

5% CPP c

5% CPP

0

18 16

Data were subjected to analysis of variance (ANOVA). Comparison of means was carried out by Duncan’s multiple-range test (Steel & Torrie, 1980).

f

d

1000

(A)

Deformation (mm)

14

d

b

12 a

10 8 6 4

(B)

T200

0

DH

2 kamaboko

2.9. Statistical analysis

e

200

2.8. Study on the effect of TGase inhibitors on protein cross-linking Surimi paste with and without CPP (0.5 g/100 g) was prepared as mentioned above. EGTA was added to the surimi paste to obtain a final concentration of 10 and 20 mmole/kg, whereas NH4Cl with a final concentration of was 125 and 250 mmole/kg was used. A control sample (without inhibitor) was also prepared. All gel samples were incubated at 40 1C for 30 min, followed by heating at 90 1C for 20 min. The gels were then subjected to analysis for the textural properties and protein patterns.

e

1200

DH

NAM paste was added with CPP (0.5 g/100 g) in presence or absence of CaCl2 (10 mmole/kg). NAM containing 0 and 10 mmole CaCl2/kg was used as the controls. Addition of CPP at a level of 0.5 g/100 g resulted in the highest breaking force and deformation (Rawdkuen et al., 2004a). For CaCl2, the level of 10 mmole/kg was found to increase the breaking force and deformation of kamaboko gels of bigeye snapper (Benjakul & Visessanguan, 2003). To study the effect of thrombin on cross-linking activity of CPP, NAM added with CPP (0.5 g/100 g) and CaCl2 (10 mmole/kg) was mixed with thrombin at various concentrations (100, 150 and 200 units/g CPP). All samples were incubated at 40 1C for 30 min. Cross-linking of proteins was visualized by SDS-PAGE using 10% running gel and 4% stacking gel. For myosin cross-linking study, myosin was diluted with buffer C to obtain the protein content of 10 mg/ml. CPP was added into myosin solution to obtain the final concentration of 3 g/100 g protein, which was equivalent to the CPP concentration used in surimi. The reaction mixtures were prepared in the same manner with those described for NAM cross-linking study. To study the effect of setting time on NAM and myosin cross-linking, sample treatment exhibiting the highest gel strength was chosen. Reaction mixture (CPP (0.5 g/100 g), CaCl2 (10 mmole/kg) and 200 units of thrombin/g CPP for NAM; CPP (3 g/100 g), CaCl2 (10 mmole/kg) and 200 units of thrombin/g CPP for myosin) was incubated at 40 1C for 0, 10, 30, 60, 90, and 120 min. All samples were analysed for protein crosslinking by SDS-PAGE.

Analysis was performed using a SPSS package (SPSS 8.0 for windows, SPSS Inc, Chicago, IL).

kamaboko

2.7. Study on the effect of CPP on NAM and myosin cross-linking

Breaking force (g)

356

Fig. 1. Breaking force (A) and deformation (B) of surimi gel added with CPP in combination with thrombin and/or CaCl2. Bars represent the standard deviation from five determinations. Different letters indicate significant differences (Po0.05). DH: directly heated gel.

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compared with the control kamaboko gel. Marked increase in breaking force was found in presence of both CaCl2 and thrombin, especially with the higher level of thrombin (T100–T200) (Po0.05). However, no differences in deformation were observed with different levels of thrombin used (P40.05). Breaking force and deformation of kamaboko gels were 73.6% and 29.6% higher than those of directly heated gel, respectively. This indicated the essential role of setting in gel strengthening. Benjakul et al. (2001) found that kamaboko gel from bigeye snapper in the presence of porcine plasma protein (0.5 g/100 g) showed the maximum breaking force and deformation with concomitant increased nondisulfide covalent bonds catalysed by both endogenous TGase in fish muscle and porcine plasma TGase. The increase in breaking force of kamaboko gel added with CPP, CaCl2 and thrombin might result from activated-factor XIII, which catalysed the covalent cross-linking of MHC via intermolecular e(g-glutamyl) lysine bond (Benjakul et al., 2001). Furthermore, addition of CaCl2 could activate endogenous TGase in bigeye snapper surimi (Benjakul, Visessanguan, & Chantarasuwan, 2004). Benjakul and Visessanguan (2003) reported that setting at either 25 or 40 1C, prior to heating at 90 1C, resulted in the increase in both breaking force and deformation and also confirmed that endogenous TGase played an important role in gel enhancement of surimi from bigeye snapper. In presence of CPP alone, it was noted that breaking force and deformation of surimi gel increased (Po0.05). The filler effect of plasma protein in surimi gel matrix was suggested to contribute to strengthening of surimi gel (Rawdkuen et al., 2004a). Foegeding, Dayton, and Allen (1986) reported that the myosin and fibrinogen interacted to form a gel, which was stronger than the gel produced by the individual protein. A fibrinogen–thrombin mixture was very reactive in forming a strong structure (Yoon, Kim, & Park, 1999). Mixing fibrinogen–thrombin into low-grade surimi-enhanced gel properties. Jiang and Lee (1992) reported that pig plasma Factor XIII was activated by thrombin and Ca2+ and activated Factor XIIIa catalysed the covalent crosslinking of MHC. The addition of crude plasma Factor XIII (including thrombin) substantially increased the gel strength of minced mackerel. Plasma TGase can catalyse the formation of glutamyl–lysine bonds in myosin, myosin/actin, myosin/fibronectin, fibrin/fibronectin and fibrin/actin (Cohen, Young–Bandala, Blankenberge, Siefring, & Siefring, 1979; Kahn & Cohen, 1981). 3.2. Effects of CPP in combination with thrombin and/or CaCl2 on protein solubility and TCA-soluble peptides of surimi gels Kamaboko gels added with CPP, CaCl2 and thrombin had the lowest protein solubility. However, no signifi-

357

Table 1 Protein solubility and TCA-soluble peptides of surimi gels added with chicken plasma protein in combination with thrombin and/or CaCl2 Samples

Solubility (%)

Tyrosine (mmole/g sample)

Kamaboko gel (K) Directly heated gel K+0.5% CPP K+10 mmole CaCl2/kg K+CPP/CaCl2 K+CPP/CaCl2/100NIH K+CPP/CaCl2/150NIH K+CPP/CaCl2/200NIH

67.1470.41acb 78.0972.76d 66.8472.59c 33.5771.15b 32.9171.11b 28.4570.98a 27.7671.31a 27.3871.48a

1.0570.01f 0.8570.02d 0.1870.02a 0.9470.02e 0.2470.01b 0.2670.02bc 0.2770.02c 0.2970.03c

Values are given as mean 7 SD from triplicate determinations. Different letters in the same column indicate significant differences (Po0.05). a

b

cant differences in solubility were observed in gels with different levels of thrombin added (P40.05) (Table 1). These results indicated that addition of CPP, CaCl2 and thrombin in combination with setting effectively induced a large number of protein–protein interactions stabilized by nondisulfide covalent bond, which cannot be destroyed by the mixture containing sodium dodecyl sulfate, urea, and bME. Nondisulfide covalent bond formed might contribute to the strengthening of gel matrix. When comparing the solubility between directly heated gel and kamaboko gels, the former had a greater solubility than the latter. Therefore, setting at 40 1C for 30 min, followed by heating at 90 1C for 20 min-induced protein cross-linking. With the addition of CPP, CaCl2 and thrombin, the solubility of kamaboko gel decreased to less than 30%. Apart from activation of factor XIII in plasma, thrombin might hydrolyse protein to some extent, resulting in the exposure of reactive groups for cross-linking induced by both endogenous and plasma TGase. TGase catalyses the conversion of soluble protein to insoluble high molecular polymers through formation of covalent cross-links (De Backer-Royer, Traore, & Meunier, 1992; Traore & Meunier, 1992). The lowest TCA-soluble peptide was obtained in kamaboko gel added with CPP (Table 1). This result suggested that CPP had inhibitory activity towards degradation of protein. The degradation probably occurred during setting at high temperature (40 1C), which was close to the temperature of gel weakening (50–60 1C) mainly caused by indigenous proteases (An, Peters, & Seymour, 1996; Kang & Lanier, 2000; Benjakul, Visessanguan, & Leelapongwattana, 2003). When comparing the degradation between directly heated gel and kamaboko gels, the former contained the lower amount of TCA-soluble peptides than the latter. Slight increase in TCA-soluble peptides in surimi gel added with thrombin was possibly owing to thrombin cleavage of an Arg37–Gly38 peptide near the amino-terminals of the a-chains of Factor XIII

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(Takahashi, Takahashi, & Putnam, 1986; Ichinose & Davie, 1988). Nevertheless, partial hydrolysis caused by thrombin might contribute to the increased cross-linking induced by TGase. 3.3. Effects of CPP in combination with thrombin and/or CaCl2 on protein pattern and microstructure of surimi gels Protein patterns of different surimi gels are depicted in Fig. 2. The kamaboko gels added with 10 mmole CaCl2/kg (lane 6A) showed the lowest intensity of MHC band when compared with other treatments. When CPP (0.5 g/100 g) was added (lane 5A), the higher amount of MHC was retained. This indicated interfering effect of CPP on MHC cross-linking. No MHC band was retained in gels added with CPP (0.5 g/100 g) in combination with CaCl2 (10 mmole/kg) and thrombin

MHC

at various concentrations (lanes 4–6B), surimi gel added with CPP and CaCl2 (lane 3B) and the gel added with 10 mmole CaCl2/kg (lane 6A). This indicated that crosslinking activity of MHC in surimi was mainly caused by endogenous TGase, which was activated by Ca2+. As a result, MHC was almost disappeared in the kamaboko gel added with CaCl2, regardless of CPP or thrombin addition. However, no marked changes in actin and tropomyosin were observed in all samples. The microstructure of bigeye snapper surimi gels prepared with different conditions was visualized by SEM as shown in Fig. 3. The kamaboko gel without additive (A) had a coarse and disordered cross-link structure with large cavities. For directly heated gel (B), more irregularly disordered and coarser fibrillar structures were observed. The fine cross-link structure of kamaboko was observed when added with 10 mmole CaCl2/kg (C). When 0.5 g CPP/100 g, 10 mmole CaCl2/ kg and 200 units of thrombin were added (D), the finer and more ordered structure became more evident. The result suggested that activation of TGase by CaCl2 addition improved the alignment and aggregation of protein, leading to the fine and ordered structure. CPP was possibly localized in the cavities, resulting in the finer structure of gel network.

Actin Tropomyosin

3.4. Effect of CPP in combination with setting on natural actomyosin cross-linking The effect of CPP in combination with CaCl2, thrombin and setting on cross-linking of bigeye snapper

(A)

2

1

3

4

5

6

MHC

Actin Tropomyosin

(B) 1

2

3

4

5

6

Fig. 2. SDS-PAGE patterns of surimi gels added with CPP in combination with thrombin and/or CaCl2. Lane 1: CPP; lane 2: surimi sol; lane 3A: directly heated gel; lane 4A: kamaboko gel; lane 5A: kamaboko gel added with 0.5 g CPP/100 g; lane 6A: kamaboko gel added with 10 mmole CaCl2/kg; lane 3B: kamaboko gel added with 0.5 g CPP/100 g and 10 mmole CaCl2/kg; lanes 4–6B: kamaboko gel added with 0.5g CPP/100g, 10 mmole CaCl2/kg and 100,150 and 200 units of thrombin/g CPP, respectively. MHC: myosin heavy chain.

Fig. 3. Electron microscopic image of surimi gels from bigeye snapper (magnification: 10,000X). A: kamaboko gel without additive; B: directly heated gel; C: kamaboko gel added with 10 mmole CaCl2/kg; D: kamaboko gel added with 0.5 g CPP/100 g, 10 mmole CaCl2/kg and 200 units of thrombin/g CPP.

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NAM is demonstrated in Fig. 4. The intensity of MHC band noticeably decreased when all NAM paste was added with CaCl2, regardless of CPP and thrombin addition. Decrease in MHC was apparently observed in NAM added with CaCl2 (lane 3). Slight decrease in MHC was obtained in the gel with CPP addition (lane 2). This result suggested that decrease in MHC in bigeye snapper NAM might cause by endogenous TGase rather than plasma TGase. Benjakul et al. (2004) reported that addition of sarcoplasmic fraction from bigeye snapper into NAM in combination with setting at 40 1C resulted in the cross-linking of MHC and higher cross-linking were observed when the higher amount of sarcoplasmic fraction and extended setting time were used. For NAM added with CPP, the presence of thrombin in the reaction mixture could induce cross-linking of MHC. Thrombin might hydrolyse and expose some reactive group of amino acid, which acts as an acyl acceptor or acyl donor in the formation of e-(g-glutamyl)lysyl crosslinking. Furthermore, activated plasma TGase possibly showed a synergetic effect with endogenous TGase on cross-linking of bigeye snapper NAM. As indicated in Fig. 5, the MHC in bigeye snapper NAM slightly decreased after 10 min of incubation with CPP, CaCl2 and thrombin at 40 1C (lane 3) and the decrease was more evident when the incubation time was extended to 2 h (lane 7). No distinct change in the density of actin was obtained in all samples. Myosin contains both acyl acceptor (g-glutamine) and acyl donor (e-lysine side chain) and is considered to be a good substrate for TGase (Gorman & Folk, 1980). Kim, Carpenter, Lanier, and Wicker (1993) reacted guinea pig liver TGase with beef actomyosin at 35 1C for 10–120 min and found that myosin monomer concen-

MHC

Actin

1

2

3

4

5

6

7

Fig. 4. SDS-PAGE patterns of NAM paste added with CPP in combination with thrombin and/or CaCl2. Lane 1: NAM; lane 2: NAM added with 0.5 g CPP/100 g; lane 3: NAM added with 10 mmole CaCl2/kg; lane 4: NAM added with 0.5 g CPP/100 g and 10 mmole CaCl2/kg: lanes 5–7: NAM added with 0.5 g CPP/100 g, 10 mmole CaCl2/kg and 100, 150, 200 units of thrombin/g CPP, respectively. MHC: myosin heavy chain.

359

MHC

Actin

1

2

3

4

5

6

7

Fig. 5. SDS-PAGE patterns of NAM paste added with CPP in combination with thrombin and/or CaCl2 as affected by incubation time at 40 1C. Lane 1: NAM; Lanes 2–7 NAM added with 0.5 g CPP/ 100 g, 10 mmole CaCl2/kg and 200 units of thrombin/g CPP incubated at 40 1C for 0, 10, 30, 60, 90 and 120 min, respectively. MHC: myosin heavy chain.

tration gradually decreased with simultaneous appearance of myosin polymers as the reaction time increased. No degradation occurred in bigeye snapper NAM with CPP addition during incubating process (data not shown). The cross-linking of MHC might have a steric hindrance to prevent proteolysis. 3.5. Effect of TGase inhibitor on textural properties and electrophoretic patterns of surimi proteins The effects of TGase inhibitors on textural properties were investigated. The lowest breaking force and deformation were observed in kamaboko gel with and without CPP when 20 mmole EGTA/kg was added (Fig. 6A and B). Breaking force of kamaboko gels decreased by 45% and 60% with the addition of 250 mmole NH4Cl/kg and 20 mmole EGTA/kg, respectively, when compared with the control kamaboko gel. Both EGTA and NH4Cl showed an inhibitory effect on gel formation in a concentration-dependent manner. During acyltransfer reaction between g-carboxyamide groups of glutamine residues and primary amine, ammonia is generated and an excess amount of ammonium ion should prevent further progress of the reaction (Ashie & Lanier, 2000). EGTA inhibits endogenous TGase activity through chelating Ca2+, a divalent cation required for the activation of tissue TGase (Folk, 1980). These results suggested that endogenous TGase played an essential role in setting of surimi gel with and without CPP addition. The higher breaking force and deformation were obtained in all samples when 0.5 g CPP/100 g was added when compared with the sample without CPP addition (Fig. 6A and B). Without TGase inhibitors, kamaboko gel added with 0.5 g CPP/100 g showed 51.7% and 7%

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Fig. 6. Breaking force (A) and deformation (B) of surimi gels added with/without CPP in the presence of TGase inhibitor. Bars represent the standard deviation from five determinations. Different letters indicate significant differences (Po0.05). DH: directly heated gel.

higher in breaking force and deformation than that without CPP addition. In the presence of both CPP and endogenous TGase inhibitor, especially 20 mmole EGTA/kg, breaking force and deformation still increased. These results revealed that CPP showed an enhancing effect on kamaboko gel from bigeye snapper, which presumably acted as filler or binder in surimi gel network. This result confirmed our previous study (Rawdkuen et al., 2004a). Regardless of CPP addition, MHC band intensity of samples mixed with EGTA and NH4Cl was greater than that of the control (without TGase inhibitor addition) (Fig. 7). From the result, intensity of MHC in kamaboko gel was more retained when CPP was added (Fig. 7B), compared with that of gel without CPP addition (Fig. 7A). This result confirmed that CPP showed an interfering effect on MHC cross-linking. Negligible TCA-soluble peptides content also revealed that proteolysis was inactivated in the sample with 0.5 g

1

2

3

4

5

6

7

Fig. 7. SDS-PAGE patterns of surimi gels without CPP (A) and with CPP (B) in the absence and presence TGase inhibitors. Lane 1: surimi; lane 2: directly heated gel; lane 3: kamaboko gel; lanes 4–5: kamaboko gel added with 10 and 20 mmole EGTA/kg, respectively; lanes 6–7: kamaboko gel added with 125 and 250 mmole NH4Cl/kg, respectively. MHC: myosin heavy chain. DH: directly heated gel.

CPP/100 g addition (data not shown). The results suggested that TGase involved in cross-linking of protein during setting of surimi sol either with or without CPP addition. 3.6. Effect of CPP in combination with setting on myosin cross-linking Myosin from bigeye snapper muscle was incubated with CPP, CaCl2, CPP/CaCl2 or CPP/CaCl2/thrombin at 40 1C for 30 min (Fig. 8). With the addition of 3.0 g CPP/100 g, no myosin cross-linking was observed for all treatments. The results indicated that CPP could not induce myosin cross-linking, even in the presence of both CaCl2 and thrombin, the factor XIII activators (Fig. 8). In the presence of CPP, CaCl2 and thrombin, no visible change in MHC intensity of myosin incubated at 40 1C was observed up to 120 min (Fig. 9). These results indicated that CPP had no cross-linking activity under the condition tested. It has been known that activation of the plasma zymogens requires the presence of both thrombin and calcium ions (Lorand & Konishi, 1964). From the result, the lack of cross-linking activity of CPP might result from

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added with CPP and CaCl2 as evidenced by the increased breaking force (Fig. 1) and lowered solubility (Table 1). Nevertheless, no cross-linking of myosin was observed with the addition of thrombin in combination with CPP and CaCl2 in pure myosin system. Thus, it was suggested that thrombin might partially hydrolyse the muscle proteins, leading to the conformational changes in fashion which endogenous TGase induced cross-linking effectively. As a result, the increased gel strength was found in the surimi, but not in pure myosin.

4. Conclusion

7

Fig. 8. SDS-PAGE pattern of myosin added with CPP in combination with thrombin and/or CaCl2. Lane 1: myosin; lane 2: myosin added with 3 g CPP/100 g; lane 3: myosin added with 10 mmole CaCl2/kg; lane 4: myosin added with 3 g CPP/100 g and 10 mmole CaCl2/kg: lanes 5–7: myosin added with 3 g CPP/100 g, 10 mmole CaCl2/kg and 100, 150, 200 units of thrombin/g CPP, respectively. MHC: myosin heavy chain.

MHC

Addition of CPP in combination with CaCl2 and setting effectively induced the cross-linking of surimi proteins as indicated by the increase in breaking force and deformation and the formation of ordered microstructure. CPP had no cross-linking activity and endogenous TGase played an important role in gel strengthening of surimi added with CPP.

Acknowledgement Financial support from Thailand Research Fund under the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0039/2544) to Saroat Rawdkuen.

References

1

2

3

4

5

6

7

Fig. 9. SDS-PAGE patterns of myosin added with CPP in combination with thrombin and/or CaCl2 as affected by incubation time at 40 1C. Lane 1: myosin; Lanes 2–7 myosin added with 3 g CPP/100 g, 10 mmole CaCl2/kg and 200 units of thrombin/g CPP incubated at 40 1C for 0, 10, 30, 60, 90 and 120 min, respectively. MHC: myosin heavy chain.

the insufficient TGase activators to convert zymogens into active enzyme. Furthermore, CPP TGase might require additional activating factors, which are different from mammal plasma TGase. Credo, Curtis, and Lorand (1978) reported that plasma Factor XIII is activated at nonphysiologically high concentration (4100 mmole/l) of CaCl2. For human placenta Factor XIII, the maximum activity was reached at about 150 mmole CaCl2/kg (De Backer-Royer, Traore, & Meunier, 1992). Cooke and Holbrook (1974) also reported that low thrombin concentration was not enough to completely convert the zymogens into Factor XIIIa. From the result, thrombin addition showed the strengthening effect on the surimi gel

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