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Chemical Interactions of Nonmuscle Proteins in the Network of Sardine (Sardina pilchardus) Muscle Gels
Lebensm.-Wiss. u.-Technol., 29, 602–608 (1997)
Chemical Interactions of Nonmuscle Proteins in the Network of Sardine (Sardina pilchardus) Muscle Gels M. C. Gomez-Guill ´ en, ´ A. J. Border´ıas and P. Montero* Departamento de Ciencia y Tecnolog´ıa de Carne y Pescado, Instituto del Fr´ıo (CSIC), Ciudad Universitaria s/n, E-28040 Madrid (Spain) (Received July 10, 1996; accepted January 10, 1997)
The present work examines the formation of different types of chemical bonding in sardine gels made with added proteins (egg white, soy, casein, gluten) at various temperatures, and possible interactions between myofibrillar and nonmuscle proteins in the gel network. The contribution of ionic and hydrogen bonds to thermal aggregation was not found to be decisive. Hydrophobic interactions in gels with egg white were significantly lower than in gels with other nonmuscle proteins. In gels with casein this protein was found to be largely nonspecifically associated with the myofibrillar proteins at high temperatures. The insolubility of the gluten proteins made it difficult to elucidate a possible interaction with the myofibrillar proteins.
©1997 Academic Press Limited Keywords: gel formation; sardine mince; chemical bonds; myofibrillar proteins; egg white; soy; casein; gluten
Introduction
Materials and Methods
For some time the conversion from sol to gel of mince homogenized with salt was attributed mainly to the formation of ionic and hydrogen bonds. It has now been recognized that the contribution of hydrophobic interactions and disulfide bonds is more important, both in the setting stage (suwari gels) (1–7), and in the high temperature gelling process (8–11). More recent works have reported the existence of a cross-linking process during setting, which is catalysed by a transglutaminase (TGase) enzyme (12–15). The contribution of different myofibrillar proteins to the sardine muscle gel network without added ingredients has been previously reported (16, 17). Other studies in this field have also been carried out by Leinot and Cheftel (18) and Roussel and Cheftel (19). Regarding the incorporation of nonmuscle proteins to fish gels, differences in the ingredient distribution pattern in the gel matrix as well as in their globular particle size (20–22) could modify gel solubility and also interfere in the arrangement of the myofibrillar proteins (23). Effective interactions have been observed in model systems between myosin and egg albumin (24) or casein (25). The aim of this work was to determine the contribution of some nonmuscle proteins to the formation of different bonds in sardine gels and to elucidate the possible interactions between myofibrillar and nonmuscle protein in the gel network.
Fish used were sardines of the species Sardina pilchardus (Walbaum) caught in June off the coast of Nantes. Fish mince was prepared following the procedure described by Gomez-Guill ´ en ´ et al. (26). Atomized-dried egg white was from SANOFI, S.A. (Barcelona, Spain). Soy protein was used in the form of a soy isolate, under product name PP 500 E from Protein Technologies International (Gerona, Spain). Atomized-dried sodium caseinate was supplied by La Pilarica, S.A. (Valencia, Spain) and wheat gluten by Levantina Agr´ıcola Industrial, S.A. (LAISA) (Barcelona, Spain), under product name VITAL ‘L’ Wheat Gluten. NaCl was supplied by PANREAC, Montplet & Esteban S.A. (Barcelona, Spain). All other chemicals used were of reagent grade.
*To whom correspondence should be addressed.
Preparation of gels Homogenization of muscle with NaCl (25 g/kg on finished product weight basis) and added nonmuscle proteins (20 g/kg) was carried out according to Gomez´ Guillen ´ et al. (26). The resulting batters were stuffed into stainless steel cylinders (inner diameter 3 cm, height 3 cm) with screw-on lids and rubber gaskets to provide a hermetic seal. At no time during this part of the process did sample temperature exceed 10 °C. Samples were heated at 35, 50, 60 and 90 °C by immersion in a waterbath for 50 min. Samples for prior setting were preincubated at 35 °C for 30 min and afterwards heated
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at 90 °C for 50 min. Immediately after heating the cylinders were placed in recipients containing ice water (0 °C) for rapid cooling of the gel.
Determination of gel solubility Studies commenced immediately the newly-made gels were sufficiently cool. According to Matsumoto (27) and Careche et al. (17), gels were treated with chemicals selected for their capacity to cleave certain kinds of bond: 0.05 mol/L NaCl (SA), 0.6 mol/L NaCl (SB), 0.6 mol/L NaCl + 1.5 mol/L urea (SC), 0.6 mol/L NaCl + 8 mol/L urea (SD) and 0.6 mol/L NaCl + 8 mol/L urea + 0.5 mol/L 2-β-mercaptoethanol (SE). Proteins were partially solubilized with these solutions in order to determine the existence of nonspecific associations (protein solubilized in SA), ionic bonds (difference between protein solubilized in SB and protein solubilized in SA), hydrogen bonds (difference between protein solubilized in SC and protein solubilized in SB), hydrophobic interactions (difference between protein solubilized in SD and protein solubilized in SC) and disulfide bonds (difference between protein solubilized in SE and protein solubilized in SD). Two grams of chopped gel were homogenized with 10 mL of each solution in an Omni-Mixer, model 17106 homogenizer (OMNI International, Waterbury, U.S.A.) for 2 min at setting 5. The resulting homogenates were stirred at 4–5 °C for 1 h, then centrifuged for 15 min at 20,000 3 g in a Cryofuge 20-3 centrifuge (Heraeus CHRIST GmbH, Germany). Protein concentration in supernatants was determined using a commercial preparation, DC Protein Assay Reagent S no 500-0116 (BIO-RAD Laboratories, CA, U.S.A.). Where necessary the solutions were dialysed previously. Results are the average of two determinations and are expressed as g soluble protein/ L of homogenate.
Electrophoresis (SDS-PAGE) Supernatants obtained with SA and SD solutions were treated according to the method of Hames (28) with a solution composed of 50 g/L 2-β-mercaptoethanol, 25 g/L SDS, 10 mmol/L Tris-HCl, 1 mmol/L EDTA and 0.02 g/L bromophenol blue. They were adjusted to a final average concentration of 2 mg/mL and then heated at 100 °C for 5 min. Electrophoresis was carried out on a Phast-System horizontal apparatus (PHARMACIA LKB Biotechnology AB, Uppsala, Sweden) using polyacrylamide gels (125 gL, PhastGel, Pharmacia LKB Biotechnology). Electrophoresis conditions were 10 mA, 250 V and 3.0 W, at 15 °C. The protein bands were stained with Coomassie brilliant blue (PhastGel Blue R, Pharmacia LKB Biotechnology). As reference for molecular weights, a standard high molecular weight reference kit (Pharmacia LKB Biotechnology) was used: ferritin half unit 220 kDa, albumin 67 kDa, catalase subunit 60 kDa, lactate dehydrogenase subunit 36 kDa and ferritin subunit 18.5 kDa.
Statistical analysis of data Two-way analysis of variance (ANOVA) was carried out. The computer program used was Statgraphics (STSC Inc. Rockville, U.S.A.). The difference between means of pairs was resolved by means of confidence intervals using a Least Significant Difference (LSD) range test. Level significance was set for P ≤ 0.05.
Results and Discussion Protein solubility: disruption of bonds Protein solubility of gels in a number of solutions selected for their capacity to disrupt certain kinds of bonds are shown in Table 1. Increase in gelling temperature resulted in a decrease of soluble protein in SA (0.05 mol/L NaCl), which at 50–90 °C was much more pronounced in the gel containing egg white. The progressive decline in solubility as a result of increased temperature is related to the disappearance of nonspecific associations. During heat treatment, proteins undergo a process of denaturation and aggregation which favours their interactions, resulting in a decline of solubility and hence of extractability (29, 30). This was more evident in the gel with egg white, denoting the formation of a structure whose components are more strongly bonded than in gels with other nonmuscle proteins. Gels containing egg white or sodium caseinate at 35–60 °C presented significantly higher protein solubility in SA than gels made with soy protein or gluten. This could be related to the small globule size, high solubility and strong tendency to distribute as small, dispersed aggregates in the gel matrix of egg white and casein (20–22). However, while in gels with egg white solubility decreased sharply on cooking at 90 °C, in gels with casein the soluble protein level remained only slightly lower than in gels heated at 35–60 °C suggesting that casein has no active part in the gel network formation. Soy protein and gluten, on the other hand, tend to form larger, less evenly distributed aggregates (20–22) which would explain why they are less soluble regardless of the heating temperature. The effect of setting at 35 °C prior to cooking at 90 °C did not substantially modify the presence of these weak nonspecific associations as compared with direct cooking, except in the gel with casein where the difference was more pronounced. Obviously nonspecific associations are not directly involved in the strengthening effect caused by the two-step heating treatment commonly used in kamaboko production (31). Solubility in SB (0.6 mol/L NaCl) as a measure of ionic bonds was very low (less than 1 g/L) at all temperatures, thus indicating very minor participation of ionic bonds in gel formation. With regard to hydrogen bonds, in general protein solubility in SC (0.6 mol/L NaCl + 1.5 mol/L urea) also showed low values. Gels with egg white were significantly less solubilized than the others at 60–90 °C, which suggests a predominance of stronger interactions in these gels. The two-step heat treatment (35 °C/90 °C)
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produced a significant (P ≤ 0.05) reduction in the presence of hydrogen bonds in gels with sodium caseinate or gluten, giving similar values to those registered for the gels with egg white or soy protein. The highest values of protein solubility were obtained with SD (0.6 mol/L NaCl + 8 mol/L urea) as a measure of hydrophobic interactions, and were exhibited by gels heated at 50–60 °C, which are typical modori temperatures. The modori phenomenon, which is appreciable rheologically as deterioration of texture, was considerably more pronounced in gels containing soy protein, casein or gluten where folding test scores dropped sharply (22). The mechanism causing this is generally poorly understood. Proteolytic activity of alkaline proteases in the muscle has been suggested as the most probable cause of gel weakening in this temperature range (32, 33). On the other hand Niwa (34) suggested that modori could come about through excessive formation of hydrophobic interactions, this would mean that the network built up at setting temperature undergoes a thermal shrinkage, releasing water and causing more uneven dispersal of the network. Direct cooking at 90 °C caused a significant decline in hydrophobic interactions, with the exception
of the gel with added egg white where solubility was still low at 60 °C. According to Miyajima (35) temperatures beyond 58 °C could weaken hydrophobic interactions by destabilizing the hydrogen bonds linking water molecules, which would interfere with hydrophobic hydration. Another and perhaps more likely explanation could be that at this cooking temperature there is a more extensive formation of stronger bonds, such as disulfide or other covalent bonds, which are not susceptible to cleavage in the extracting solution (36). Disulfide bonds in a relatively high quantity were detected in gels with added egg white at 60 and 90 °C (Table 1). This could be related to the fact that egg white tends to form thermostable gels with extensive cross-linking by disulfide bonds (24, 37). In contrast, less than 1 g/L of soluble protein in SE was obtained for the other gels at 90 °C. These low values could mean that the experimental conditions employed for measuring disulfide bonds were not sensitive enough and only registered appreciable data when these bonds were present in a large quantity. Gels containing soy protein, sodium caseinate or gluten generally exhibited more hydrophobic interactions than gels with egg white. These nonmuscle proteins had a stronger tendency to
Table 1 Soluble protein of sardine gels solubilized with 0.05 mol/L NaCl (nonspecific associations), 0.6 mol/L NaCl (ionic bonds), 0.6 mol/L + 1.5 mol/L urea (hydrogen bonds), 0.6 mol/L NaCl + 8 mol/L urea (hydrophobic interactions) and 0.6 mol/L NaCl + 8 mol/L urea + 0.5 mol/L -mercaptoethanol (disulfide bonds) Soluble protein (g/L) Sample
35 °C
50 °C
Nonspecific associations Egg white Soy Casein Gluten
5.37 a/x 3.26 b/x 5.71 a/x 3.00 b/x
4.57 a/y 1.74 b/y 5.44 c/x 2.76 d/x
Ionic bonds Egg white Soy Casein Gluten
0.60 a/x 0.27 b/x 0.12 c/x 0.89 d/x
Hydrogen bonds Egg white Soy Casein Gluten
2.20 a/x 2.72 a/x 4.16 b/x 2.62 a/x
Hydrophobic interactions Egg white Soy Casein Gluten
4.01 a/x 6.40 b/x 6.06 c/x 6.41 b/x
Disulfide bonds Egg white Soy Casein Gluten
0.50 a/x 0.89 b/x 0.26 c/x 0.92 b/x
60 °C
90 °C
35/90 °C
2.67 a/z 1.80 b/yz 5.61 c/x 2.52 d/y
1.28 a/v 1.95 b/z 4.90 c/y 2.23 d/z
1.34 a/v 1.43 a/z 3.22 b/z 1.59 a/v
0.31 ab/y 0.45 a/y 0.23 c/y 0.08 b/y
0.57 a/xy 0.32 a/x 0.16 b/x 0.39 a/z
0.50 a/xy 0.48 a/y 0.15 b/x 0.26 c/z
0.46 a/y 0.57 b/y 0.10 c/x 0.41 d/yz
0.56 a/y 2.85 b/x 2.60 b/yz 0.47 a/x
0.56 a/y 1.59 b/y 2.97 c/z 2.41 c/x
0.90 a/y 1.27 b/z 2.27 c/z 2.85 d/y
0.56 a/y 0.75 b/z 1.07 c/yv 0.73 b/z
12.60 a/y 13.88 b/y 11.49 a/y 15.80 c/y
5.80 a/z 15.21 b/z 11.31 c/y 14.37 b/y
3.47 a/xz 9.71 b/v 8.06 c/z 8.55 c/z
2.74 a/v 7.24 b/x 4.68 c/v 4.72 c/v
0.24 a/y 0.54 b/y 0.70 c/y 0.13 d/y
2.66 a/v 0.32 b/y 0.51 c/z 0.28 b/y
3.29 a/u 0.58 b/y 0.95 c/y 0.70 b/x
0.52 a/x 0.47 a/y 0.30 b/x 0.18 c/y
Different letters a, b, c etc. indicate significant differences among gels containing the various nonmuscle proteins for each type of bond. Different letters x, y, z etc. indicate significant differences among heating treatments for each type of bond.
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aggregate via hydrophobic interactions (4, 38–40), but as is shown further below, they also interfere to some extent in the cross-linking of the myofibrillar proteins. Hydrophobic interactions were especially low both in the suwari (35 °C) and in the heat-set (35 °C/90 °C) gels. Since very little formation of disulfide bonds was detected (due in part to low sensitivity of the experimental technique), it could be assumed that other types of covalent bonds predominate. At setting temperatures such covalent bonds have been attributed to the action of transglutaminase (TGase) enzymes (12, 13). In gels made with the two-step treatment therefore, the bonds formed at setting temperatures apparently still remained, and increased only slightly after cooking.
Electrophoretic study Electrophoretic profiles of the fraction soluble in SA (0.05 mol/L NaCl) and in SD (0.6 mol/L NaCl + 8 mol/L urea) of gels with added egg white made at 35, 90 and 35/90 °C are shown in Fig. 1. In the SA solubilized fraction from the suwari gel (35 °C) (Fig. 1a) the bands corresponding to actin, tropomyosin, troponins and other low molecular weight (MW) proteins were clearly apparent, as well as two new bands of 42 kDa and 76 kDa, which in previous assays were found to correspond to ovalbumin and conalbumin, respectively. Ma
Fig. 1 Electrophoretic profiles of the fractions soluble in SA (0.05 mol/L NaCl) and in SD (0.6 mol/L NaCl + 8 mol/L urea) of gels with egg white made at 35, 90 and 35/90 °C. (a) SA (35 °C); (b) SD (35 °C); (c) SA (90 °C); (d) SD (90 °C); (e) SD (35/90 °C)
and Holme (41) reported two similar bands in electrophoretic profiles of native egg white protein. The considerable presence of egg white proteins in this fraction confirmed that these were chiefly responsible for the high level of nonspecific associations referred to above (Table 1). This suggests that at the given temperature the egg white was weakly linked to the gel matrix. On solubilizing with SD (Fig. 1b) a new band appeared in the sample application zone which corresponded to an aggregate of proteins (Ag) polymerized by bonds stronger than hydrophobic interactions. This aggregate was not seen in the SA fraction because it remained entirely in the precipitate. The small number of hydrophobic interactions detected at this temperature (Table 1) and the absence of the MHC band in these conditions of solubilization indicate that the MHC was completely polymerized by disulfide or other covalent bonds. In gels directly cooked at 90 °C, the main visible band appearing in the SA fraction (Fig. 1c) presented similar MW to that of tropomyosin, suggesting that tropomyosin is not directly involved in gel formation. These results are consistent with the findings of Samejima et al. (42) and Jimenez-Colmenero ´ et al. (43). There were also minor traces of MHC and a polypeptide of 89 kDa which could be a product of proteolysis. In the SD fraction (Fig. 1d) both the actin band and the MHC band were prominent, which suggests that they became partially involved in the gel network by means of both hydrogen and hydrophobic bonds. Nevertheless, the presence of a large aggregate in the sample application zone (Ag) as well as in the interphase between the stacking and resolving gels, together with the absence of albumin and conalbumin bands, suggests that the egg white was completely aggregated and may have interacted to some extent with the bulk of the MHC through disulfide bonds or other covalent bonds. Foegeding et al. (24) found that myosin and albumin in a model system interacted to form a gel matrix at temperatures beyond 80 °C, this being the temperature at which albumin undergoes sufficient heat alteration. The affinity of egg white for MHC at high temperature may be the reason why a proportion of the MHC continued to interact by means of hydrogen bonds or hydrophobic interactions. In gels set at 35 °C and cooked immediately at 90 °C (Fig. 1e) the aggregate (Ag) was less perceptible, thus suggesting that the bulk of the MHC together with the egg white proteins was considerably polymerized by disulfide or other covalent bonds still remaining in the insoluble fraction. The electrophoretic profiles of gels made with addition of soy protein are shown in Fig. 2. In the SA soluble fraction of the suwari gel (Fig. 2a) some bands appeared which preliminary results showed to correspond to different protein fractions of the soy isolate. This would indicate that at the given temperature a considerable part of the added nonmuscle protein was nonspecifically associated. On solubilizing with SD (Fig. 2b), however, a new fraction of soy isolate appeared, and the MHC band was also faintly visible. This would explain the higher protein solubility in SD
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(as a measure of hydrophobic interactions) (Table 1) recorded in the suwari gel with soy protein as compared to the gel with egg white. In the SD solubilized fraction of gels cooked directly at 90 °C (Fig. 2d) the MHC band, the actin band and the soy isolate fractions appeared more strongly marked than in the suwari gel (Fig. 2b), indicating considerable participation of all these proteins in the formation of the definitive gel via hydrogen bonds or hydrophobic interactions. In the heat-set gel (35/90 °C) (Fig. 2e) the MHC was completely polymerized, and the aggregate (Ag) and actin band were less visible than in directly cooked gels, indicating greater involvement of these myofibrillar proteins in gel formation by means of bonds stronger than hydrophobic interactions. A considerable part of the soy proteins remained in the SD soluble fraction and was largely responsible for the fact that protein solubility in SD (as a measure of hydrophobic interactions) was higher in the gel with soy protein than in the others. In the gel with casein made at 35 °C the bands corresponding to MHC and casein were not clearly visible in the electrophoretic profiles of the SA and SD fractions (Fig. 3a,b). Thus, MHC was found to be completely polymerized by bonds stronger than hydrophobic interactions, such as disulfide bonds or covalent bonds, the latter possibly resulting from TGase activity,
which could be responsible for the covalent linkage between the MHC and the casein (25). In gels directly cooked at 90 °C, two proteins of 30 kDa and 25 kDa, which are the molecular weights of α- and β-casein, appeared in the electrophoretic profile of the SA soluble fraction (Fig. 3c). A considerable part of the casein, then, seems not to have interacted with the myofibrillar proteins in the gel matrix. No bond corresponding to MHC was observed in either the SA or SD solubilized fractions (Fig. 3c,d), indicating that polymerization was complete apart from casein. The reason for the low capacity of casein to interact with muscle proteins at high temperature may be that the component molecules of casein are highly amphipatic, with a strong tendency to associate in the form of micelles by means of hydrophobic interactions and hydrogen bonds. Moreover, casein has very few sulphydryl groups and hence little capacity to form disulfide bonds with myofibrillar proteins (40, 44). In the gel made with the two-step treatment (Fig. 3e), the casein bands appeared less intense than in the directly cooked gel, from which it was assumed that following the setting phase part of the casein remained in association with the MHC, linked by bonds stronger than hydrophobic interactions. The electrophoretic profiles of gels made with added gluten (Fig. 4) showed no band that might correspond
Fig. 2 Electrophoretic profiles of the fractions soluble in SA (0.05 mol/L NaCl) and in SD (0.6 mol/L NaCl + 8 mol/L urea) of gels with soy protein made at 35, 90 and 35/90 °C. (a) SA (35 °C); (b) SD (35 °C); (c) SA (90 °C); (d) SD (90 °C); (e) 35/90 °C)
Fig. 3 Electrophoretic profiles of the fractions soluble in SA (0.05 mol/L NaCl) and in SD (0.6 mol/L NaCl + 8 mol/L urea) of gels with sodium caseinate made at 35, 90 and 35/90 °C. (a) SA (35 °C); (b) SD (35 °C); (c) SA (90 °C); (d) SD (90 °C); (e) SD (35/90 °C)
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to this nonmuscle protein. Gluten is composed of proteins of high molecular weight and is characterized by its high degree of insolubility; it was therefore assumed that it formed large aggregates, which would be retained in the precipitate along with the bulk of the MHC. As with the other ingredients, tropomyosin was scarcely involved in formation of the gel network and actin appeared to interact chiefly via hydrophobic interactions or hydrogen bonds in the gel directly cooked at 90 °C.
Conclusions Gels made with addition of egg white presented a network characterized by the predominance of stronger bonding than in the gels with added soy protein, casein or gluten. The modori phenomenon at 50–60 °C was apparent in all gels and largely related to an increase of hydrophobic interactions. In cooked gels (90 °C) and in pre-set gels (35/90 °C) the egg white proteins were completely polymerized by stronger bonds than hydrophobic interactions, whereas these interactions were those mainly responsible for the aggregation of soy proteins, casein and gluten. On the other hand, a considerable part of sodium caseinate remained nonspecifically associated to the gel network.
Fig. 4 Electrophoretic profiles of the fractions soluble in SA (0.05 mol/L NaCl) and in SD (0.6 mol/L NaCl + 8 mol/L urea) of gels with gluten made at 35, 90 and 35/90 °C. (a) SA (35 °C); (b) SD (35 °C); (c) SA (90 °C); (d) SD (90 °C); (e) SD (35/90 °C)
Acknowledgements This research was financed by the Comision ´ Interministerial de Ciencia y Tecnologia (CICyT) under project ALI-910899-CO3-01 (1991/1994).
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30 CAMOU, J. P., SEBRANEK, J. G. AND OLSON, D. G. Effects of heating rate and protein concentration on gel strength and water loss of muscle protein gel. Journal of Food Science, 54, 850–854 (1989) 31 OKADA, M. Ashi and its reinforcement. In: OKADA, M., KINUMAKI, T. AND YOKOSEKI, M. (Eds), Gyoniku Neriseihin [Kneaded Seafood Products]. Tokyo: KoseisyaKoseikaku, pp. 189–212 (1981) 32 LANIER, T. C., LIN, T. S., HAMANN, D. D. AND THOMAS, F. B. Effects of alkaline proteinase in minced fish on texture of heat processed gels. Journal of Food Chemistry, 46, 1643–1645 (1981) 33 MAKINODAN, Y., KITAGAWA, T., TOYOHARA, H. AND SHIMIZU, Y. Participation of muscle alkaline proteinase in himodori of kamaboko. Nippon Suisan Gakkaishi, 53, 99–101 (1987) 34 NIWA, E. Chemistry of surimi gelation. In: LANIER, T. C. AND LEE, C. M. (Eds), Surimi Technology. New York: Marcel Dekker, pp. 389–427 (1992) 35 MIYAJIMA, K. Hydration and hydrophobic bonding. In: NAKAGAKI, M. (Ed), Structure and Physical Properties of Water. Tokyo: Nankodo, pp. 83–109 (1974) 36 LEE, H. G. AND LANIER, T. C. The role of covalent crosslinking in the texturizing of muscle protein sols. Journal of Muscle Foods, 6, 125–138 (1995) 37 EGELANDSDAL, B. Heat-induced gelling in solutions of ovalbumin. Journal of Food Science, 45, 570–573/581 (1980) 38 SCHMIDT, D. G. Association of caseins and casein micelle structure. In: FOX, P. F. (Ed), Developments in Dairy Chemistry — 1. London, New York: Elsevier Applied Science Publishers, p. 61 (1982) 39 RHA, C. K. AND PRADIPASENA, P. Viscosity of proteins. In: MITCHELL, J. R. AND LEDWARD, D. A. (Eds), Functional Properties of Food Macromolecules. London, New York: Elsevier Applied Science Publishers, pp. 72–120 (1986) 40 KINSELLA, J. E., WHITEHEAD, D. M., BRADY, J. AND BRINGE, N. A. Milk proteins: possible relationship of structure and function. In: FOX, P. F. (Ed), Development in Dairy Chemistry – 4: Functional milk proteins. London, New York: Elsevier Applied Science Publishers, p. 55 (1989) 41 MA, C. Y. AND HOLME, J. Effect of chemical modifications on some physicochemical properties and heat coagulation of egg albumin. Journal of Food Science, 47, 1454–1459 (1982) 42 SAMEJIMA, K., EGELANDSDAL, B. AND FRETHEIM, K. Heat gelation properties and protein extractability of beef myofibrils. Journal of Food Science, 50, 1540–1543/1555 (1985) ´ -COLMENERO, F., CARECHE, J., CARBALLO, J. AND 43 JIMENEZ COFRADES, S. Influence of thermal treatment on gelation of actomyosin from different myosystems. Journal of Food Science, 59, 211–215/220 (1994) 44 FOX, P. F. AND MULVIHILL, D. M. Casein. In: HARRIS, P. (Ed), Food Gels. London, New York: Elsevier Applied Science Publishers, pp. 121–173 (1990)
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Chemical Interactions of Nonmuscle Proteins in the Network of Sardine (Sardina pilchardus) Muscle Gels M. C. Gomez-Guill ´ en, ´ A. J. Border´ıas and P. Montero* Departamento de Ciencia y Tecnolog´ıa de Carne y Pescado, Instituto del Fr´ıo (CSIC), Ciudad Universitaria s/n, E-28040 Madrid (Spain) (Received July 10, 1996; accepted January 10, 1997)
The present work examines the formation of different types of chemical bonding in sardine gels made with added proteins (egg white, soy, casein, gluten) at various temperatures, and possible interactions between myofibrillar and nonmuscle proteins in the gel network. The contribution of ionic and hydrogen bonds to thermal aggregation was not found to be decisive. Hydrophobic interactions in gels with egg white were significantly lower than in gels with other nonmuscle proteins. In gels with casein this protein was found to be largely nonspecifically associated with the myofibrillar proteins at high temperatures. The insolubility of the gluten proteins made it difficult to elucidate a possible interaction with the myofibrillar proteins.
©1997 Academic Press Limited Keywords: gel formation; sardine mince; chemical bonds; myofibrillar proteins; egg white; soy; casein; gluten
Introduction
Materials and Methods
For some time the conversion from sol to gel of mince homogenized with salt was attributed mainly to the formation of ionic and hydrogen bonds. It has now been recognized that the contribution of hydrophobic interactions and disulfide bonds is more important, both in the setting stage (suwari gels) (1–7), and in the high temperature gelling process (8–11). More recent works have reported the existence of a cross-linking process during setting, which is catalysed by a transglutaminase (TGase) enzyme (12–15). The contribution of different myofibrillar proteins to the sardine muscle gel network without added ingredients has been previously reported (16, 17). Other studies in this field have also been carried out by Leinot and Cheftel (18) and Roussel and Cheftel (19). Regarding the incorporation of nonmuscle proteins to fish gels, differences in the ingredient distribution pattern in the gel matrix as well as in their globular particle size (20–22) could modify gel solubility and also interfere in the arrangement of the myofibrillar proteins (23). Effective interactions have been observed in model systems between myosin and egg albumin (24) or casein (25). The aim of this work was to determine the contribution of some nonmuscle proteins to the formation of different bonds in sardine gels and to elucidate the possible interactions between myofibrillar and nonmuscle protein in the gel network.
Fish used were sardines of the species Sardina pilchardus (Walbaum) caught in June off the coast of Nantes. Fish mince was prepared following the procedure described by Gomez-Guill ´ en ´ et al. (26). Atomized-dried egg white was from SANOFI, S.A. (Barcelona, Spain). Soy protein was used in the form of a soy isolate, under product name PP 500 E from Protein Technologies International (Gerona, Spain). Atomized-dried sodium caseinate was supplied by La Pilarica, S.A. (Valencia, Spain) and wheat gluten by Levantina Agr´ıcola Industrial, S.A. (LAISA) (Barcelona, Spain), under product name VITAL ‘L’ Wheat Gluten. NaCl was supplied by PANREAC, Montplet & Esteban S.A. (Barcelona, Spain). All other chemicals used were of reagent grade.
*To whom correspondence should be addressed.
Preparation of gels Homogenization of muscle with NaCl (25 g/kg on finished product weight basis) and added nonmuscle proteins (20 g/kg) was carried out according to Gomez´ Guillen ´ et al. (26). The resulting batters were stuffed into stainless steel cylinders (inner diameter 3 cm, height 3 cm) with screw-on lids and rubber gaskets to provide a hermetic seal. At no time during this part of the process did sample temperature exceed 10 °C. Samples were heated at 35, 50, 60 and 90 °C by immersion in a waterbath for 50 min. Samples for prior setting were preincubated at 35 °C for 30 min and afterwards heated
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at 90 °C for 50 min. Immediately after heating the cylinders were placed in recipients containing ice water (0 °C) for rapid cooling of the gel.
Determination of gel solubility Studies commenced immediately the newly-made gels were sufficiently cool. According to Matsumoto (27) and Careche et al. (17), gels were treated with chemicals selected for their capacity to cleave certain kinds of bond: 0.05 mol/L NaCl (SA), 0.6 mol/L NaCl (SB), 0.6 mol/L NaCl + 1.5 mol/L urea (SC), 0.6 mol/L NaCl + 8 mol/L urea (SD) and 0.6 mol/L NaCl + 8 mol/L urea + 0.5 mol/L 2-β-mercaptoethanol (SE). Proteins were partially solubilized with these solutions in order to determine the existence of nonspecific associations (protein solubilized in SA), ionic bonds (difference between protein solubilized in SB and protein solubilized in SA), hydrogen bonds (difference between protein solubilized in SC and protein solubilized in SB), hydrophobic interactions (difference between protein solubilized in SD and protein solubilized in SC) and disulfide bonds (difference between protein solubilized in SE and protein solubilized in SD). Two grams of chopped gel were homogenized with 10 mL of each solution in an Omni-Mixer, model 17106 homogenizer (OMNI International, Waterbury, U.S.A.) for 2 min at setting 5. The resulting homogenates were stirred at 4–5 °C for 1 h, then centrifuged for 15 min at 20,000 3 g in a Cryofuge 20-3 centrifuge (Heraeus CHRIST GmbH, Germany). Protein concentration in supernatants was determined using a commercial preparation, DC Protein Assay Reagent S no 500-0116 (BIO-RAD Laboratories, CA, U.S.A.). Where necessary the solutions were dialysed previously. Results are the average of two determinations and are expressed as g soluble protein/ L of homogenate.
Electrophoresis (SDS-PAGE) Supernatants obtained with SA and SD solutions were treated according to the method of Hames (28) with a solution composed of 50 g/L 2-β-mercaptoethanol, 25 g/L SDS, 10 mmol/L Tris-HCl, 1 mmol/L EDTA and 0.02 g/L bromophenol blue. They were adjusted to a final average concentration of 2 mg/mL and then heated at 100 °C for 5 min. Electrophoresis was carried out on a Phast-System horizontal apparatus (PHARMACIA LKB Biotechnology AB, Uppsala, Sweden) using polyacrylamide gels (125 gL, PhastGel, Pharmacia LKB Biotechnology). Electrophoresis conditions were 10 mA, 250 V and 3.0 W, at 15 °C. The protein bands were stained with Coomassie brilliant blue (PhastGel Blue R, Pharmacia LKB Biotechnology). As reference for molecular weights, a standard high molecular weight reference kit (Pharmacia LKB Biotechnology) was used: ferritin half unit 220 kDa, albumin 67 kDa, catalase subunit 60 kDa, lactate dehydrogenase subunit 36 kDa and ferritin subunit 18.5 kDa.
Statistical analysis of data Two-way analysis of variance (ANOVA) was carried out. The computer program used was Statgraphics (STSC Inc. Rockville, U.S.A.). The difference between means of pairs was resolved by means of confidence intervals using a Least Significant Difference (LSD) range test. Level significance was set for P ≤ 0.05.
Results and Discussion Protein solubility: disruption of bonds Protein solubility of gels in a number of solutions selected for their capacity to disrupt certain kinds of bonds are shown in Table 1. Increase in gelling temperature resulted in a decrease of soluble protein in SA (0.05 mol/L NaCl), which at 50–90 °C was much more pronounced in the gel containing egg white. The progressive decline in solubility as a result of increased temperature is related to the disappearance of nonspecific associations. During heat treatment, proteins undergo a process of denaturation and aggregation which favours their interactions, resulting in a decline of solubility and hence of extractability (29, 30). This was more evident in the gel with egg white, denoting the formation of a structure whose components are more strongly bonded than in gels with other nonmuscle proteins. Gels containing egg white or sodium caseinate at 35–60 °C presented significantly higher protein solubility in SA than gels made with soy protein or gluten. This could be related to the small globule size, high solubility and strong tendency to distribute as small, dispersed aggregates in the gel matrix of egg white and casein (20–22). However, while in gels with egg white solubility decreased sharply on cooking at 90 °C, in gels with casein the soluble protein level remained only slightly lower than in gels heated at 35–60 °C suggesting that casein has no active part in the gel network formation. Soy protein and gluten, on the other hand, tend to form larger, less evenly distributed aggregates (20–22) which would explain why they are less soluble regardless of the heating temperature. The effect of setting at 35 °C prior to cooking at 90 °C did not substantially modify the presence of these weak nonspecific associations as compared with direct cooking, except in the gel with casein where the difference was more pronounced. Obviously nonspecific associations are not directly involved in the strengthening effect caused by the two-step heating treatment commonly used in kamaboko production (31). Solubility in SB (0.6 mol/L NaCl) as a measure of ionic bonds was very low (less than 1 g/L) at all temperatures, thus indicating very minor participation of ionic bonds in gel formation. With regard to hydrogen bonds, in general protein solubility in SC (0.6 mol/L NaCl + 1.5 mol/L urea) also showed low values. Gels with egg white were significantly less solubilized than the others at 60–90 °C, which suggests a predominance of stronger interactions in these gels. The two-step heat treatment (35 °C/90 °C)
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produced a significant (P ≤ 0.05) reduction in the presence of hydrogen bonds in gels with sodium caseinate or gluten, giving similar values to those registered for the gels with egg white or soy protein. The highest values of protein solubility were obtained with SD (0.6 mol/L NaCl + 8 mol/L urea) as a measure of hydrophobic interactions, and were exhibited by gels heated at 50–60 °C, which are typical modori temperatures. The modori phenomenon, which is appreciable rheologically as deterioration of texture, was considerably more pronounced in gels containing soy protein, casein or gluten where folding test scores dropped sharply (22). The mechanism causing this is generally poorly understood. Proteolytic activity of alkaline proteases in the muscle has been suggested as the most probable cause of gel weakening in this temperature range (32, 33). On the other hand Niwa (34) suggested that modori could come about through excessive formation of hydrophobic interactions, this would mean that the network built up at setting temperature undergoes a thermal shrinkage, releasing water and causing more uneven dispersal of the network. Direct cooking at 90 °C caused a significant decline in hydrophobic interactions, with the exception
of the gel with added egg white where solubility was still low at 60 °C. According to Miyajima (35) temperatures beyond 58 °C could weaken hydrophobic interactions by destabilizing the hydrogen bonds linking water molecules, which would interfere with hydrophobic hydration. Another and perhaps more likely explanation could be that at this cooking temperature there is a more extensive formation of stronger bonds, such as disulfide or other covalent bonds, which are not susceptible to cleavage in the extracting solution (36). Disulfide bonds in a relatively high quantity were detected in gels with added egg white at 60 and 90 °C (Table 1). This could be related to the fact that egg white tends to form thermostable gels with extensive cross-linking by disulfide bonds (24, 37). In contrast, less than 1 g/L of soluble protein in SE was obtained for the other gels at 90 °C. These low values could mean that the experimental conditions employed for measuring disulfide bonds were not sensitive enough and only registered appreciable data when these bonds were present in a large quantity. Gels containing soy protein, sodium caseinate or gluten generally exhibited more hydrophobic interactions than gels with egg white. These nonmuscle proteins had a stronger tendency to
Table 1 Soluble protein of sardine gels solubilized with 0.05 mol/L NaCl (nonspecific associations), 0.6 mol/L NaCl (ionic bonds), 0.6 mol/L + 1.5 mol/L urea (hydrogen bonds), 0.6 mol/L NaCl + 8 mol/L urea (hydrophobic interactions) and 0.6 mol/L NaCl + 8 mol/L urea + 0.5 mol/L -mercaptoethanol (disulfide bonds) Soluble protein (g/L) Sample
35 °C
50 °C
Nonspecific associations Egg white Soy Casein Gluten
5.37 a/x 3.26 b/x 5.71 a/x 3.00 b/x
4.57 a/y 1.74 b/y 5.44 c/x 2.76 d/x
Ionic bonds Egg white Soy Casein Gluten
0.60 a/x 0.27 b/x 0.12 c/x 0.89 d/x
Hydrogen bonds Egg white Soy Casein Gluten
2.20 a/x 2.72 a/x 4.16 b/x 2.62 a/x
Hydrophobic interactions Egg white Soy Casein Gluten
4.01 a/x 6.40 b/x 6.06 c/x 6.41 b/x
Disulfide bonds Egg white Soy Casein Gluten
0.50 a/x 0.89 b/x 0.26 c/x 0.92 b/x
60 °C
90 °C
35/90 °C
2.67 a/z 1.80 b/yz 5.61 c/x 2.52 d/y
1.28 a/v 1.95 b/z 4.90 c/y 2.23 d/z
1.34 a/v 1.43 a/z 3.22 b/z 1.59 a/v
0.31 ab/y 0.45 a/y 0.23 c/y 0.08 b/y
0.57 a/xy 0.32 a/x 0.16 b/x 0.39 a/z
0.50 a/xy 0.48 a/y 0.15 b/x 0.26 c/z
0.46 a/y 0.57 b/y 0.10 c/x 0.41 d/yz
0.56 a/y 2.85 b/x 2.60 b/yz 0.47 a/x
0.56 a/y 1.59 b/y 2.97 c/z 2.41 c/x
0.90 a/y 1.27 b/z 2.27 c/z 2.85 d/y
0.56 a/y 0.75 b/z 1.07 c/yv 0.73 b/z
12.60 a/y 13.88 b/y 11.49 a/y 15.80 c/y
5.80 a/z 15.21 b/z 11.31 c/y 14.37 b/y
3.47 a/xz 9.71 b/v 8.06 c/z 8.55 c/z
2.74 a/v 7.24 b/x 4.68 c/v 4.72 c/v
0.24 a/y 0.54 b/y 0.70 c/y 0.13 d/y
2.66 a/v 0.32 b/y 0.51 c/z 0.28 b/y
3.29 a/u 0.58 b/y 0.95 c/y 0.70 b/x
0.52 a/x 0.47 a/y 0.30 b/x 0.18 c/y
Different letters a, b, c etc. indicate significant differences among gels containing the various nonmuscle proteins for each type of bond. Different letters x, y, z etc. indicate significant differences among heating treatments for each type of bond.
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aggregate via hydrophobic interactions (4, 38–40), but as is shown further below, they also interfere to some extent in the cross-linking of the myofibrillar proteins. Hydrophobic interactions were especially low both in the suwari (35 °C) and in the heat-set (35 °C/90 °C) gels. Since very little formation of disulfide bonds was detected (due in part to low sensitivity of the experimental technique), it could be assumed that other types of covalent bonds predominate. At setting temperatures such covalent bonds have been attributed to the action of transglutaminase (TGase) enzymes (12, 13). In gels made with the two-step treatment therefore, the bonds formed at setting temperatures apparently still remained, and increased only slightly after cooking.
Electrophoretic study Electrophoretic profiles of the fraction soluble in SA (0.05 mol/L NaCl) and in SD (0.6 mol/L NaCl + 8 mol/L urea) of gels with added egg white made at 35, 90 and 35/90 °C are shown in Fig. 1. In the SA solubilized fraction from the suwari gel (35 °C) (Fig. 1a) the bands corresponding to actin, tropomyosin, troponins and other low molecular weight (MW) proteins were clearly apparent, as well as two new bands of 42 kDa and 76 kDa, which in previous assays were found to correspond to ovalbumin and conalbumin, respectively. Ma
Fig. 1 Electrophoretic profiles of the fractions soluble in SA (0.05 mol/L NaCl) and in SD (0.6 mol/L NaCl + 8 mol/L urea) of gels with egg white made at 35, 90 and 35/90 °C. (a) SA (35 °C); (b) SD (35 °C); (c) SA (90 °C); (d) SD (90 °C); (e) SD (35/90 °C)
and Holme (41) reported two similar bands in electrophoretic profiles of native egg white protein. The considerable presence of egg white proteins in this fraction confirmed that these were chiefly responsible for the high level of nonspecific associations referred to above (Table 1). This suggests that at the given temperature the egg white was weakly linked to the gel matrix. On solubilizing with SD (Fig. 1b) a new band appeared in the sample application zone which corresponded to an aggregate of proteins (Ag) polymerized by bonds stronger than hydrophobic interactions. This aggregate was not seen in the SA fraction because it remained entirely in the precipitate. The small number of hydrophobic interactions detected at this temperature (Table 1) and the absence of the MHC band in these conditions of solubilization indicate that the MHC was completely polymerized by disulfide or other covalent bonds. In gels directly cooked at 90 °C, the main visible band appearing in the SA fraction (Fig. 1c) presented similar MW to that of tropomyosin, suggesting that tropomyosin is not directly involved in gel formation. These results are consistent with the findings of Samejima et al. (42) and Jimenez-Colmenero ´ et al. (43). There were also minor traces of MHC and a polypeptide of 89 kDa which could be a product of proteolysis. In the SD fraction (Fig. 1d) both the actin band and the MHC band were prominent, which suggests that they became partially involved in the gel network by means of both hydrogen and hydrophobic bonds. Nevertheless, the presence of a large aggregate in the sample application zone (Ag) as well as in the interphase between the stacking and resolving gels, together with the absence of albumin and conalbumin bands, suggests that the egg white was completely aggregated and may have interacted to some extent with the bulk of the MHC through disulfide bonds or other covalent bonds. Foegeding et al. (24) found that myosin and albumin in a model system interacted to form a gel matrix at temperatures beyond 80 °C, this being the temperature at which albumin undergoes sufficient heat alteration. The affinity of egg white for MHC at high temperature may be the reason why a proportion of the MHC continued to interact by means of hydrogen bonds or hydrophobic interactions. In gels set at 35 °C and cooked immediately at 90 °C (Fig. 1e) the aggregate (Ag) was less perceptible, thus suggesting that the bulk of the MHC together with the egg white proteins was considerably polymerized by disulfide or other covalent bonds still remaining in the insoluble fraction. The electrophoretic profiles of gels made with addition of soy protein are shown in Fig. 2. In the SA soluble fraction of the suwari gel (Fig. 2a) some bands appeared which preliminary results showed to correspond to different protein fractions of the soy isolate. This would indicate that at the given temperature a considerable part of the added nonmuscle protein was nonspecifically associated. On solubilizing with SD (Fig. 2b), however, a new fraction of soy isolate appeared, and the MHC band was also faintly visible. This would explain the higher protein solubility in SD
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(as a measure of hydrophobic interactions) (Table 1) recorded in the suwari gel with soy protein as compared to the gel with egg white. In the SD solubilized fraction of gels cooked directly at 90 °C (Fig. 2d) the MHC band, the actin band and the soy isolate fractions appeared more strongly marked than in the suwari gel (Fig. 2b), indicating considerable participation of all these proteins in the formation of the definitive gel via hydrogen bonds or hydrophobic interactions. In the heat-set gel (35/90 °C) (Fig. 2e) the MHC was completely polymerized, and the aggregate (Ag) and actin band were less visible than in directly cooked gels, indicating greater involvement of these myofibrillar proteins in gel formation by means of bonds stronger than hydrophobic interactions. A considerable part of the soy proteins remained in the SD soluble fraction and was largely responsible for the fact that protein solubility in SD (as a measure of hydrophobic interactions) was higher in the gel with soy protein than in the others. In the gel with casein made at 35 °C the bands corresponding to MHC and casein were not clearly visible in the electrophoretic profiles of the SA and SD fractions (Fig. 3a,b). Thus, MHC was found to be completely polymerized by bonds stronger than hydrophobic interactions, such as disulfide bonds or covalent bonds, the latter possibly resulting from TGase activity,
which could be responsible for the covalent linkage between the MHC and the casein (25). In gels directly cooked at 90 °C, two proteins of 30 kDa and 25 kDa, which are the molecular weights of α- and β-casein, appeared in the electrophoretic profile of the SA soluble fraction (Fig. 3c). A considerable part of the casein, then, seems not to have interacted with the myofibrillar proteins in the gel matrix. No bond corresponding to MHC was observed in either the SA or SD solubilized fractions (Fig. 3c,d), indicating that polymerization was complete apart from casein. The reason for the low capacity of casein to interact with muscle proteins at high temperature may be that the component molecules of casein are highly amphipatic, with a strong tendency to associate in the form of micelles by means of hydrophobic interactions and hydrogen bonds. Moreover, casein has very few sulphydryl groups and hence little capacity to form disulfide bonds with myofibrillar proteins (40, 44). In the gel made with the two-step treatment (Fig. 3e), the casein bands appeared less intense than in the directly cooked gel, from which it was assumed that following the setting phase part of the casein remained in association with the MHC, linked by bonds stronger than hydrophobic interactions. The electrophoretic profiles of gels made with added gluten (Fig. 4) showed no band that might correspond
Fig. 2 Electrophoretic profiles of the fractions soluble in SA (0.05 mol/L NaCl) and in SD (0.6 mol/L NaCl + 8 mol/L urea) of gels with soy protein made at 35, 90 and 35/90 °C. (a) SA (35 °C); (b) SD (35 °C); (c) SA (90 °C); (d) SD (90 °C); (e) 35/90 °C)
Fig. 3 Electrophoretic profiles of the fractions soluble in SA (0.05 mol/L NaCl) and in SD (0.6 mol/L NaCl + 8 mol/L urea) of gels with sodium caseinate made at 35, 90 and 35/90 °C. (a) SA (35 °C); (b) SD (35 °C); (c) SA (90 °C); (d) SD (90 °C); (e) SD (35/90 °C)
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to this nonmuscle protein. Gluten is composed of proteins of high molecular weight and is characterized by its high degree of insolubility; it was therefore assumed that it formed large aggregates, which would be retained in the precipitate along with the bulk of the MHC. As with the other ingredients, tropomyosin was scarcely involved in formation of the gel network and actin appeared to interact chiefly via hydrophobic interactions or hydrogen bonds in the gel directly cooked at 90 °C.
Conclusions Gels made with addition of egg white presented a network characterized by the predominance of stronger bonding than in the gels with added soy protein, casein or gluten. The modori phenomenon at 50–60 °C was apparent in all gels and largely related to an increase of hydrophobic interactions. In cooked gels (90 °C) and in pre-set gels (35/90 °C) the egg white proteins were completely polymerized by stronger bonds than hydrophobic interactions, whereas these interactions were those mainly responsible for the aggregation of soy proteins, casein and gluten. On the other hand, a considerable part of sodium caseinate remained nonspecifically associated to the gel network.
Fig. 4 Electrophoretic profiles of the fractions soluble in SA (0.05 mol/L NaCl) and in SD (0.6 mol/L NaCl + 8 mol/L urea) of gels with gluten made at 35, 90 and 35/90 °C. (a) SA (35 °C); (b) SD (35 °C); (c) SA (90 °C); (d) SD (90 °C); (e) SD (35/90 °C)
Acknowledgements This research was financed by the Comision ´ Interministerial de Ciencia y Tecnologia (CICyT) under project ALI-910899-CO3-01 (1991/1994).
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