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Effect of porcine plasma protein and setting on gel properties of surimi produced from fish caught in Thailand
ARTICLE IN PRESS
Lebensm.-Wiss. u.-Technol. 37 (2004) 177–185
Effect of porcine plasma protein and setting on gel properties of surimi produced from fish caught in Thailand Soottawat Benjakula,*, Wonnop Visessanguanb, Chakkawat Chantarasuwana b
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 (BIOTEC), 113 Paholyothin Rd., Klong 1, Klong Luang, Pathumthani 12120, Thailand
Received 6 January 2003; accepted 24 July 2003
Abstract Effects of porcine plasma protein (PPP) and high temperature setting on gel properties of surimi from bigeye snapper, bigeye croaker, threadfin bream and barracuda were investigated. PPP was effective in increasing breaking force and deformation of kamaboko gels set at 40 C for 30 min and heated at 90 C for 20 min. The optimum levels of PPP were 0.5, 0.5, 1.5 and 1.5 g/100 g and the optimum setting times were 2, 1.5, 1.5 and 2 h for bigeye snapper, bigeye croaker, threadfin bream and barracuda surimi, respectively. However, the addition of PPP significantly decreased whiteness (Po0:05). An increase in gel-forming ability of surimi with PPP coincided with a decrease in solubility in mixture of SDS, urea and b-mercaptoethanol, indicating the formation of nondisulfide covalent bond induced by both endogenous and plasma transglutaminase. The results supported that PPP improve the gelation of surimi in combination with setting. r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Porcine; Plasma; Blood; Setting; Suwari; Surimi; Gelation; Tropical fish
1. Introduction Surimi gel is a three-dimensional muscle protein network. Textural characteristics expressed in terms of gel strength is the primary determinant for surimi quality and price (Lanier, 1992). The improvement of textural quality of surimi or surimi based products has been attempted by addition of protein additives (Chung & Lee, 1990; Morrissey, Wu, Lin, & An, 1993; Park, 1994; Benjakul, Visessanguan, & Srivilai, 2001a). Additionally, starch and other polysaccharides have been used as ingredient in surimi (Kim & Lee, 1987; Niwa, Tsujimoto, & Kanoh, 1992). For surimi processing, wheat and soybean proteins as well as starch are used as additives not only to increase gel strength but also to increase the volume (Yamashita, 1991; Yoon & Lee, 1993). To increase the gel strength of surimi, gelstrengthening ingredients such as ascorbic acid or dehydroascorbic acid were found to increase the gel strength of surimi due to the oxidation of sulfhydryl group in the protein matrix (Yoshinaka, Shiraishi, & *Corresponding author. Tel.: +66-74-286334; fax: +66-74-212889. E-mail address: [email protected] (S. Benjakul).
Ikeda, 1972; Lee, Lee, Chung, & Lavey, 1992). Protein additives have been also used as proteinase inhibitor to improve the gel strength of surimi by controlling proteolytic activities during surimi seafood production (Morrissey et al., 1993). The proteolytic degradation of myofibrillar proteins has a detrimental effect on surimi quality and substantially lowers the gel strength (An, Peters, & Seymour, 1996). The most commonly used food grade inhibitors are beef plasma protein, egg white and potato powder (Morrissey et al., 1993). Degree of proteinase inhibition was dependent upon the types of additives used (Morrissey et al., 1993; Reppond & Babbitt, 1993; Weerasinghe, Morrissey, & An, 1996). Apart from the use of additives, gel strengthening of surimi can be achieved by subjecting sols to setting at the temperatures ranging from 0 C to 40 C prior to heating (Kamath, Lanier, Foegeding, & Hamann, 1992; An et al., 1996). During setting, myosin heavy chain (MHC) undergoes polymerization via formation of nonsulfide covalent cross-links, catalysed by an endogenous transglutaminase (TGase) (Kimura et al., 1991; Kumazawa, Numazawa, Seguro, & Motoki, 1995; Benjakul & Visessanguan, 2003).
0023-6438/$30.00 r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2003.07.002
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Porcine blood is an abundant byproduct in the slaughtering process in Thailand at an estimated amount of 30,000 metric tons per year (Benjakul et al., 2001a). Blood plasma contains a variety of bioactive compounds including proteinase inhibitor and plasma transglutaminase (Benjakul et al., 2001a b). Porcine plasma at a level of 0.5 g/100 g was found to increase the breaking force and deformation of bigeye snapper surimi (Benjakul et al., 2001a). The fractionation of bioactive components in porcine plasma was an effective means to concentrate those compounds and minimize the discoloration caused by residual pigment in plasma (Benjakul, Visessanguan, & Srivilai, 2001c). Although PPP at a level of 0.5 g/100 g effectively increased the gel strength of surimi from bigeye snapper (Benjakul et al., 2001a), no information on the use of PPP in surimi produced from other fish species caught in Thailand. The gelling characteristic of surimi could vary with both intrinsic and extrinsic factors (Benjakul, Visessanguan, Ishizaki, & Tanaka, 2001d). Therefore, the effect of PPP addition on gelling characteristic of different surimi could vary with different surimi which require different optimum condition for PPP application. The appropriate use of PPP in combination with setting would be one of effective means to enhance the gel strength of surimi. The objective of this investigation was to study the effect of PPP in combination with setting on gel properties and changes in myofibrillar proteins in surimi produced from four tropical fish species, widely present in Thailand.
2. Materials and methods 2.1. Chemicals Sodium dodecyl sulfate (SDS), urea, trichloroacetic acid (TCA) and sodium hydroxide were purchased from Riedel-deHaen (Seelze, Germany). Trisodium citrate, bmercaptoethanol (b-ME) and N,N,N0 ,N0 -tetramethyl ethylene diamine (TEMED)were obtained from Sigma Co. (St. Louis, MO, USA). 2.2. Preparation of porcine plasma protein (PPP) Porcine blood was collected from a slaughter house in Hat Yai, Thailand. The blood was mixed with trisodium citrate solution (3.8 g/100 ml) at a ratio of 1:9 (v/v) to prevent blood coagulation and transported to the Department of Food Technology, Prince of Songkla University, Hat Yai. The blood was then centrifuged at 1000g for 30 min at 4 C using a Sorvall Model RC-B Plus centrifuge (Newtown, CT, USA) to remove red blood cells. The resultant supernatant was freeze-dried and kept at 4 C until used. The dry powder was referred to as porcine plasma protein (PPP).
2.3. Surimi and surimi gel preparation Frozen surimi (grade A) produced from threadfin bream (Nemipterus bleekeri), bigeye snapper (Priacanthus tayenus), baracuda (Sphyraena jello) and bigeye croaker (Pennahai macrophthalmus) were purchased from Man A Frozen Foods Co., Ltd. (Songkhla, Thailand) and kept at 20 C until used. To prepare the gel, frozen surimi was tempered for 30 min in the running water (25 C). The surimi was then cut into small pieces with an approximate thickness of 1 cm. The surimi was placed in the mixer (National Model MK-K77, Tokyo, Japan) and PPP at different concentrations (0, 0.25, 0.5, 0.75, 1, 1.5, 2 and 2.5 g/ 100 g) was added in combination with CaCl2 (50 mmol/ kg). The moisture was then adjusted to 80 g/100 g and salt (2.5 g/100 g; surimi weight basis) was added. The mixture was chopped for 5 min at 4 C and its temperature was kept below 10 C. The surimi sol was stuffed into the polyvinylidine casing (2.5 cm diameter) and both ends were closed tightly and subjected to setting at 40 C in a water bath (Memmert, Schwabach, Germany) for 2, 3, 1 and 1.5 h for bigeye snapper, bigeye croaker, threadfin bream and barracuda surimi, respectively (Benjakul, Visessanguan, & Chantarasuwan, unpublished data). After setting for designated time, the gels were heated at 90 C for 20 min in a water bath. The gels were then cooled in iced water (0–1 C) and stored at 4 C for 24 h before analysis. The gels obtained were referred to as ‘kamaboko gels’. PPP at a concentration, which rendered the highest breaking force and deformation, was chosen for further study. To investigate the effect of setting time on gel properties, surimi sol added with PPP at an optimum concentration was subjected to setting at 40 C for different time (0, 0.5, 1, 1.5, 2, 2.5, or 3 h), followed by heating and cooling as previously described and stored at 4 C for 24 h prior to analysis. 2.4. Texture analysis The breaking force (gel strength) and deformation (elasticity/deformability) were measured with a 5-mm diameter cylindrical plunger at 60 mm/min deformation rate using a texture analyser (Model TA-XT2, Stable Micro Systems, Surrey, England). Five cylinder-shaped samples of 2.5 cm in length were prepared and equilibrated at room temperature before testing. 2.5. Whiteness measurement Whiteness of surimi gels was measured using colorimeter JP7100F (Juki Corporation, Tokyo, Japan). The measurement was performed in five replications. The whiteness was calculated using the following
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2.9. Protein determination
equation (NFI, 1991): Whiteness ¼ 100 ½ð100 L Þ2 þ a þ b 1=2 : 2
2
2.6. Solubility studies The solubility of surimi gels in 20 mM Tris-HCl, pH 8.0 containing SDS (1 g/100 ml), urea (8 M) and b-ME (2 g/100 ml) was determined as described by Chawla, Venugopol and Nair (1996). The sample (1 g) was homogenized in 20 ml of solution for 1 min using homogenizer (IKA Labortechnik, Selangor, Malaysia). The homogenate was heated in boiling water (100 C) for 2 min and stirred at room temperature for 4 h. The resulting homogenate was centrifuged at 10,000g for 30 min using Sorvall Model RC-B plus centrifuge (Newtown, CT, USA). Protein in the supernatant (10 ml) was precipitated by the addition of trichloroacetic acid (TCA) (50 g/100 ml) to a final concentration of 10 g/100 ml. The mixture was kept at 4 C for 18 h and then centrifuged at 10,000g for 30 min. The precipitate was washed with TCA (10 g/100 ml) and solubilized in 0.5 M NaOH. To obtain the total amount of protein, gels were directly solubilized in 0.5 M NaOH. The protein content was measured using the Biuret test (Robinson & Hodgen, 1940). The solubility was expressed as percent of the total protein. 2.7. Determination of TCA soluble peptides TCA soluble peptides were determined according to the method described by Morrissey et al. (1993). Surimi gel (3 g) was homogenized with 27 ml of TCA (5 g/ 100 ml). The homogenate was kept in ice for 1 h and centrifuged at 5000g for 5 min. Soluble peptides in supernatant were measured and expressed as mmol tyrosine/g. 2.8. SDS-polyacrylamide gel electrophoresis (SDS-page) The protein pattern of surimi gel was analysed on SDS-PAGE according to the method of Laemmli (1970). To prepare the protein sample, 27 ml of SDS solution (5 g/100 ml) heated to 85 C was added into the sample (3 g). The mixture was then homogenized using homogenizer for 2 min. The homogenate was incubated at 85 C for 1 h to dissolve total proteins. The samples were centrifuged at 3500g for 20 min to remove undissolved debris. Protein concentration was determined according to the method of Lowry, Rosebrough, Farr, and Randall (1951) using bovine serum albumin as standard. SDS-PAGE gel was made of 10% running gel and 4% stacking gel. After separation, the proteins were fixed and stained with Coomassie Blue R-250.
Protein concentration was measured by the method of Lowry et al. (1951) using bovine serum albumin as standard. 2.10. Statistical analysis Analysis of variance (ANOVA) was performed to analyse the effect of PPP concentration and setting time in a completely randomized design. Mean comparisons were run by Duncan’s multiple range test (Steel & Torrie, 1980).
3. Results and discussion 3.1. Effect of PPP concentration on surimi gel properties Breaking force and deformation of kamaboko gels from four fish species added with different levels of PPP in combination with CaCl2 (50 mmol/kg) are shown in Fig. 1. Breaking force and deformation of gels generally increased as PPP was added up to 0.5 g/100 g for bigeye snapper and bigeye croaker surimi and up to 1.5 g/100 g for threadfin bream and barracuda surimi, respectively (Po0:05). Thereafter, breaking force of bigeye snapper surimi gel significantly decreased as PPP levels increased (Po0:05), however no marked changes were found in other surimi gels (P > 0:05). At a level of 0.5 g PPP/ 100 g, breaking force of gels from bigeye snapper and bigeye croaker surimi increased by 58.6% and 24.4%, respectively. For gels from threadfin bream and barracuda surimi added with 1.5 g PPP/100 g, breaking force increased by 62.2 and 79.1%, respectively. Deformation of surimi gels with the optimum level of PPP increased by 11.0 to 23.7%. The increased amount of PPP resulted in the decrease in gel-forming ability as observed by the decrease in breaking force. However, no changes in deformation of surimi gel from all species, except bigeye croaker, were observed with an increasing PPP concentration (P > 0:05). Because of the inhibitory activity of PPP against protease (Visessanguan, Benjakul, & An, 2000), PPP showed a great potential as surimi gel enhancer, however PPP should be used at optimum concentration since its components interfere with gelation process. Blood plasma comprises three main proteins including albumin, globulin and fibrinogen (Raeker & Johnson, 1995). These proteins at appropriate levels possibly enhanced protein–protein interaction, thereby strengthening gel network. In view of the optimum level of PPP varying from species to species, the interaction appears to be species specific. An excessive amount of PPP caused the dilution of muscle proteins, which is a major contributor for gel formation. As a consequence, it might interfere with the formation
ARTICLE IN PRESS S. Benjakul et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 177–185 1800 1600
Breaking force (g)
1400 1200 1000 800 600 400 200 0 0
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1
1.5
2
2.5
PPP concentration (%) 20
Deformation (mm)
15
10
5
0 0
0.5
1
1.5
2
2.5
PPP concentration (%)
Fig. 1. Breaking force and deformation of kamaboko gels added with different levels of PPP. Bars indicate the standard deviation from five determinations. Bigeye snapper (J), Bigeye croaker (W), Threadfin bream ( ), and Barracuda (m).
of a three-dimensional gel network between myosin molecules. Yamashita, Araki, and Seki (1996) report that the addition of casein at a level of 1 and 5 g/100 g decreased myosin content by 9% and 32%, respectively and resulted in the decreased gel strength of walleye pollack surimi. The interference of myosin–myosin interaction may be a major cause of the inferior gelation when casein was added in the surimi (Yamashita et al., 1996). PPP, a water-soluble protein, may interfere with protein–protein interaction (Yoon, Lee, & Hufnagel, 1991). In general, optimum PPP levels added into surimi varied, depending upon the species. From the result, optimal PPP level for gel enhancement of surimi from bigeye snapper and bigeye croaker was less than that of surimi from threadfin bream and barracuda. This was probably related to different intrinsic factors such as the presence of endogenous enzymes, e.g. proteinases and transglutaminase as well as the molecular properties of myofibrillar proteins. Endogenous proteinases, especially heat activated and heat stable proteinases, have been known to play a detrimental role in surimi gel
quality (Morrissey et al., 1993; Benjakul, Visessanguan, & Leelapongwattana, 2003). The most active proteinases in fish muscle, which can soften the surimi gel, vary with species (An et al., 1996: Choi, Cho, & Lanier, 1999). Since different surimi possibly contained the different proteinase activity, especially myofibril-associated proteinases, which still remained in the surimi after washing process, different level of PPP was required to inhibit the proteolytic activity. PPP showed the inhibitory activity toward trypsin, papain, digestive proteinases and modori-inducing proteinase from bigeye snapper (Benjakul, Visessanguan, & Srivilai, 2001b). The inhibitory component in PPP was identified as a protein with apparent MW of 60,000–63,000 Da on the inhibitory activity-stained gel (Benjakul & Visessanguan, 2000). Whiteness of kamaboko gels with different levels of PPP is depicted in Fig. 2. Whiteness of all kamaboko gels decreased steadily as PPP level increased. At the optimum level of PPP for each surimi gel strengthening, the whiteness decreased by 1.8%, 0.8%, 5.4% and 4.4% for surimi from bigeye snapper, bigeye croaker, threadfin bream and barracuda, respectively. Benjakul et al. (2001a) also reported that addition of PPP at a higher level to surimi from bigeye snapper resulted in the decrease in whiteness. Nevertheless, no effect of PPP fraction I-S (an enriched plasma transglutaminase fraction) on whiteness of surimi gel was observed, even added at higher concentrations (Benjakul et al., 2001c). Changes in solubility of kamaboko gels with PPP at different levels are shown in Fig. 3. A decrease in solubility was observed as PPP increased up to the optimum level and a further increase in PPP resulted in an increase in solubility to varying degree. The decrease in solubility mostly coincided with the increased breaking force and deformation. The subsequent increase in 90
85
Whiteness
180
80
75
70 0
0.5
1
1.5
2
2.5
PPP concentration (%)
Fig. 2. Whiteness of kamaboko gels added with different levels of PPP. Bars indicate the standard deviation from five determinations. Bigeye snapper (J), Bigeye croaker (W), Threadfin bream ( ), and Barracuda (m).
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myosin–myosin interaction that was induced by TGase. Although PPP at a higher concentration inhibited proteolysis more effectively as indicated by the decrease in TCA-soluble peptides (data not shown), a decrease in nondisulfide cross-linking was found as indicated by increased solubility. Thus, the balance between proteolytic inhibitory effect and the interfering effect on protein–protein interaction of PPP may be the important factor determining surimi gel enhancement. From the result, it can be inferred that PPP, a nonmuscle protein, should be added at an appropriate level to avoid the interfering effect on myofibrillar proteins, which are essential for gelation. The electrophoretic pattern of kamaboko gels with different levels of PPP is shown in Fig. 4. Myosin heavy chain (MHC) in directly heated gel (lane 3) decreased to some extent, compared to that found in surimi sol (lane 1). For the control kamaboko gel (without PPP and CaCl2) (lane 2) of surimi from all species, except threadfin bream, no MHC band was remained. Kamaboko gels from bigeye snapper, bigeye croaker and barracuda without (lane 4) and with PPP addition (lane 5–8) contained no MHC. This indicated that surimi from those species exhibited an excellent setting capability. As a result, no MHC was remained, even with the higher amount of PPP added. However, MHC in kamaboko gel from threadfin bream was retained more with the increasing amount of PPP. It suggests that threadfin bream surimi possibly did not have the good setting response, when compared to other species. As a consequence, PPP at a higher level might cause the interfering effect on myosin polymerization induced by TGase, resulting in more retained MHC.
solubility was accompanied by the decrease in breaking force. The solution containing SDS, urea and bmercaptoethanol was used to solubilize protein by cleaving all bonds, except nondisulfide covalent bond, particularly e-(g-glutamyl) lysine linkage (Benjakul et al., 2001c). The decrease in solubility thus indicates the formation of nondisulfide covalent bond that was induced by endogenous TGase and plasma TGase. TGase has been known to play a crucial role in e-(gglutamyl) lysine linkage formation in surimi gel (Kumazawa et al., 1995). From the result, the increase in solubility observed in surimi with an excessive amount of PPP possibly resulted from the interference with
100
Solubility (%)
80 60 40 20 0 0
0.5
1 1.5 PPP concentration (%)
2
181
2.5
Fig. 3. Solubility of kamaboko gels added with different levels of PPP. Bars indicate the standard deviation from triplicate determinations. Bigeye snapper (J), Bigeye croaker (W), Threadfin bream ( ), and Barracuda (m).
MHC
Actin
(A)
1
2
3
4
5
6
7
8
9
(B) 1
2
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4
5
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7
8 9
(C)
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2
3
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6
7
8 9
(D) 1
2
3
4
5
6
7
8
9
Fig. 4. SDS-PAGE pattern of kamaboko gels added with different levels of PPP. A: Bigeye snapper; B: bigeye croaker; C: threadfin bream; D: barracuda; lane 1: surimi sol; lane 2: control (No CaCl2 and PPP, setting at 40 C, followed by heating at 90 C); lane 3: directly heated gel (No CaCl2 and PPP, heating at 90 C); lane 4, 5, 6, 7, 8: kamaboko gels added with PPP at a level of 0, 0.5, 1.0, 1.5 and 2.5 g/100 g, respectively; lane 9: PPP.
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182
90
Breaking force of surimi gel with PPP at the optimum level in combination with 50 mM CaCl2 increased with setting time, especially in the first 1–1.5 h (Fig. 5). The optimum setting time varied with species. The optimum setting times were 2, 1.5, 1.5 and 2 h for bigeye snapper, croaker, threadfin bream and barracuda surimi, respectively, which corresponded to increased breaking force by 123.5%, 167.7%, 163.9% and 186.6%, compared to directly heated gel (without setting). A similar trend was observed with deformation. The differences in setting time required for surimi among species owe to the different intrinsic properties of different muscle, including the thermal stability, endogenous proteinases and TGase. For bigeye snapper and threadfin bream surimi, the decreased breaking force was observed with extended setting time (Po0:05). For bigeye croaker and barracuda, no marked changes in breaking force were observed beyond the optimum setting time (P > 0:05).
85
Whiteness
3.2. Effect of setting time on gel properties of surimi with PPP added
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75
70 0
0.5
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Setting time (h)
Fig. 6. Whiteness of surimi gels added with PPP and subjected to setting for different times. Bars indicate the standard deviation from five determinations. Bigeye snapper (J), Bigeye croaker (W), Threadfin bream ( ), and Barracuda (m).
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Fig. 7. Solubility of surimi gels added with PPP and subjected to setting for different times. Bars indicate the standard deviation from triplicate determinations. Bigeye snapper (J), Bigeye croaker (W), Threadfin bream ( ), and Barracuda (m).
Setting time (h) 20
15 Deformation (mm)
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Setting time (h)
Fig. 5. Breaking force and deformation of surimi gels added with PPP and subjected to setting for different times. Bars indicate the standard deviation from five determinations. Bigeye snapper (J), Bigeye croaker (W), Threadfin bream ( ), and Barracuda (m).
Changes in whiteness of surimi gels with PPP and CaCl2 subjected to different setting times are shown in Fig. 6. Setting time did not affect the changes in whiteness of surimi gels from all species studied. The solubility of surimi gels from all species decreased as the setting time increased up to 1.5 h of setting time (Po0:05) (Fig. 7). However, beyond 1.5 h setting, the solubility slightly increased for all species with the largest increase for barracuda. With a sufficient setting time, proteins likely oriented themselves and made reactive lysine or glutamine residues exposed for e-(gglutamyl) lysine linkage formation induced by TGase, resulting in the decreased solubility. A decreased solubility with a concomitant increase in breaking force was generally observed. The differences in solubility were possibly caused by the differences in TGase activity
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as well as protein conformation. TGase activity varied among fish species and cross-linking of myosin heavy chain induced by TGase was regulated by the conformation of substrate actomyosin (Seki et al., 1990; Kishi, Nozawa, & Seki, 1991; Araki & Seki, 1993; Benjakul & Visessanguan, 2003). Numakura et al. (1985) found that solubility of Alaska pollack surimi paste in solution containing SDS, urea and b-mercaptoethanol decreased with prolonged setting at 20 C and 30 C. The increase in solubility in kamaboko gel subjected to extended setting time was possibly associated with proteolysis. Polymerized proteins could be hydrolysed by heat-activated proteinases in surimi, leading to the softening of gel network previously formed during
setting (Takeda & Seki, 1996). Kamath et al. (1992) found that proteolysis in croaker paste increased with increasing temperature of setting, especially in the temperature range of 40–50 C. As a result, the gradual decrease in breaking force was found with kamaboko gels subjected to setting for extended time. TCA-soluble peptides increased as the setting time increased (Po0:05) (Table 1). Though PPP, which was reported to exhibit the proteinase inhibitory activity, was present in the surimi, proteolysis still occurred, particularly with the increasing setting time. This was postulated that PPP amount used was not sufficient for total inhibition of proteinases in surimi. From the result, the increase in TCA-soluble peptide was coincidental with the increase in solubility, especially when the setting time increased (Fig. 7). Proteinases have been known to induce modori as a result of the proteolysis of myofibrils (An et al., 1996; Benjakul et al., 2003). Electrophoretic patterns of kamaboko gels with PPP at an optimum level and set for various times is shown in Fig. 8. No marked changes in MHC were observed with directly heated gel without prior setting (lane 2). For kamaboko gel of surimi from all species except threadfin bream, MHC decreased markedly as the setting time increased. With extended setting time, no MHC band remained, possibly due to the higher degree of polymerization. From the result, no MHC band was observed in kamaboko gel of surimi from bigeye croaker and barracuda after 1 h setting. However, no marked changes in MHC were found in kamaboko gel from threadfin bream subjected to a longer setting time. This was presumed that threadfin bream surimi had a poorer setting capability, compared to other species.
Table 1 TCA-soluble peptide (mmol tyrosine/g) in surimi gel added with PPP and subjected to setting for different times Setting time (h)
Bigeye snapper
Bigeye croaker
Threadfin bream
Barracuda
Surimi sol 0 0.5 1 1.5 2 2.5 3
0.6370.03aab 0.8470.01b 0.8770.04b 1.0570.06c 1.0770.05cd 1.1270.02de 1.1270.04de 1.1870.01e
0.4970.02a 0.6470.03ab 0.7570.06bc 0.8170.03cd 0.9670.09de 1.0770.08ef 1.2370.21f 1.1470.02f
0.8770.18a 1.0870.02b 1.1270.04c 1.2170.03d 1.3770.02e 1.3670.01e 1.3670.04e 1.4170.01f
0.7370.03a 0.8670.01b 0.9770.04c 0.9970.06c 1.0870.06d 1.1770.03e 1.2170.04e 1.3270.04f
183
a
Mean7SD from triplicate determinations. Different letters in the same column indicate significant differences (Po0:05). b
MHC
Actin
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1
2
3
4
5
6
7
8
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2
3
4
5
(C)
1
2
3
4
5
6
7
8
(D) 1
2
3
4
5
6
6
7
7
8
8
Fig. 8. SDS-PAGE pattern of surimi gels added with PPP and subjected to setting for different times. A: bigeye snapper; B: bigeye croaker; C: threadfin bream; D: barracuda; lane 1: surimi sol; lane 2, 3, 4, 5, 6, 7, 8: setting time of 0, 0.5, 1. 1.5, 2, 2.5 and 3, respectively.
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4. Conclusion Porcine plasma protein at an appropriate level in combination with setting at 40 for a proper time effectively enhanced surimi gel strength. The optimum conditions varied from species to species. Generally, an excessive amount of PPP and extended setting caused the decrease in gel strength of surimi.
Acknowledgements Authors would like to express their sincere thank to International Foundation for Science for Project No. E/ 2878-2 and Prince of Songkla University for the financial support.
References An, H., Peters, M. Y., & Seymour, T. A. (1996). Roles of endogenous enzymes in surimi gelation. Trends in Food Science and Technology, 7, 321–326. Araki, H., & Seki, N. (1993). Comparison of reactivity of transglutamianse to various fish actomyosin. Nippon Suisan Gakkaishi, 59, 711–716. Benjakul, S., & Visessanguan, W. (2000). Pig plasma protein: potential use as proteinase inhibitor for surimi manufacture: Inhibitory activity and the active component. Journal of the Science of Food and Agriculture, 80, 1351–1356. Benjakul, S., & Visessanguan, W. (2003). Transglutaminase-mediated setting in bigeye snapper surimi. Food Research International, 36, 253–266. Benjakul, S., Visessanguan, W., & Srivilai, C. (2001a). Gel properties of bigeye snapper (Priacanthus tayenus) surimi as affected by setting and porcine plasma proteins. Journal of Food Quality, 24, 453–471. Benjakul, S., Visessanguan, W., & Srivilai, C. (2001b). Porcine plasma protein as proteinase inhibitor in bigeye snapper (Priacanthus tayenus) muscle and surimi. Journal of the Science of Food and Agriculture, 81, 1039–1046. Benjakul, S., Visessanguan, W., & Srivilai, C. (2001c). Porcine plasma proteins as gel enhancer in bigeye snapper (Priacanthus tayenus) surimi. Journal of Food Biochemistry, 25, 285–306. Benjakul, S., Visessanguan, W., Ishizaki, S., & Tanaka, M. (2001d). Differences in gelation characteristics of natural actomyosin from two species of bigeye snapper, Priacanthus tayenus and Priacanthus macracanthus. Journal of Food Science, 66, 1311–1318. Benjakul, S., Visessanguan, W., & Leelapongwattana, K. (2003). Purification and characterization of heat-stable alkaline proteinase from bigeye snapper (Priacanthus macracanthus) muscle. Comparative Biochemistry and Physiology Part B, 134, 579–591. Chawla, S. P., Venugopol, V., & Nair, P. M. (1996). Gelation of proteins from washed muscle of threadfin bream (Nemipterus japonicus) under mild acidic conditions. Journal of Food Science, 54, 362–366. Choi, Y. J., Cho, Y. J., & Lanier, T. C. (1999). Purification and characterization of alkaline proteinase from Atlantic menhaden muscle. Journal of Food Science, 64, 772–775. Chung, K. H., & Lee, C. M. (1990). Relationships between physicochemical properties of nonfish protein and textural properties of protein-incorporated surimi gel. Journal of Food Science, 55, 972–975.
Kamath, G. G., Lanier, T. C., Foegeding, E. A., & Hamann, D. D. (1992). Nondisulfide covalent cross-linking of myosin heavy chain in ‘‘setting’’ of Alaska pollock and Atlantic croaker surimi. Journal of Food Biochemistry, 16, 151–172. Kim, J. M., & Lee, C. M. (1987). Effect of starch on textural properties of surimi gel. Journal of Food Science, 52, 722–725. Kimura, I. M., Sugimoto, M., Toyoda, K., Seki, N., Arai, K., & Fujita, T. (1991). A study on the cross-links reaction of myosin in kamaboko ‘‘suwari’’ gels. Nippon Suisan Gakkaishi, 57, 1386–1396. Kishi, H., Nozawa, H., & Seki, N. (1991). Reactivity of muscle transglutaminase on carp myofibrils and myosin B. Nippon Suisan Gakkaishi, 57, 1203–1210. Kumazawa, Y., Numazawa, T., Seguro, K., & Motoki, M. (1995). Suppression of surimi gel setting by transglutaminase inhibitors. Journal of Food Science, 60, 715–717. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature, 227, 680–685. Lanier, T. C. (1992). Measurement of surimi composition and junctional properties. In T. C. Lanier, & C. M. Lee (Eds.), Surimi technology (pp. 123–166). New York: Marcel Dekker, Inc. Lee, H. G., Lee, C. M., Chung, K. H., & Lavey, S. A. (1992). Sodium ascorbate affects surimi gel-forming properties. Journal of Food Science, 57, 1343–1347. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with Folin phenol reagent. The Journal of Biological Chemistry, 193, 256–275. Morrissey, M. T., Wu, J. W., Lin, D. D., & An, H. (1993). Effect of food grade protease inhibitor on autolysis and gel strength of surimi. Journal of Food Science, 58, 1050–1054. NFI, (1991). A manual of standard methods for measuring, specifying the properties of surimi. Washington, DC: National Fisheries Institute. Niwa, E., Tsujimoto, K., & Kanoh, S. (1992). Kamaboko gelstrengthening effect of polyuronides and other polysaccharides. Nippon Suisan Gakkaishi, 58, 85–88. Numakura, T., Seki, N., Kimura, I., Toyoda, K., Fugita, T., Takama, K., & Arai, K. (1985). Cross-linking reaction of myosin in the fish paste during setting (suwari). Bulletin of the Japanese Society of Scientific Fisheries, 51, 1559–1565. Park, J. W. (1994). Functional protein additives in surimi gels. Journal of Food Science, 59, 525–527. Raeker, M., & Johnson, L. A. (1995). Thermal and functional properties of bovine blood plasma and egg white proteins. Journal of Food Science, 60, 685–690. Reppond, K. D., & Babbitt, J. K. (1993). Protease inhibitors affect physical properties of arrowtooth flounder and walleye pollock surimi. Journal of Food Science, 58, 96–98. Robinson, H. W., & Hodgen, C. G. (1940). The biuret reaction in the determination of serum protein. I. A study of the condition necessary for the production of the stable color which bears a quantitative relationship to the protein concentration. The Journal of Biological Chemistry, 135, 707–725. Seki, N., Uno, H., Lee, N. H., Kimura, I., Toyoda, K., Fujita, T., & Arai, K. (1990). Transglutaminase activity in Alaska pollack muscle and surimi and its reaction with myosin B. Nippon Suisan Gakkaishi, 56, 125–132. Steel, R. G. D., & Torrie, J.H. (1980). Principles and procedures of statistics; A biometrical approach (2nd ed.) (633pp). New York: McGraw-Hill. Takeda, H., & Seki, N. (1996). Enzyme-catalyzed cross-linking and degradation of myosin heavy chain in walleye pollack surimi paste during setting. Fisheries Science, 62, 462–467. Visessanguan, W., Benjakul, S., & An, H. (2000). Porcine plasma proteins as a surimi protease inhibitor: Effects on actomyosin gelation. Journal of Food Science, 65, 607–611.
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Effect of porcine plasma protein and setting on gel properties of surimi produced from fish caught in Thailand Soottawat Benjakula,*, Wonnop Visessanguanb, Chakkawat Chantarasuwana b
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 (BIOTEC), 113 Paholyothin Rd., Klong 1, Klong Luang, Pathumthani 12120, Thailand
Received 6 January 2003; accepted 24 July 2003
Abstract Effects of porcine plasma protein (PPP) and high temperature setting on gel properties of surimi from bigeye snapper, bigeye croaker, threadfin bream and barracuda were investigated. PPP was effective in increasing breaking force and deformation of kamaboko gels set at 40 C for 30 min and heated at 90 C for 20 min. The optimum levels of PPP were 0.5, 0.5, 1.5 and 1.5 g/100 g and the optimum setting times were 2, 1.5, 1.5 and 2 h for bigeye snapper, bigeye croaker, threadfin bream and barracuda surimi, respectively. However, the addition of PPP significantly decreased whiteness (Po0:05). An increase in gel-forming ability of surimi with PPP coincided with a decrease in solubility in mixture of SDS, urea and b-mercaptoethanol, indicating the formation of nondisulfide covalent bond induced by both endogenous and plasma transglutaminase. The results supported that PPP improve the gelation of surimi in combination with setting. r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Porcine; Plasma; Blood; Setting; Suwari; Surimi; Gelation; Tropical fish
1. Introduction Surimi gel is a three-dimensional muscle protein network. Textural characteristics expressed in terms of gel strength is the primary determinant for surimi quality and price (Lanier, 1992). The improvement of textural quality of surimi or surimi based products has been attempted by addition of protein additives (Chung & Lee, 1990; Morrissey, Wu, Lin, & An, 1993; Park, 1994; Benjakul, Visessanguan, & Srivilai, 2001a). Additionally, starch and other polysaccharides have been used as ingredient in surimi (Kim & Lee, 1987; Niwa, Tsujimoto, & Kanoh, 1992). For surimi processing, wheat and soybean proteins as well as starch are used as additives not only to increase gel strength but also to increase the volume (Yamashita, 1991; Yoon & Lee, 1993). To increase the gel strength of surimi, gelstrengthening ingredients such as ascorbic acid or dehydroascorbic acid were found to increase the gel strength of surimi due to the oxidation of sulfhydryl group in the protein matrix (Yoshinaka, Shiraishi, & *Corresponding author. Tel.: +66-74-286334; fax: +66-74-212889. E-mail address: [email protected] (S. Benjakul).
Ikeda, 1972; Lee, Lee, Chung, & Lavey, 1992). Protein additives have been also used as proteinase inhibitor to improve the gel strength of surimi by controlling proteolytic activities during surimi seafood production (Morrissey et al., 1993). The proteolytic degradation of myofibrillar proteins has a detrimental effect on surimi quality and substantially lowers the gel strength (An, Peters, & Seymour, 1996). The most commonly used food grade inhibitors are beef plasma protein, egg white and potato powder (Morrissey et al., 1993). Degree of proteinase inhibition was dependent upon the types of additives used (Morrissey et al., 1993; Reppond & Babbitt, 1993; Weerasinghe, Morrissey, & An, 1996). Apart from the use of additives, gel strengthening of surimi can be achieved by subjecting sols to setting at the temperatures ranging from 0 C to 40 C prior to heating (Kamath, Lanier, Foegeding, & Hamann, 1992; An et al., 1996). During setting, myosin heavy chain (MHC) undergoes polymerization via formation of nonsulfide covalent cross-links, catalysed by an endogenous transglutaminase (TGase) (Kimura et al., 1991; Kumazawa, Numazawa, Seguro, & Motoki, 1995; Benjakul & Visessanguan, 2003).
0023-6438/$30.00 r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2003.07.002
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Porcine blood is an abundant byproduct in the slaughtering process in Thailand at an estimated amount of 30,000 metric tons per year (Benjakul et al., 2001a). Blood plasma contains a variety of bioactive compounds including proteinase inhibitor and plasma transglutaminase (Benjakul et al., 2001a b). Porcine plasma at a level of 0.5 g/100 g was found to increase the breaking force and deformation of bigeye snapper surimi (Benjakul et al., 2001a). The fractionation of bioactive components in porcine plasma was an effective means to concentrate those compounds and minimize the discoloration caused by residual pigment in plasma (Benjakul, Visessanguan, & Srivilai, 2001c). Although PPP at a level of 0.5 g/100 g effectively increased the gel strength of surimi from bigeye snapper (Benjakul et al., 2001a), no information on the use of PPP in surimi produced from other fish species caught in Thailand. The gelling characteristic of surimi could vary with both intrinsic and extrinsic factors (Benjakul, Visessanguan, Ishizaki, & Tanaka, 2001d). Therefore, the effect of PPP addition on gelling characteristic of different surimi could vary with different surimi which require different optimum condition for PPP application. The appropriate use of PPP in combination with setting would be one of effective means to enhance the gel strength of surimi. The objective of this investigation was to study the effect of PPP in combination with setting on gel properties and changes in myofibrillar proteins in surimi produced from four tropical fish species, widely present in Thailand.
2. Materials and methods 2.1. Chemicals Sodium dodecyl sulfate (SDS), urea, trichloroacetic acid (TCA) and sodium hydroxide were purchased from Riedel-deHaen (Seelze, Germany). Trisodium citrate, bmercaptoethanol (b-ME) and N,N,N0 ,N0 -tetramethyl ethylene diamine (TEMED)were obtained from Sigma Co. (St. Louis, MO, USA). 2.2. Preparation of porcine plasma protein (PPP) Porcine blood was collected from a slaughter house in Hat Yai, Thailand. The blood was mixed with trisodium citrate solution (3.8 g/100 ml) at a ratio of 1:9 (v/v) to prevent blood coagulation and transported to the Department of Food Technology, Prince of Songkla University, Hat Yai. The blood was then centrifuged at 1000g for 30 min at 4 C using a Sorvall Model RC-B Plus centrifuge (Newtown, CT, USA) to remove red blood cells. The resultant supernatant was freeze-dried and kept at 4 C until used. The dry powder was referred to as porcine plasma protein (PPP).
2.3. Surimi and surimi gel preparation Frozen surimi (grade A) produced from threadfin bream (Nemipterus bleekeri), bigeye snapper (Priacanthus tayenus), baracuda (Sphyraena jello) and bigeye croaker (Pennahai macrophthalmus) were purchased from Man A Frozen Foods Co., Ltd. (Songkhla, Thailand) and kept at 20 C until used. To prepare the gel, frozen surimi was tempered for 30 min in the running water (25 C). The surimi was then cut into small pieces with an approximate thickness of 1 cm. The surimi was placed in the mixer (National Model MK-K77, Tokyo, Japan) and PPP at different concentrations (0, 0.25, 0.5, 0.75, 1, 1.5, 2 and 2.5 g/ 100 g) was added in combination with CaCl2 (50 mmol/ kg). The moisture was then adjusted to 80 g/100 g and salt (2.5 g/100 g; surimi weight basis) was added. The mixture was chopped for 5 min at 4 C and its temperature was kept below 10 C. The surimi sol was stuffed into the polyvinylidine casing (2.5 cm diameter) and both ends were closed tightly and subjected to setting at 40 C in a water bath (Memmert, Schwabach, Germany) for 2, 3, 1 and 1.5 h for bigeye snapper, bigeye croaker, threadfin bream and barracuda surimi, respectively (Benjakul, Visessanguan, & Chantarasuwan, unpublished data). After setting for designated time, the gels were heated at 90 C for 20 min in a water bath. The gels were then cooled in iced water (0–1 C) and stored at 4 C for 24 h before analysis. The gels obtained were referred to as ‘kamaboko gels’. PPP at a concentration, which rendered the highest breaking force and deformation, was chosen for further study. To investigate the effect of setting time on gel properties, surimi sol added with PPP at an optimum concentration was subjected to setting at 40 C for different time (0, 0.5, 1, 1.5, 2, 2.5, or 3 h), followed by heating and cooling as previously described and stored at 4 C for 24 h prior to analysis. 2.4. Texture analysis The breaking force (gel strength) and deformation (elasticity/deformability) were measured with a 5-mm diameter cylindrical plunger at 60 mm/min deformation rate using a texture analyser (Model TA-XT2, Stable Micro Systems, Surrey, England). Five cylinder-shaped samples of 2.5 cm in length were prepared and equilibrated at room temperature before testing. 2.5. Whiteness measurement Whiteness of surimi gels was measured using colorimeter JP7100F (Juki Corporation, Tokyo, Japan). The measurement was performed in five replications. The whiteness was calculated using the following
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equation (NFI, 1991): Whiteness ¼ 100 ½ð100 L Þ2 þ a þ b 1=2 : 2
2
2.6. Solubility studies The solubility of surimi gels in 20 mM Tris-HCl, pH 8.0 containing SDS (1 g/100 ml), urea (8 M) and b-ME (2 g/100 ml) was determined as described by Chawla, Venugopol and Nair (1996). The sample (1 g) was homogenized in 20 ml of solution for 1 min using homogenizer (IKA Labortechnik, Selangor, Malaysia). The homogenate was heated in boiling water (100 C) for 2 min and stirred at room temperature for 4 h. The resulting homogenate was centrifuged at 10,000g for 30 min using Sorvall Model RC-B plus centrifuge (Newtown, CT, USA). Protein in the supernatant (10 ml) was precipitated by the addition of trichloroacetic acid (TCA) (50 g/100 ml) to a final concentration of 10 g/100 ml. The mixture was kept at 4 C for 18 h and then centrifuged at 10,000g for 30 min. The precipitate was washed with TCA (10 g/100 ml) and solubilized in 0.5 M NaOH. To obtain the total amount of protein, gels were directly solubilized in 0.5 M NaOH. The protein content was measured using the Biuret test (Robinson & Hodgen, 1940). The solubility was expressed as percent of the total protein. 2.7. Determination of TCA soluble peptides TCA soluble peptides were determined according to the method described by Morrissey et al. (1993). Surimi gel (3 g) was homogenized with 27 ml of TCA (5 g/ 100 ml). The homogenate was kept in ice for 1 h and centrifuged at 5000g for 5 min. Soluble peptides in supernatant were measured and expressed as mmol tyrosine/g. 2.8. SDS-polyacrylamide gel electrophoresis (SDS-page) The protein pattern of surimi gel was analysed on SDS-PAGE according to the method of Laemmli (1970). To prepare the protein sample, 27 ml of SDS solution (5 g/100 ml) heated to 85 C was added into the sample (3 g). The mixture was then homogenized using homogenizer for 2 min. The homogenate was incubated at 85 C for 1 h to dissolve total proteins. The samples were centrifuged at 3500g for 20 min to remove undissolved debris. Protein concentration was determined according to the method of Lowry, Rosebrough, Farr, and Randall (1951) using bovine serum albumin as standard. SDS-PAGE gel was made of 10% running gel and 4% stacking gel. After separation, the proteins were fixed and stained with Coomassie Blue R-250.
Protein concentration was measured by the method of Lowry et al. (1951) using bovine serum albumin as standard. 2.10. Statistical analysis Analysis of variance (ANOVA) was performed to analyse the effect of PPP concentration and setting time in a completely randomized design. Mean comparisons were run by Duncan’s multiple range test (Steel & Torrie, 1980).
3. Results and discussion 3.1. Effect of PPP concentration on surimi gel properties Breaking force and deformation of kamaboko gels from four fish species added with different levels of PPP in combination with CaCl2 (50 mmol/kg) are shown in Fig. 1. Breaking force and deformation of gels generally increased as PPP was added up to 0.5 g/100 g for bigeye snapper and bigeye croaker surimi and up to 1.5 g/100 g for threadfin bream and barracuda surimi, respectively (Po0:05). Thereafter, breaking force of bigeye snapper surimi gel significantly decreased as PPP levels increased (Po0:05), however no marked changes were found in other surimi gels (P > 0:05). At a level of 0.5 g PPP/ 100 g, breaking force of gels from bigeye snapper and bigeye croaker surimi increased by 58.6% and 24.4%, respectively. For gels from threadfin bream and barracuda surimi added with 1.5 g PPP/100 g, breaking force increased by 62.2 and 79.1%, respectively. Deformation of surimi gels with the optimum level of PPP increased by 11.0 to 23.7%. The increased amount of PPP resulted in the decrease in gel-forming ability as observed by the decrease in breaking force. However, no changes in deformation of surimi gel from all species, except bigeye croaker, were observed with an increasing PPP concentration (P > 0:05). Because of the inhibitory activity of PPP against protease (Visessanguan, Benjakul, & An, 2000), PPP showed a great potential as surimi gel enhancer, however PPP should be used at optimum concentration since its components interfere with gelation process. Blood plasma comprises three main proteins including albumin, globulin and fibrinogen (Raeker & Johnson, 1995). These proteins at appropriate levels possibly enhanced protein–protein interaction, thereby strengthening gel network. In view of the optimum level of PPP varying from species to species, the interaction appears to be species specific. An excessive amount of PPP caused the dilution of muscle proteins, which is a major contributor for gel formation. As a consequence, it might interfere with the formation
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Fig. 1. Breaking force and deformation of kamaboko gels added with different levels of PPP. Bars indicate the standard deviation from five determinations. Bigeye snapper (J), Bigeye croaker (W), Threadfin bream ( ), and Barracuda (m).
of a three-dimensional gel network between myosin molecules. Yamashita, Araki, and Seki (1996) report that the addition of casein at a level of 1 and 5 g/100 g decreased myosin content by 9% and 32%, respectively and resulted in the decreased gel strength of walleye pollack surimi. The interference of myosin–myosin interaction may be a major cause of the inferior gelation when casein was added in the surimi (Yamashita et al., 1996). PPP, a water-soluble protein, may interfere with protein–protein interaction (Yoon, Lee, & Hufnagel, 1991). In general, optimum PPP levels added into surimi varied, depending upon the species. From the result, optimal PPP level for gel enhancement of surimi from bigeye snapper and bigeye croaker was less than that of surimi from threadfin bream and barracuda. This was probably related to different intrinsic factors such as the presence of endogenous enzymes, e.g. proteinases and transglutaminase as well as the molecular properties of myofibrillar proteins. Endogenous proteinases, especially heat activated and heat stable proteinases, have been known to play a detrimental role in surimi gel
quality (Morrissey et al., 1993; Benjakul, Visessanguan, & Leelapongwattana, 2003). The most active proteinases in fish muscle, which can soften the surimi gel, vary with species (An et al., 1996: Choi, Cho, & Lanier, 1999). Since different surimi possibly contained the different proteinase activity, especially myofibril-associated proteinases, which still remained in the surimi after washing process, different level of PPP was required to inhibit the proteolytic activity. PPP showed the inhibitory activity toward trypsin, papain, digestive proteinases and modori-inducing proteinase from bigeye snapper (Benjakul, Visessanguan, & Srivilai, 2001b). The inhibitory component in PPP was identified as a protein with apparent MW of 60,000–63,000 Da on the inhibitory activity-stained gel (Benjakul & Visessanguan, 2000). Whiteness of kamaboko gels with different levels of PPP is depicted in Fig. 2. Whiteness of all kamaboko gels decreased steadily as PPP level increased. At the optimum level of PPP for each surimi gel strengthening, the whiteness decreased by 1.8%, 0.8%, 5.4% and 4.4% for surimi from bigeye snapper, bigeye croaker, threadfin bream and barracuda, respectively. Benjakul et al. (2001a) also reported that addition of PPP at a higher level to surimi from bigeye snapper resulted in the decrease in whiteness. Nevertheless, no effect of PPP fraction I-S (an enriched plasma transglutaminase fraction) on whiteness of surimi gel was observed, even added at higher concentrations (Benjakul et al., 2001c). Changes in solubility of kamaboko gels with PPP at different levels are shown in Fig. 3. A decrease in solubility was observed as PPP increased up to the optimum level and a further increase in PPP resulted in an increase in solubility to varying degree. The decrease in solubility mostly coincided with the increased breaking force and deformation. The subsequent increase in 90
85
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80
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70 0
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Fig. 2. Whiteness of kamaboko gels added with different levels of PPP. Bars indicate the standard deviation from five determinations. Bigeye snapper (J), Bigeye croaker (W), Threadfin bream ( ), and Barracuda (m).
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myosin–myosin interaction that was induced by TGase. Although PPP at a higher concentration inhibited proteolysis more effectively as indicated by the decrease in TCA-soluble peptides (data not shown), a decrease in nondisulfide cross-linking was found as indicated by increased solubility. Thus, the balance between proteolytic inhibitory effect and the interfering effect on protein–protein interaction of PPP may be the important factor determining surimi gel enhancement. From the result, it can be inferred that PPP, a nonmuscle protein, should be added at an appropriate level to avoid the interfering effect on myofibrillar proteins, which are essential for gelation. The electrophoretic pattern of kamaboko gels with different levels of PPP is shown in Fig. 4. Myosin heavy chain (MHC) in directly heated gel (lane 3) decreased to some extent, compared to that found in surimi sol (lane 1). For the control kamaboko gel (without PPP and CaCl2) (lane 2) of surimi from all species, except threadfin bream, no MHC band was remained. Kamaboko gels from bigeye snapper, bigeye croaker and barracuda without (lane 4) and with PPP addition (lane 5–8) contained no MHC. This indicated that surimi from those species exhibited an excellent setting capability. As a result, no MHC was remained, even with the higher amount of PPP added. However, MHC in kamaboko gel from threadfin bream was retained more with the increasing amount of PPP. It suggests that threadfin bream surimi possibly did not have the good setting response, when compared to other species. As a consequence, PPP at a higher level might cause the interfering effect on myosin polymerization induced by TGase, resulting in more retained MHC.
solubility was accompanied by the decrease in breaking force. The solution containing SDS, urea and bmercaptoethanol was used to solubilize protein by cleaving all bonds, except nondisulfide covalent bond, particularly e-(g-glutamyl) lysine linkage (Benjakul et al., 2001c). The decrease in solubility thus indicates the formation of nondisulfide covalent bond that was induced by endogenous TGase and plasma TGase. TGase has been known to play a crucial role in e-(gglutamyl) lysine linkage formation in surimi gel (Kumazawa et al., 1995). From the result, the increase in solubility observed in surimi with an excessive amount of PPP possibly resulted from the interference with
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Fig. 3. Solubility of kamaboko gels added with different levels of PPP. Bars indicate the standard deviation from triplicate determinations. Bigeye snapper (J), Bigeye croaker (W), Threadfin bream ( ), and Barracuda (m).
MHC
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Fig. 4. SDS-PAGE pattern of kamaboko gels added with different levels of PPP. A: Bigeye snapper; B: bigeye croaker; C: threadfin bream; D: barracuda; lane 1: surimi sol; lane 2: control (No CaCl2 and PPP, setting at 40 C, followed by heating at 90 C); lane 3: directly heated gel (No CaCl2 and PPP, heating at 90 C); lane 4, 5, 6, 7, 8: kamaboko gels added with PPP at a level of 0, 0.5, 1.0, 1.5 and 2.5 g/100 g, respectively; lane 9: PPP.
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90
Breaking force of surimi gel with PPP at the optimum level in combination with 50 mM CaCl2 increased with setting time, especially in the first 1–1.5 h (Fig. 5). The optimum setting time varied with species. The optimum setting times were 2, 1.5, 1.5 and 2 h for bigeye snapper, croaker, threadfin bream and barracuda surimi, respectively, which corresponded to increased breaking force by 123.5%, 167.7%, 163.9% and 186.6%, compared to directly heated gel (without setting). A similar trend was observed with deformation. The differences in setting time required for surimi among species owe to the different intrinsic properties of different muscle, including the thermal stability, endogenous proteinases and TGase. For bigeye snapper and threadfin bream surimi, the decreased breaking force was observed with extended setting time (Po0:05). For bigeye croaker and barracuda, no marked changes in breaking force were observed beyond the optimum setting time (P > 0:05).
85
Whiteness
3.2. Effect of setting time on gel properties of surimi with PPP added
80
75
70 0
0.5
1
1.5
2
2.5
3
Setting time (h)
Fig. 6. Whiteness of surimi gels added with PPP and subjected to setting for different times. Bars indicate the standard deviation from five determinations. Bigeye snapper (J), Bigeye croaker (W), Threadfin bream ( ), and Barracuda (m).
100
1800
80
Solubility (%)
1600
Breaking force (g)
1400 1200 1000
60
40
800
20
600 400
0 200
0
1
1.5
2
2.5
3
Setting time (h)
0 0
0.5
1
1.5
2
2.5
3
Fig. 7. Solubility of surimi gels added with PPP and subjected to setting for different times. Bars indicate the standard deviation from triplicate determinations. Bigeye snapper (J), Bigeye croaker (W), Threadfin bream ( ), and Barracuda (m).
Setting time (h) 20
15 Deformation (mm)
0.5
10
5
0 0
0.5
1
1.5
2
2.5
3
Setting time (h)
Fig. 5. Breaking force and deformation of surimi gels added with PPP and subjected to setting for different times. Bars indicate the standard deviation from five determinations. Bigeye snapper (J), Bigeye croaker (W), Threadfin bream ( ), and Barracuda (m).
Changes in whiteness of surimi gels with PPP and CaCl2 subjected to different setting times are shown in Fig. 6. Setting time did not affect the changes in whiteness of surimi gels from all species studied. The solubility of surimi gels from all species decreased as the setting time increased up to 1.5 h of setting time (Po0:05) (Fig. 7). However, beyond 1.5 h setting, the solubility slightly increased for all species with the largest increase for barracuda. With a sufficient setting time, proteins likely oriented themselves and made reactive lysine or glutamine residues exposed for e-(gglutamyl) lysine linkage formation induced by TGase, resulting in the decreased solubility. A decreased solubility with a concomitant increase in breaking force was generally observed. The differences in solubility were possibly caused by the differences in TGase activity
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as well as protein conformation. TGase activity varied among fish species and cross-linking of myosin heavy chain induced by TGase was regulated by the conformation of substrate actomyosin (Seki et al., 1990; Kishi, Nozawa, & Seki, 1991; Araki & Seki, 1993; Benjakul & Visessanguan, 2003). Numakura et al. (1985) found that solubility of Alaska pollack surimi paste in solution containing SDS, urea and b-mercaptoethanol decreased with prolonged setting at 20 C and 30 C. The increase in solubility in kamaboko gel subjected to extended setting time was possibly associated with proteolysis. Polymerized proteins could be hydrolysed by heat-activated proteinases in surimi, leading to the softening of gel network previously formed during
setting (Takeda & Seki, 1996). Kamath et al. (1992) found that proteolysis in croaker paste increased with increasing temperature of setting, especially in the temperature range of 40–50 C. As a result, the gradual decrease in breaking force was found with kamaboko gels subjected to setting for extended time. TCA-soluble peptides increased as the setting time increased (Po0:05) (Table 1). Though PPP, which was reported to exhibit the proteinase inhibitory activity, was present in the surimi, proteolysis still occurred, particularly with the increasing setting time. This was postulated that PPP amount used was not sufficient for total inhibition of proteinases in surimi. From the result, the increase in TCA-soluble peptide was coincidental with the increase in solubility, especially when the setting time increased (Fig. 7). Proteinases have been known to induce modori as a result of the proteolysis of myofibrils (An et al., 1996; Benjakul et al., 2003). Electrophoretic patterns of kamaboko gels with PPP at an optimum level and set for various times is shown in Fig. 8. No marked changes in MHC were observed with directly heated gel without prior setting (lane 2). For kamaboko gel of surimi from all species except threadfin bream, MHC decreased markedly as the setting time increased. With extended setting time, no MHC band remained, possibly due to the higher degree of polymerization. From the result, no MHC band was observed in kamaboko gel of surimi from bigeye croaker and barracuda after 1 h setting. However, no marked changes in MHC were found in kamaboko gel from threadfin bream subjected to a longer setting time. This was presumed that threadfin bream surimi had a poorer setting capability, compared to other species.
Table 1 TCA-soluble peptide (mmol tyrosine/g) in surimi gel added with PPP and subjected to setting for different times Setting time (h)
Bigeye snapper
Bigeye croaker
Threadfin bream
Barracuda
Surimi sol 0 0.5 1 1.5 2 2.5 3
0.6370.03aab 0.8470.01b 0.8770.04b 1.0570.06c 1.0770.05cd 1.1270.02de 1.1270.04de 1.1870.01e
0.4970.02a 0.6470.03ab 0.7570.06bc 0.8170.03cd 0.9670.09de 1.0770.08ef 1.2370.21f 1.1470.02f
0.8770.18a 1.0870.02b 1.1270.04c 1.2170.03d 1.3770.02e 1.3670.01e 1.3670.04e 1.4170.01f
0.7370.03a 0.8670.01b 0.9770.04c 0.9970.06c 1.0870.06d 1.1770.03e 1.2170.04e 1.3270.04f
183
a
Mean7SD from triplicate determinations. Different letters in the same column indicate significant differences (Po0:05). b
MHC
Actin
(A)
1
2
3
4
5
6
7
8
(B) 1
2
3
4
5
(C)
1
2
3
4
5
6
7
8
(D) 1
2
3
4
5
6
6
7
7
8
8
Fig. 8. SDS-PAGE pattern of surimi gels added with PPP and subjected to setting for different times. A: bigeye snapper; B: bigeye croaker; C: threadfin bream; D: barracuda; lane 1: surimi sol; lane 2, 3, 4, 5, 6, 7, 8: setting time of 0, 0.5, 1. 1.5, 2, 2.5 and 3, respectively.
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4. Conclusion Porcine plasma protein at an appropriate level in combination with setting at 40 for a proper time effectively enhanced surimi gel strength. The optimum conditions varied from species to species. Generally, an excessive amount of PPP and extended setting caused the decrease in gel strength of surimi.
Acknowledgements Authors would like to express their sincere thank to International Foundation for Science for Project No. E/ 2878-2 and Prince of Songkla University for the financial support.
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