Contribution of Sarcoplasmic Proteins to Myofibrillar Proteins Gelation

Authors

  • Ali Jafarpour,

    1. Authors Jafarpour and Gorczyca are with the School of Applied Sciences, RMIT Univ., Melbourne, Victoria, 3001 Australia. Author Jafarpour is also with the Dept. of Fishery, Faculty of Animal Science and Fishery, Sari Agricultural Sciences and Natural Resources Univ. (SANRU), Mazandaran, Iran. Direct inquiries to author Jafarpour (E-mail: ali.jafarpour@rmit.edu.au and a.jafarpour@sanru.ac.ir).
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  • Elisabeth M. Gorczyca

    1. Authors Jafarpour and Gorczyca are with the School of Applied Sciences, RMIT Univ., Melbourne, Victoria, 3001 Australia. Author Jafarpour is also with the Dept. of Fishery, Faculty of Animal Science and Fishery, Sari Agricultural Sciences and Natural Resources Univ. (SANRU), Mazandaran, Iran. Direct inquiries to author Jafarpour (E-mail: ali.jafarpour@rmit.edu.au and a.jafarpour@sanru.ac.ir).
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Abstract

Abstract:  Surimi, a refined protein extract, is produced by solubilizing myofibrillar proteins during the comminuting and salting stages of manufacturing. The resulting paste gels on heating to produce kamaboko or a range of analog shellfish such as crab claw, filament sticks, fish mushroom, and so on. The myosin molecule is the major myofibrillar protein in gelation. It is believed that washing steps during the traditional surimi process play an important role in enhancing the gel properties of the resultant kamaboko by removing water-soluble (sarcoplasmic, Sp-P) proteins. By contrast, some researchers claim that retaining Sp-P or adding it into the surimi gel network not only does not interfere with the action of myofibrillar proteins during the sol–gel transition step but also improves the gel characteristics of the resultant kamaboko. It seems that retention of Sp-P or their addition into raw surimi does enhance the textural properties of kamaboko gel perhaps by functioning as a proteinase inhibitor, particularly against trypsin and trypsin-like proteinases but this depends on the type of applied surimi process. Among different types of Sp-P, it has been claimed that some proteins such as endogenous transglutaminase (TGase) play a more important role than other Sp-P in bond formation, by catalyzing the cross-linking of myosin heavy chain (MHC) molecules during low-temperature setting of surimi, resulting a more elastic kamaboko gel.

Introduction

Surimi gel, as concentrated myofibrillar proteins (Mf-P) and its subsequent thermally treated product (known as kamaboko) is considered a viscoelastic gel system. Tejada (1994), quoting from Tanaka (1981), defined a gel structure as “a form of matter intermediate between a solid and a liquid, consisting of strands or chains cross-linked to create a continuous network immersed in a liquid medium. Hence, gels are space-filling, three-dimensional structures.” In order to attain an orderly thermo-irreversible myofibrillar gel network, energy is needed to be supplied by heating of surimi paste at 90 °C. However, in order to enhance the extent of myofibrillar gelation process, it needs that the surimi paste to be set at a specific time and temperature. Setting is responsible for the formation of a partial gel network by a gradual sol–gel transition process mainly at low temperature (<40 °C), due to the enzymatic action of endogenous transglutaminase. This process causes an enhancement of the textural properties of the myofibrillar gel network, specifically the elasticity of its final product (Suzuki 1981; Sano and others 1988).

Surimi gel is assessed mainly in terms of color and texture (Hamann and MacDonald 1992; Kim BY and others 2005; Lauro and others 2005). The textural quality of surimi and kamaboko is measured in a number of ways including: force and stress; deformation and strain; rheological tests (using either small strains such as stress relaxation and oscillatory dynamics or large strains such as axial compression and torsion tests), and empirical tests (such as puncture test and texture profile analysis TPA; Kim YS and others 2005).

Each of these methods ostensibly measures a different textural characteristic of surimi and kamaboko. However, the basis of all these measurements is to measure the extent to which gelling has occurred, which is a key characteristic of surimi and kamaboko (Szczesniak 1963; Breen 1975; Lee and Toledo 1976; Salfi and others 1985; Lee and Chung 1989; Pons and Fiszman 1996; Shie and Park 1999; Bourne 2002; Yoshioka and Yamada 2002; Wiles and others 2004). The quality of the surimi and kamaboko gel depends on a number of factors, such as species (Hastings and others 1990; Reppond and others 1995; Klesk and others 2000; Ramirez and others 2000; Benjakul and others 2002; Kristinsson and others 2005; Jafarpour and Gorczyca 2008), processing variables (Hsu 1990) such as washing agents (Benjakul and others 2004; Jafarpour and others 2008), moisture content (Reppond and others 1995; Reppond and Babbitt 1997), salt and pH (Alvarez and others 1995; Ni and others 2001), processing methods (Perez-Mateos and others 2004; Jafarpour and Gorczyca 2008) such as washing with plain water in conventional method, modified conventional method by replacing centrifugation instead of manual dewatering, and acid- and alkali-aided solubilization processes.

The mechanism of gelling has been thoroughly reviewed and discussed by Stone and Stanley (1992). These authors and some others (Sato and Tsuchiya 1970; Nakayama and Sato 1971; Grabowska and Sikorski 1976) have clearly shown that Mf-P, specifically myosin and actomyosin, have the main roles in fish protein gelling. Myosin is the main portion of Mf-P that is responsible for the gelling characteristics of the actomyosin gel. The essential attribute of myosin is its heavy chain portion. Tropomyosin is another portion of actomyosin but seems to have no effect on the Mf-P gelation process. To form the gels, the myofibrils must be solubilized to F-actin and myosin, followed by repolymerization to actomyosin to form an actomyosin sol produced by the dispersed myofibrillar protein, which retains water (Tejada 1994). Hence, as the Mf-P gel entraps water molecules and forms a continuous protein–water cross-linked matrix, it is considered a hydrogel system.

Sarcoplasmic proteins (Sp-P) are another major component of fish muscle (up to 35%). Okada (1964) believed that this group of proteins do not provide an elastic gel after heating, and interfere with the gel-forming ability of the Mf-P if they are not removed by washing during surimi processing. However, there is no consensus on the role of Sp-P, as some authors (Okada 1964; Nakayama and Sato 1971; Nakagawa and Nagayama 1988; Nakagawa and others 1988b, 1988a, 1989) believe that Sp-P negatively impact on the surimi gel texture, and some authors (Kim and Park 2001; Park and others 2003a; Jafarpour and Gorczyca 2009) claiming an improvement in surimi gelation process by addition of Sp-P into suimi paste.

The conventional method for processing involves 1 to 3 or more water washing cycles, which results in the loss of Sp-P. One of the main aims of these washing steps is to remove Sp-P as well as blood, fat, skin, scales, and other remnants. The historical reason why washing was seen as a necessary step in surimi processing was the need to extend the storage time of washed mince by removing the water-soluble Sp-P. These were regarded as detrimental to the gelling quality of the resultant surimi and kamaboko. This was thereafter supported by researchers who concluded that Sp-P had a negative effect on the gelling process (Nakagawa and others 1989; Morioka and Shimizu 1990; Ko and Hwang 1995a, 1995b; Kim and others 1996). A second reason for washing was to avoid darkening of the surimi produced, particularly if dark-fleshed fish were used (Reppond and others 1995; Jiang and others 1998; Hultin and Kelleher 2000). With the advent of the pH-shift (acid- or alkaline-aided) approach to surimi processing (Nishioka and others 1983; Hultin and Kelleher 1997) and the adding back of the Sp-P (Morioka and Shimizu 1990; Morioka and others 1992; Morioka and Shimizu 1992, 1993; Ko and Hwang 1995a, 1995b; Nowsad and others 1995a, 1995b; Park and others 2003a; Kim YS and others 2005; Jafarpour and Gorczyca 2009), it is possible to use data from these studies to resolve the issue of whether Sp-P enhance or inhibit the gelling quality of surimi gel.

The purpose of this article is to review these studies to elucidate the controversial role of Sp-P in the textural characteristics of surimi gel. An understanding of the important characteristics (location, quantity, extractability, and so on) of Sp-P will clarify the role the Sp-P have in gel formation as well as explain some of the contradictory results obtained in the literature. Hence, the 1st section will review some Sp-P characteristics such as their definition, quantity, function, and extractability, and in the 2nd section the role of Sp-P in enhancing and inhibiting the gel characteristics of surimi gel will be discussed.

General Issues Associated with Sp-P

Scopes (1970) identified Sp-P based on the extraction methodology, when he stated that “an extract of pre-rigor muscle made, for instance, with a dilute Tris buffer to ensure that the pH of the homogenate does not fall below 7.0, and centrifuged at 15000 to 20000 ×g, can be defined as an extract of sarcoplasmic proteins.” He defined Sp-P according to their function, that is “to carry out the normal function of cellular metabolism” and so the many enzymes responsible for energy production in the muscle are included as Sp-P; these include enzymes, such as those involved in glycolysis, the citrate cycle, and oxidative phosphorylation. Toyohara and others (1983) reported the presence in fish muscle of other enzymes such as cathepsin A, cathepsin D, a sub-endopeptidase, a calcium-dependent cysteine proteinase known as calpain and its specific inhibitor, calpastatin, and even a trypsin inhibitor. An examination of the location of Sp-P in the cell may help in understanding of the role (positive/negative) of these proteins in gel formation.

The Distribution of the Sp-P Molecular Weights (MWs)

Our knowledge of the MW distribution of Sp-P can explain the behavior of Sp-P during gel formation. According to Asghar and others (1985), Sp-P are of relatively low MW, globular or rod-shaped, and have low viscosity. The Sp-P listed in Table 1 have MW ranging from 18 kDa to 360 kDa, with about 54% (of 55 mg/g) of the Sp-P having MW >120 kDa. This distribution of MW does not seem to be consistent with Asghar and others' (1985) description of “relatively low molecular weight” for Sp-P. This discrepancy can be explained because the MW of each protein in its quaternary form is reported in Table 1, rather than the MW of each of the subunits in the quaternary structure. For example, the stated MW for aldolase (ALD) is 160 kDa, which is categorized as high MW, whereas the MW of each of the 4 subunits of ALD is only 40 kDa. Although Table 1 is not a complete list of Sp-P, Scopes (1970) found that the enzyme with the highest concentration (20%) was glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme with a MW of 146 kDa. Furthermore, only some 6 glycolytic enzymes accounted for about 63% of the Sp-P, with 4 of these enzymes having high (>140 kDa) MW.

Table 1–.  Relative proportion of glycolytic and associated enzymes (adapted from Scopes 1970]).
Type of protein Sarcoplasmic proteinsQuantity (mg/g)Molecular weight (KDa)
  1. aMolecular weight sourced from Moosavi−nasab (2003).

  2. V: variable molecular weights; −: information not available.

Glyceraldehyde phosphate dehydrogenase12.0146
Aldolase6.0160
Creatine kinase5.082
Enolase5.047.3
Lactate dehydrogenase4.0140
Pyruvate kinase3.0237
Phosphorylase b2.5180
Triose phosphate isomerase2.045
Phosphoglucomutase1.562
“F protein”1.535
Phosphoglycerate kinase1.294
Phosphoglucose isomerase1.0130
Phosphofructokinase1.0360
Phosphoglycerate mutase1.065
α-Glycerophosphate dehydrogenase0.560
Myokinase0.521
MyoglobinApproximately 0.518
AMP deaminase0.2300
Others6.6V
Total55 
 QuantityMolecular
Myofibrillar proteins(mg/g)weighta (KDa)
Myosin (M)65MHC (200)
   and MLC (20)
actin2543
Tropomyosin (Tm)Approximately 15α-TM (34)
   and β-TM (36)
Troponin (Tn)Approximately 4Tn T (31), Tn I (21)
   and Tn C (18)
α-ActininApproximately 395
C-Proteins135
OthersApproximately 8V
Total115 

Jafarpour and Gorczyca (2008) stated that during the alkali-aided method of preparing surimi, after the 2nd centrifugation only those Sp-P with MW < 60 kDa were present in the supernatant and the rest (MW > 60 kDa) were retained inside the surimi paste. This is in agreement with Scopes’ statement (1970) that Sp-P with high MW are difficult to extract with plain water as they are naturally salt soluble inside the muscle. Subsequently, the gel quality (breaking force, breaking distance, and gel strength) of the resultant surimi and kamaboko gels was significantly (P < 0.05) greater than that of the control (traditionally prepared surimi). One possible explanation is that specific Sp-P contribute to gelling while other Sp-P cannot form links to assist with gel formation, provided the ionic strength is kept equal; however, it does not mean that only Sp-P with higher MWs contribute to the enhancement of surimi gel strength. Significantly, Morioka and Shimizu (1993) found a reasonable correlation (0.81) between “jelly” strength and specific Sp-P; in particular those with MW of 94 kDa, 40 kDa, and 26 kDa (Macfarlane and others 1977) whereas other Sp-P of different MWs could not be correlated with “jelly” strength.

The Amount of Sp-P Present in the Muscle and Their Extractability

According to Scopes (1970) the estimated concentration of Sp-P was about 16% in both the myofibril and the sarcoplasm around the myofibril, but the concentration varies with the fish species. Using a muscle cross section with a light microscope, Scopes (1970) estimated the “volume ratio” of myofibrils and sarcoplasm to be 3:1 in situ. It is well known, however, that Sp-P are not evenly distributed throughout fish muscle. Furthermore, dark-fleshed fish contain a greater amount of Sp-P in their muscle than white-fleshed species. For example, the amount of Sp-P is 650 to 800 mg/100 g of muscle in the dark-fleshed Pacific mackerel and sardines, almost 3 times greater than that found in the light-fleshed species such as Alaska Pollock, with about 187 g/100 g of muscle. Shimizu and others (1976) noted that it is difficult to produce a surimi with a “good” gel from dark muscle.

The extractability of the Sp-P may also affect gel quality. Scopes (1970) pointed out that dark-muscle fish contain, in the sarcoplasmic fraction, a “higher ratio of high molecular weight” proteins, which are difficult to extract with water, despite their classification as “water soluble.” This is because the proteins occur naturally in a salt solution in the tissue; that is, they are salt soluble and the addition of water during washing reduces the salt concentration and makes them less extractable (Shimizu and Ikeda 1979; Hultin and Kelleher 2000). Sp-P and Mf-P can be separated by their solubility, as Sp-P are soluble at low ionic strength whereas Mf-P are soluble in “more concentrated salt solutions” (Scopes 1970) with ionic strengths greater than 0.3 M (Hultin and Kelleher 2000). However, Shimizu and others (1976) claimed that optimal extraction of Sp-P from either dark or white muscle fish species occurred at I= 0.05. Also, the extractability of glycolytic enzymes from red sea bream, Pacific mackerel, and carp indicated that the extent of extraction was dependent on the concentration of KCl “up to 0.1 M, irrespective of fish species” (Nakagawa and others 1988a).

In 1983 Nishioka and others were involved in a study to isolate Sp-P from leachate water produced during surimi manufacturing by the pH-shifting technique. According to their results, 20 min of either acidification or alkalization (and holding for another 20 min after neutralizing at 25 °C) was the optimum treatment with a Sp-P recovery level of 94.4%. The efficiency of the recovery was dependent on the muscle type (dark compared with white); the recovery of 97% to 98% Sp-P from the dark-muscle Frigate mackerel and sardine was greater than the 70% to 72% Sp-P recovery from white-muscle fish species such as Flathead and Grub fish (Nishioka and others 1983).

Ionic strength may also play a part in Sp-P extraction and hence gel strength. Shimizu and others (1976) found a positive linear relationship between extractability of water-soluble proteins and the amount of dark muscle in 18 fish species by raising the ionic strength (I) from 0 to 0.5. An increase in the ionic strength from 0 to 0.05 at a ratio of 1:30 (muscle to extractant) resulted in a 24% increase in extracted protein from sardines, which have a greater accumulation of Sp-P in their dark muscle than in their white muscle, whereas the percentage increase was only about 4.0% for Alaska pollack; a white-muscle demersal fish species that has less water-soluble Sp-P (Figure 1). Even though, Shimizu and others (1976) stated that Sp-P from the dark-muscle fish species precipitated if the ionic strength was decreased to less than 0.1, however, Shimizu and Ikeda (1979) claimed that the “rate of precipitation” was a function of Mf-P level rather than ionic strength. Accordingly, in the presence of Mf-P in dilute solution (1:22.5 press juice:deionized water) the “rate of precipitation” of Sp-P increased from 5% to 6%, 58% in the absence and presence of Mf-P, respectively. They hypothesized that at low ionic strength, the extraction of Sp-P from the dark-muscle fish species was reduced mainly by the interaction between myogen and Mf-P, not the high globulin content of the Sp-P. Quoting from Nakagawa and Nagayama (1988), the extractability of proteins such as Sp-P cannot necessarily be directly equated with their solubility. In addition, some Sp-P although soluble in a specific solvent, may not be extractable from muscle tissue in that same solvent because they may be located in a site inaccessible to the solvent or are bound rather than “free” (Hultin and Kelleher 2000).

Figure 1–.

Extractability of proteins of muscle homogenate from 3 species of dark and light muscle fish in the low ionic strength region (adapted from Shimizu and others [1992]).

Evidence for Inhibitory Effects of Sp-P on Gel Formation

The presence of some Sp-P can potentially destroy the integrity of Mf-P, specifically during the postmortem period. Some studies (Table 2) involving different fish species have concluded that the presence of some Sp-P in the raw surimi is detrimental to the qualities of the resultant kamaboko gel prepared by conventional methods.

Table 2–.  Inhibitory role of sarcoplasmic proteins on the kamaboko gel properties based on the type of applied method.
NrApplied methodFish speciesMeasured parameter(s)ConclusionAuthor(s)
1Washing compared with nonwashingArrow-tooth flounder and the Saury-pikeJelly strength and expressible waterThe well-washed meat formed an elastic jelly with a high jelly strength and little expressible water, while the control (unwashed meat) gave an easily crushable-brittle jellyOkada (1964)
2 Pacific coast species such as Lingcod, Pacific Cod, and so onTexture elasticity moisture contentWashing treatment caused better gel-forming ability in some species which are being divided into 3 groups (excellent, good, and poor grade)Kudo and others (1973)
3 Red sea bream, Pacific mackerel, and CarpGel strength (g cm)Gel strength of kamaboko from washed mince was 3 times greater than the one from nonwashedNakagawa and others (1989)
4 Pacific Mackerel“Jelly” strength (g cm)It was noted that at the same water content, washing Pacific mackerel mince improved the gel strength of the resultant kamaboko by a factor of 5 over unwashed minceMorioka and Shimizu (1990)
5 Milk fishGel strength (g mm)There was an improvement (1.4 to 2.0 times) in the kamaboko gel strength produced from washed mince compared with that produced from unwashed minceKo and Hwang (1995a, 1995b)
6 Catfish minceL value, penetration forceThe lack of washing of catfish mince had a negative impact on the textural properties of resultant kamabokoKim and others (1996)

In these studies, a range of techniques was used to assess the surimi gel quality in terms of jelly strength, breaking force, breaking distance, penetration force, expressible water, L value, and whiteness. For example, using Arrow-tooth flounder (Atheresthes stomias) and Saury-pike (Scomberosox saurus), Okada (1964) demonstrated that when fish muscle was washed (1 to 10 times) an “elastic jelly with high 'jelly' strength and little (33% to 41%) expressible water” was produced. Unwashed muscle produced, by contrast, only an “easily crushable brittle jelly” with more expressible water (54% to 64%). In another study on 4 fish species: Rockfish (Sebasted spp.), Starry flounder (Platichthys stellatus), Puget sound hake (Merluccius productus), and Spiny dogfish (Squalus acanthias), Kudo and others (1973) concluded that washing fish mince with cold water was an effective method for removal of water-soluble proteins and resulted in a firmer gel compared with gels from unwashed mince. At the same water content, washing Pacific mackerel mince improved the gel strength of the resultant kamaboko by a factor of 5 over unwashed mince (Morioka and Shimizu 1990). Ko and Hwang (1995a, 1995b) found an improvement (1.4 to 2.0 times) in the kamaboko gel strength produced from washed milkfish (Chanos chanos) mince compared with that produced from unwashed mince within a moisture range of 78% to 88%. Kim and others (1996) also reported that not washing catfish mince reduced the desirable textural properties of the resultant kamaboko.

The explanations given for the improvement in textural quality of kamaboko after washing vary and have included the following:

  • 1that the jelly strength improved because washing removed interfering components (Sp-P, Okada 1964; Kudo and others 1973) and fat (Okada 1964);
  • 2that washing increased the concentration of myosin (Okada 1964);
  • 3that some sarcoplasmic enzymes, specifically ALD and GAPDH bind with fish myofibrils (Nakagawa and others 1989), specifically with actomyosin, making it less available for cross-linking (Shimizu and Ikeda 1979) and that this interaction results in weaker gel formation;
  • 4that the presence of cathepsin L and heat-stable alkaline proteases, and their action on Mf-P by breaking the long polymer chain into short polymer chains in the unwashed mince is responsible for the lower hardness and breaking force in the gel compared with the gel produced from washed mince (Kim and others 1996). This issue has been thoroughly reviewed by An and others (1996).

Some support was given to explanations (1) and (4) by a study by Nakagawa and others (1989), who demonstrated a general inverse relationship between specific Sp-P, namely ALD and to a lesser extent GAPDH, and the gel strength of kamaboko processed from 3 fish species: red sea bream (Pagrus mojor), Pacific mackerel (Scomber japonicus), and carp (Cyprinus carpio). As the ALD concentration was decreased by washing in solutions of increasing salt concentration (from 0 to 0.15 M) at neutral pH, the gel strength of samples increased. Any increase in the strength of surimi gels can be directly attributed to the absence of some Sp-P, not presence of more Mf-P, since Mf-P only start to go into solution once the ionic strength exceeds 0.15 M and increases to 0.96 M NaCl.

Furthermore, Okada (1964) reported that when heated and deproteinized wash water was added to the raw surimi paste, it showed no impact on surimi gel strength, but by addition of wash water containing undegraded Sp-P, the texture quality of resultant surimi gel was increased as it formed a firmer gel with less expressible water (48%) compared with the deproteinized samples that produced a gel with expressible water of approximately 60% (Table 3).

Table 3.  Table 3–Effect of water-soluble extractives added to washed meat Arrow-tooth flounder (Atheresthes stomias) on jelly-forming ability (adapted from Okada [1964]).
AdditivesProperties of kamaboko gel
Appearance“Jelly” strength Expressible water (%)
  1. aDeproteinization carried out by removing precipitated protein after heating of the wash water.

  2. bThese ingredients were dissolved in hydrochloric acid solution and then separated.

Whole extractivesBrittle20859.7
Deproteinized extractivesaElastic30548.4
Inorganic constituentsbElastic29550.3
Water (control)Elastic26852.9

Together, these studies lead to the conclusion that either the presence of Sp-P in unwashed fish mince or the addition of Sp-P solution in the surimi gel network affected the desirable kamaboko gel properties.

Evidence for Enhancement of Gel Formation by Sp-P, Specifically TGase

Unlike the evidence for the inhibitory effect of Sp-P on gel strength, discussed in the previous section, several studies involving different fish species have concluded that the presence of Sp-P in surimi enhances the textural characteristics of the resulting kamaboko prepared by conventional methods (Table 4). Washing trials demonstrating that Sp-P are not inhibitory to desirable textural characteristics are limited to those carried out by Morioka and Shimizu (1993). Surprisingly, these researchers reported that washing of Pacific mackerel mince reduced the gel strength by a factor of about 1.5 compared with that produced from unwashed mince at the same myofibril content. By contrast, Seki and others (1990) demonstrated that the addition of a soluble extract from Alaska Pollock muscle and surimi to carp myosin B incubated at 25 °C greatly stimulated the cross-linking reaction of myosin heavy chains (MHCs). These authors indicated that such enhancement was not observed if the soluble extract was heated and attributed the difference to the “presence of an active enzyme, transglutaminase, in the extract.” This enzyme catalyzes the acyl transition between the γ-carboxyamide group of the glutamine residue, as an acyl donor, and the amino group of the ɛ-lysine residue, as an acyl receptor, mainly during low-temperature setting, and forms a ɛ-(γ-glutamyl)lysine linkage that is resistant to proteolysis.

Table 4–.  Enhancive role of sarcoplasmic proteins on the surimi gel properties based on the type of applied method.
NrApplied methodFish speciesMeasured parameter(s)ConclusionAuthor(s)
 1Washing compared with nonwashingThreadfin breamJelly strength (g cm)For the same myofibrillar content, the nonwashed gave gels approximately 1.5 times stronger than the washed oneMorioka and Shimizu (1990)
 2Heat-coagulability of Sp-PYellow tuna, Pacific mackerel, Carp, and so onSDS-PAGE and solubility curveSp-P components bound to the actomyosin molecules one after another at their coagulation temperatures and remained suspended in the solutionMorioka and Shimizu (1992)
 3 Pacific mackerel, Sardine, and so onJelly strength (g cm)Sp-P with molecular weights of 94-, and 40-kDa showed a good correlation with the jelly strengthMacfarlane and others (1977)
 4Acid–alkaline methodRockfish kamabokoColor, storage modulusThe retention of sarcoplasmic proteins at the gel network did not interfere with the gel characteristic of surimiYongsawatdugul and Park (2004)
 5  Color and gel strengthThey found no indication that the sarcoplasmic proteins interfered with gel formation, and there is further evidence that sarcoplasmic proteins may actually enhance gelationHultin and Kelleher (2000)
 6 Atlantic croakerBreaking force (g) Deformation (mm)Kamaboko gel from alkaline-aide method showed significantly (P < 0.05) greater breaking force but equal deformationPerez-Mateos and others (2004)
 7 Common carpWhiteness, breaking force (g) and breaking distance (mm)Resultant kamaboko had lower breaking force and breaking distance compared with that from conventional methodJafarpour and Gorczyca (2008)
 8Added microbial TGaseAtlantic croakerGel strength (g cm)Induced the higher gel strength and deformability compare to the conventionally washed surimiPerez-Mateos and others (2004)
 9Added Sp-PThreadfin breamGel strength (g cm)“Sp-P does not interfere with gel formation of Mf-P.”Morioka and Shimizu (1990)
10 MilkfishGel strength (g cm)The gel strength in both washed and unwashed meat paste was increasedKo and Hwang (1995a, 1995b)
11 Spanish mackerelBreaking force, MHCClarified that the advantage of the presence of sarcoplasmic proteins is due to endogenous transglutaminase (TGase)Nowsad and others (1995a)
12 Japanese Jack mackerelColor and Punch testThe addition of sarcoplasmic protein increased firmness of the resultant gelPark and others (2003a)
13 Alaska PollockGel strength and temperature weep testSp-P significantly increased the breaking force and slightly decreased the breaking distance compared to the control (no sarcoplasmic protein addition)Kim YS and others 2005)
14 Common carpBreaking force (g) and breaking distance (mm)Addition of Sp-P caused a significant increase in both breaking force and breaking distanceJafarpour and Gorczyca (2009)

The most convincing evidence for enhancement of gel characteristics by Sp-P has come from experiments in which Sp-P have been added back to the raw surimi. Morioka and Shimizu (1990) showed that the addition of Sp-P from Pacific mackerel into threadfin bream myofibril at a ratio of 1:3 (Sp-P:Mf-P) resulted in the kamaboko having greater (approximately 54%) gel strength than that of the control (without added Sp-P, Table 5). This positive effect of Sp-P on gel strength was also demonstrated by Ko and Hwang (1995a, 1995b) in an experiment using washed and unwashed milkfish to which Sp-P (3.2 mg/g) was added. The gel strengths of the resultant kamaboko were increased for both washed and unwashed surimi by 4% and 21%, respectively, when compared with the control to which no Sp-P were added (only water to maintain a volume balance). Nowsad and others (1995b) also found that the breaking force of the suwari gel made from Alaska Pollock, carp, horse mackerel, sardine, and Spanish mackerel was improved by the addition of 3% to 4% Sp-P at the same water content level. The authors noted that the cross-linking of MHC molecules in the suwari gel had increased with the addition of Sp-P mainly because of the “enzymic action of TGase,” which mediated the cross-linking reaction of MHC and subsequent increase in percentage of cross-linked myosin heavy chains (CMHC) while reducing the MHC molecules. The role of TGase on protein gelation has been widely investigated by many researchers (Folk 1980; Seki and others 1990; Araki and Seki 1993; Joseph and others 1994; Yasueda and others 1994; Nowsad and others 1995a; Yongsawatdigul and others 2002; Worratao and Yongsawatdigul 2005). In 2007, Yongsawatdigul and Piyadhammaviboon reported that Sp-P concentrate increased cross-linking of MHC and tropon in tilapia surimi, mainly due to the catalytic activity of TGase. Yongsawatdigul and Hemung (2010) reported that some fractions of Sp-P act as a proteinase inhibitor, mainly against trypsin and/or trypsin-like proteinases, thus preventing MHC degradation, and consequently improved lizardfish surimi textural properties when set at 37 to 40 °C. This issue was endorsed in a study conducted by Hu and others (2010) who stated that cysteine protease cathepsin L is responsible for surimi gel disintegration during heating process.

Table 5–.  Effect of native and heat-coagulated sarcoplasmic proteins on gel-forming of threadfin bream myofibrils (adapted from Morioka and Shimizu [1990]).
AdditivesPuncture force (g)Puncture depth (cm)Gel strength (g cm)
  1. Sp-P was prepared from Pacific mackerel by homogenizing its dorsal muscle with 5 volumes of phosphate buffer (I= 0.05, pH = 7.0) and concentrating the extract obtained to 85% water content. Myofibrils were prepared from threadfin bream by homogenizing its dorsal muscle with 5 volumes of buffer (0.09 M KCl, 5mM EDTA, 0.039 M borate buffer, pH = 7.0), washing with the same buffer solution 4 times, dehydration to 88% water content, and adding 5% sucrose.

  2. aWater was added to myofibril (84% moisture, 5% sucrose) instead of Sp-P.

  3. bNative Sp-P added gel (Mf:Sp-P ratio of 3:1).

  4. cHeat coagulated Sp-P added gel (Mf:Sp-P ration of 3:1).

Controla55.20.4424.3
+Sp-Pb67.20.5637.6
+DSp-Pc39.60.4317.0

Some of the statements (or explanations) given by the various authors for the above-mentioned results include the following:

  • 1that Sp-P do not interfere with gel formation of Mf-P (Morioka and Shimizu 1990);
  • 2that Sp-P contribute positively to gel strength, possibly by cross-linking with MHC (Nowsad and others 1995a, 1995b);
  • 3that the alkali-aided method of producing surimi renders the muscle proteins more accessible as a substrate for TGase as well as to other protein–protein interactions (Perez-Mateos and others 2004);
  • 4that Sp-P binds to actomyosin, with the resulting complex remaining suspended even after high speed (12000 × g) centrifugation (Morioka and Shimizu 1992);
  • 5that the Sp-P either act as proteinase inhibitors and prevent the degradation of MHC or catalyze the cross-linking of Mf-P and thereby enhance the textural characteristics of surimi gel (Piyadhammaviboon and Yongsawatdigul 2009, 2010).

Macfarlane and others (1977) noted that the “jelly” strength of Sp-P could be correlated with (i) heat coagulation of specific Sp-P and (ii) Sp-P with high MWs (94 kDa, 40 kDa, and 26 kDa) with a correlation coefficient of 0.81. Morioka and Shimizu (1992) demonstrated that the coagulation temperature of Sp-P from white muscle was specific to a fish species. For example, 91% of Sp-P from the white muscle of sardine coagulated at 60 °C, whereas at the same temperature only 29% of the Sp-P from the white muscle of Tilapia coagulated. More protein remained in solution than expected when a solution of actomyosin:Sp-P (2:1) was heated, particularly over the temperature range of 60 to 80 °C. Moreover, Karthikeyan and others (2004) stated that different fractions of Sp-P have different impacts on thermal gelation of Mf-P; those with a MW of 36 to 55 kDa had the greatest effect on enhancement of viscoelastic properties, particularly storage modulus, whereas those with greater MW (97 kDa) showed lesser effects. In another study, Miyaguchi and others (2007) reported that only GAPDH (37 kDa), enhanced gel strength among different fractions of Sp-P such as phosphorylase b (PHb, 97 kDa), enolase (EN, 43 kDa), actin (AC, 45 kDa), and phosphoglycerate mutase (PGM, 30 kDa), and other proteins such as bovine plasma, egg white, and soy protein isolate. This was attributed to a greater dissociation of actomyosin at lower temperature (during setting process) and more effective interaction between GAPDH and Mf-P after heating.

Point (4) mentioned above summarizes the significance of these results. However, the authors (Jafarpour and Gorczyca) disagree with the conclusion (IV) of Morioka and Shimizu (1992), since if the Sp-P reacted with actomyosin then the MW of the complex would be expected to increase and hence be removed by centrifugation, provided that the concentrations of actomyosin and Sp-P exceeded those critical for gel formation. If the respective solutions were too dilute, then it is possible that the proteins would remain in solution after heating because no gel was formed.

The enhancement of the surimi gel strength has been demonstrated by results of different experimental methods, including: washing compared with nonwashing, adding the heat-treated wash water compared with unheated wash water, acid- and alkali-aided methods compared with the conventional method, and the addition of isolated Sp-P into the surimi compared with no Sp-P addition (Table 3). Of all the methods, the most conclusive evidence was obtained by adding isolated Sp-P into the fish mince and evaluating the gel characteristics of the resultant surimi and kamaboko. Other studies on the effect of endogenous transglutaminase on the surimi gel characteristics (Seki and others 1990; Nowsad and others 1995a, 1995b; An and others 1996) have shown that the increase in kamaboko gel strength can be partially attributed to endogenous TGase activity. In 1993, Araki and Seki determined the reactivity of TGase to various fish actomyosins in terms of polymerization rate of MHC. An and others (1996) reviewing the role of endogenous enzymes on surimi gelation, stated that transglutaminase function as a catalyzer agent in order to facilitate the acyl-transfer reactions between “the γ-carboxyamide group of peptide-bound glutamine” and “the ɛ-amino group of peptide-bound lysine” resulting in their cross-linking and hence improvement in gel strength of surimi. Now there is a question mark; is TGase enzyme the only predominant contributive to the interaction of protein molecules in order to enhance the gel formation of surimi? To answer this question, Banlue and others (2010) carried out a study on the effect of KBrO3 on gel forming of surimi with and without TGase inhibitor, and interestingly stated that addition of KBrO3 is capable of increasing of surimi gel strength mainly via oxidation of sulfhydryl compounds to disulfide bounds of MHC and inactivation of proteinase during heat setting, but “its contribution seems not to be cooperative with that of TGase.” However, this issue needs to be investigated extensively.

An explanation is needed as to why similar experiments have led to contradictory conclusions with respect to the role of Sp-P in gel formation during surimi processing. The enhancive effects of either retaining Sp-P in the surimi or adding back Sp-P are largely associated with surimi that has been produced by the pH-shift method. The 1st item that needs to be demonstrated is that the surimi produced by the conventional method is not identical in all respects to the surimi produced by the pH-shift method in terms of composition and particularly integrity of the myofilaments (myofibrils). Once that has been shown, it needs to be demonstrated that the enhancive effect is not simply due to a difference in ionic concentration in the 2 systems. The role of ionic strength on extractability of Sp-P has been discussed earlier in this study. It also needs to be confirmed that the thick filaments from the pH-shift method are similar to conventionally processed thick filaments. This is discussed below.

Retained Sp-P by pH-shift Treatment and Its Contribution to Mf-P Gelation

In 1983, Nishioka and others used the pH-shifting technique to separate water-soluble proteins, lost during the dewatering stage of traditional surimi processing. By 1997 Hultin and Kelleher had developed and patented a processing method that also resulted in the recovery of proteins, specifically Sp-P from the dark muscle of fish species. This recovered Sp-P could then be used for further experimentation. Some studies used the pH-shift technique to isolate Mf-P in order to make surimi and found that the acid–alkali-aided method resulted in the retention of more Sp-P. Some researchers subsequently reported positive effects of retaining Sp-P on the surimi gel network whereas others reported the negative effects. These results are not compatible with the role of Sp-P in gelation of Mf-P or surimi gel prepared by the conventional method, as Mf-P and Sp-P subjected to pH treatment are in different states of structure and conformation from native proteins. It is not correct to assume that the 3 methods of preparing surimi produce an identical product, and that the sarcoplasmic fractions obtained from the various methods of surimi production are likewise similar.

Hence, differences found in the gelation properties and other physical measurements of the resulting surimi and kamaboko gels cannot be attributed to the presence or absence of Sp-P unless there is proof that the myofibrillar and sarcoplasmic fractions from the 3 surimi production methods are the same. The pH-shift acid and alkali treatments induce major conformational changes to the proteins in the Mf-P and the Sp-P. Given the extreme pH values to which the system is exposed prior to the separation of the Sp-P (pH 2.0 in the case of the acid process, pH > 9.0 in the alkaline process), it would be most surprising if the swelling of proteins at these pH changes does not impart permanent structural changes and that these changes affect their reactivity. It is almost certain that the thick filaments in particular will be damaged when the pH is either dropped to 2 or raised to pH > 9.0. At these pH values, the proteins swell significantly compared to neutral pH. Once the thick filaments are broken up, myosin will readily go into solution when 2% salt is added to the system, resulting in the pH-adjusted surimi having a greater gelling strength than conventionally processed surimi.

Another concern about the pH-adjusted system is that the ionic strength must be greater than for conventionally processed surimi, as there is no process to remove the salts naturally present in fish sarcoplasm, whereas there is in the conventional process. Moreover, the ionic strength is increased further by the addition of acid or alkali and their subsequent neutralization in the pH-adjusted method. Myofibrillar protein solubility increases with sodium chloride concentration until the salt concentration reaches 0.96 M. Generally, the more myosin that is in solution the greater is the gel strength. Furthermore, Yongsawatdigul and Hemung (2010) reported that by application of pH 3 and pH 12 treatments on threadfin bream Sp-P, the water holding capacity of acid- and alkaline-treated Sp-P was increased about 6.5 and 5.5 folds, respectively, in comparison with the crude counterpart. This implies that Sp-P goes under significant structural and conformational changes upon treatment with either extreme acid or alkaline pH followed by neutralization to pH 7.0.

Evidence for the Negative Role of Retained Sp-P

Park and others (2003b) stated that retention of Sp-P from Japanese Jack mackerel, Pacific mackerel, White croaker, Yellow croaker, and black spotted croaker resulted in lower gel strength (breaking force and deformation) in the raw surimi network by the alkali-aided method than in the surimi and kamaboko produced by the conventional method from the same species. Moreover, Jafarpour and Gorczyca (2008) who modified the conventional method of making surimi by replacing decanting by centrifugation during washing steps reported the same trend with common carp surimi gel. Accordingly, after the 2nd centrifugation in alkaline-aided method, some Sp-P with heavy MW (>60 kDa) were retained in the surimi gel network, whereas most low MW fractions were removed in the supernatant. The possible explanation is that the retention of these types of Sp-P in the surimi gel network specifically those of heat-stable alkaline proteases, which are responsible for degradation of MHC, could reduce the extent of myofibrillar gelation process and consequently result in lower gel strength.

Evidence for the Positive Role of Retained Sp-P

The gel enhancement effect of Sp-P has been demonstrated by several authors (Nishioka and others 1983; Hultin and Kelleher 1997, 2000; Park and others 2003a, 2003b; Perez-Mateos and others 2004) who used the alkali–acid-aided approach (without added Sp-P) to prepare surimi. Huiltin and Kelleher in 2000 claimed that the yield and color of the surimi prepared by the acid–alkali methods were superior to those produced by the conventional method. With respect to texture, they found no evidence that Sp-P interfered with gel formation. However, given the extensive swelling that occurs to the proteins at the pH values utilized for the acid and alkaline treatments, the thick filaments may have been disrupted in both these treatments compared to conventionally processed surimi. One would therefore expect to see an increase in gel strength with pH-shift produced surimi and not only due to the presence of Sp-P.

Park and others (2003a) showed that in surimi produced by the pH-shifting approach, increasing the concentration of Sp-P (up to 8%) increased the breaking force (hardness) and breaking distance (deformation) of the gels. A similar enhancement of Sp-P on the functional properties of kamaboko gel was reported by Kim YS and others (2005) and Jafarpour and Gorczyca (2009). According to Yongsawatdigul and Piyadhammaviboon (2007) such an enhancive effect of Sp-P on Mf-P gelation can be attributed to the presence of TGase and its action in catalyzing the cross-linking of the “acyl transfer reaction, in which the γ-carboxyamide groups of glutamine residues in proteins or peptides act as acyl donors, and primary amino groups, including ɛ-amino groups of lysine residues, either as protein-bound or free lysine, act as the acyl acceptors.” The formation of consequent peptide bonds results in enhancement of textural properties such as elasticity of protein gels (Piyadhammaviboon and Yongsawatdigul 2009, 2010)

The proteinase inhibitory function of Sp-P is another positive role of Sp-P in Mf-P gelation. Jafarpour and Gorczyca (2009) added freeze-dried Sp-P from common carp to threadfin bream surimi at different levels (0% to 35% w/w) and observed that at temperatures between 45 °C and 50 °C, G dramatically decreased and the depth of the G graph was inversely associated with the concentration of added Sp-P. Surimi with added Sp-P underwent a smaller reduction in G at a lower temperature compared with the control treatment at this stage. Such a phenomenon can most probably be related to proteinase inhibition by added Sp-P. Furthermore, in 2010 Piyadhammaviboon and Yongsawatdigul reported that threadfin bream Sp-P added into lizardfish surimi improved the textural properties of resultant surimi gel, mainly because of inhibition by Sp-P of proteinase enzymes such as trypsin but not papain or chymotrypsin at 45 °C. However, such Sp-P inhibition is species dependent.

Conclusion

The effect of Sp-P on surimi and kamaboko gel characteristics is likely to be a function of the method used to prepare the surimi. For instance, removing Sp-P, specifically its heat-stable alkaline proteases, by washing mostly enhances the gel properties of the resulting traditional kamaboko, whereas retaining the Sp-P produced by the acid–alkali method and adding them back to the surimi improves the gel characteristics, but not always. The enhancement of surimi gel strength in the acid–alkali solubilization method cannot be merely attributed to the presence of Sp-P, as ionic strength in pH-adjusted systems is greater than for conventionally processed surimi. This is because there is no process to remove the salts naturally present in fish sarcoplasm in the pH-adjustment method of producing surimi, whereas there is for the conventional process. Moreover, the ionic strength is increased further by the addition of acid or alkali and their subsequent neutralization in the pH-adjusted method. Myofibrillar protein solubility increases with sodium chloride concentration until the salt concentration reaches 0.96 M. Generally, the more myosin that is in solution, the greater is the gel strength. To clarify the role of Sp-P on strengthening of surimi gel, a comparison is needed between surimi produced in the conventional manner with that produced by the pH-adjusted produced method at iso-ionic concentrations before accepting that all the differences between the systems can be traced to the presence or absence of the Sp-P fraction. Adding Sp-P improves the gel characteristics of the resulting kamaboko, mainly due either to catalytic action of TGase in cross-linking the MHC or to Sp-P proteinase inhibition.

The mechanism of the gelling process and the characteristics of Sp-P, especially their location in the muscle and surimi gel, need to be fully understood. Further research should add Sp-P to the surimi gel while keeping the ratio of Sp-P to Mf-P the same as in fish muscle and examine the rheology and microstructure of the resulting surimi and kamaboko. Furthermore, staining myosin, for instance, and one or more of the Sp-P with a specific fluorescent antibody and examining resulting gels with confocal microscopy would show the positions of the Sp-P in the final gels. Another recommendation is to use sequential ultrafiltration (UF) to remove various-sized fractions from the sarcoplasm to examine the effects of various Sp-P on gel strength. Conducting these experiments would elucidate the apparently contradictory effects of Sp-P on surimi and kamaboko gel strength.

Acknowledgment

The authors are thankful to Prof. S. Benjakul, Prof T. Lanier, and Prof. J. Yongsawatdigul for reviewing this manuscript and sharing their knowledge to enhance its scientific quality. Also, authors would like to acknowledge from Prof. Ann Lawrie from school of Applied Science at RMIT Univ., Melbourne, Australia for proof-reading of this manuscript and for help with English expression.

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