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Chemical Interactions of Nonmuscle Proteins in the Network of Sardine (Sardina pilchardus) Muscle Gels M. C. G ´ omez-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 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 inter- actions 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 non- muscle protein in the gel network. Materials and Methods Fish used were sardines of the species Sardina pilchar- dus (Walbaum) caught in June off the coast of Nantes. Fish mince was prepared following the procedure described by G ´ omez-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) (Barce- lona, 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. 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 G ´ omez- Guill ´ en 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 water- bath for 50 min. Samples for prior setting were preincubated at 35 °C for 30 min and afterwards heated *To whom correspondence should be addressed. Lebensm.-Wiss. u.-Technol., 29, 602–608 (1997) 0023-6438/97/060602 + 07 $25.00/0/fs970239 ©1997 Academic Press Limited 602

Chemical Interactions of Nonmuscle Proteins in the Network of Sardine (Sardina pilchardus) Muscle Gels

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Page 1: Chemical Interactions of Nonmuscle Proteins in the Network of Sardine (Sardina pilchardus) Muscle Gels

Chemical Interactions of Nonmuscle Proteins in theNetwork of Sardine (Sardina pilchardus) Muscle Gels

M. C. Gomez-Guillen, A. J. Borderıas and P. Montero*

Departamento de Ciencia y Tecnologıa de Carne y Pescado, Instituto del Frıo (CSIC), Ciudad Universitarias/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 (eggwhite, soy, casein, gluten) at various temperatures, and possible interactions between myofibrillar and nonmuscle proteins in thegel network. The contribution of ionic and hydrogen bonds to thermal aggregation was not found to be decisive. Hydrophobicinteractions in gels with egg white were significantly lower than in gels with other nonmuscle proteins. In gels with casein thisprotein was found to be largely nonspecifically associated with the myofibrillar proteins at high temperatures. The insolubility ofthe 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

For some time the conversion from sol to gel of mincehomogenized with salt was attributed mainly to theformation of ionic and hydrogen bonds. It has now beenrecognized that the contribution of hydrophobic inter-actions and disulfide bonds is more important, both inthe setting stage (suwari gels) (1–7), and in the hightemperature gelling process (8–11). More recent workshave reported the existence of a cross-linking processduring setting, which is catalysed by a transglutaminase(TGase) enzyme (12–15). The contribution of differentmyofibrillar proteins to the sardine muscle gel networkwithout added ingredients has been previouslyreported (16, 17). Other studies in this field have alsobeen carried out by Leinot and Cheftel (18) andRoussel and Cheftel (19).Regarding the incorporation of nonmuscle proteins tofish gels, differences in the ingredient distributionpattern in the gel matrix as well as in their globularparticle size (20–22) could modify gel solubility andalso interfere in the arrangement of the myofibrillarproteins (23). Effective interactions have beenobserved in model systems between myosin and eggalbumin (24) or casein (25).The aim of this work was to determine the contributionof some nonmuscle proteins to the formation ofdifferent bonds in sardine gels and to elucidate thepossible interactions between myofibrillar and non-muscle protein in the gel network.

Materials and Methods

Fish used were sardines of the species Sardina pilchar-dus (Walbaum) caught in June off the coast of Nantes.Fish mince was prepared following the proceduredescribed by Gomez-Guillen et al. (26).Atomized-dried egg white was from SANOFI, S.A.(Barcelona, Spain). Soy protein was used in the form ofa soy isolate, under product name PP 500 E fromProtein Technologies International (Gerona, Spain).Atomized-dried sodium caseinate was supplied by LaPilarica, S.A. (Valencia, Spain) and wheat gluten byLevantina Agrıcola Industrial, S.A. (LAISA) (Barce-lona, Spain), under product name VITAL ‘L’ WheatGluten. NaCl was supplied by PANREAC, Montplet &Esteban S.A. (Barcelona, Spain). All other chemicalsused were of reagent grade.

Preparation of gelsHomogenization of muscle with NaCl (25 g/kg onfinished product weight basis) and added nonmuscleproteins (20 g/kg) was carried out according to Gomez-Guillen et al. (26).The resulting batters were stuffed into stainless steelcylinders (inner diameter 3 cm, height 3 cm) withscrew-on lids and rubber gaskets to provide a hermeticseal. At no time during this part of the process didsample temperature exceed 10 °C. Samples wereheated at 35, 50, 60 and 90 °C by immersion in a water-bath for 50 min. Samples for prior setting werepreincubated at 35 °C for 30 min and afterwards heated*To whom correspondence should be addressed.

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at 90 °C for 50 min. Immediately after heating thecylinders were placed in recipients containing ice water(0 °C) for rapid cooling of the gel.

Determination of gel solubilityStudies commenced immediately the newly-made gelswere sufficiently cool. According to Matsumoto (27)and Careche et al. (17), gels were treated with chemicalsselected for their capacity to cleave certain kinds ofbond: 0.05 mol/L NaCl (SA), 0.6 mol/L NaCl (SB), 0.6mol/L NaCl + 1.5 mol/L urea (SC), 0.6 mol/L NaCl + 8mol/L urea (SD) and 0.6 mol/L NaCl + 8 mol/Lurea + 0.5 mol/L 2-â-mercaptoethanol (SE). Proteinswere partially solubilized with these solutions in orderto determine the existence of nonspecific associations(protein solubilized in SA), ionic bonds (differencebetween protein solubilized in SB and protein solubi-lized in SA), hydrogen bonds (difference betweenprotein solubilized in SC and protein solubilized in SB),hydrophobic interactions (difference between proteinsolubilized in SD and protein solubilized in SC) anddisulfide bonds (difference between protein solubilizedin SE and protein solubilized in SD). Two grams ofchopped gel were homogenized with 10 mL of eachsolution in an Omni-Mixer, model 17106 homogenizer(OMNI International, Waterbury, U.S.A.) for 2 min atsetting 5. The resulting homogenates were stirred at 4–5°C for 1 h, then centrifuged for 15 min at 20,000 3 g ina Cryofuge 20-3 centrifuge (Heraeus CHRIST GmbH,Germany). Protein concentration in supernatants wasdetermined using a commercial preparation, DC Pro-tein Assay Reagent S no 500-0116 (BIO-RAD Labora-tories, CA, U.S.A.). Where necessary the solutions weredialysed previously. Results are the average of twodeterminations and are expressed as g soluble protein/L of homogenate.

Electrophoresis (SDS-PAGE)Supernatants obtained with SA and SD solutions weretreated according to the method of Hames (28) with asolution composed of 50 g/L 2-â-mercaptoethanol, 25g/L SDS, 10 mmol/L Tris-HCl, 1 mmol/L EDTA and0.02 g/L bromophenol blue. They were adjusted to afinal average concentration of 2 mg/mL and thenheated at 100 °C for 5 min. Electrophoresis was carriedout on a Phast-System horizontal apparatus (PHAR-MACIA LKB Biotechnology AB, Uppsala, Sweden)using polyacrylamide gels (125 gL, PhastGel, Pharma-cia LKB Biotechnology). Electrophoresis conditionswere 10 mA, 250 V and 3.0 W, at 15 °C. The proteinbands were stained with Coomassie brilliant blue(PhastGel Blue R, Pharmacia LKB Biotechnology). Asreference for molecular weights, a standard highmolecular weight reference kit (Pharmacia LKB Bio-technology) was used: ferritin half unit 220 kDa,albumin 67 kDa, catalase subunit 60 kDa, lactatedehydrogenase subunit 36 kDa and ferritin subunit 18.5kDa.

Statistical analysis of dataTwo-way analysis of variance (ANOVA) was carriedout. The computer program used was Statgraphics(STSC Inc. Rockville, U.S.A.). The difference betweenmeans of pairs was resolved by means of confidenceintervals using a Least Significant Difference (LSD)range test. Level significance was set for P ≤ 0.05.

Results and Discussion

Protein solubility: disruption of bondsProtein solubility of gels in a number of solutionsselected for their capacity to disrupt certain kinds ofbonds are shown in Table 1.Increase in gelling temperature resulted in a decreaseof soluble protein in SA (0.05 mol/L NaCl), which at50–90 °C was much more pronounced in the gelcontaining egg white. The progressive decline in sol-ubility as a result of increased temperature is related tothe disappearance of nonspecific associations. Duringheat treatment, proteins undergo a process of denatura-tion and aggregation which favours their interactions,resulting in a decline of solubility and hence ofextractability (29, 30). This was more evident in the gelwith egg white, denoting the formation of a structurewhose components are more strongly bonded than ingels with other nonmuscle proteins.Gels containing egg white or sodium caseinate at 35–60°C presented significantly higher protein solubility inSA than gels made with soy protein or gluten. Thiscould be related to the small globule size, high solubilityand strong tendency to distribute as small, dispersedaggregates in the gel matrix of egg white and casein(20–22). However, while in gels with egg white sol-ubility decreased sharply on cooking at 90 °C, in gelswith casein the soluble protein level remained onlyslightly lower than in gels heated at 35–60 °C suggestingthat casein has no active part in the gel networkformation. 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 solubleregardless of the heating temperature.The effect of setting at 35 °C prior to cooking at 90 °Cdid not substantially modify the presence of these weaknonspecific associations as compared with direct cook-ing, except in the gel with casein where the differencewas more pronounced. Obviously nonspecific associa-tions are not directly involved in the strengtheningeffect caused by the two-step heating treatment com-monly used in kamaboko production (31).Solubility in SB (0.6 mol/L NaCl) as a measure of ionicbonds was very low (less than 1 g/L) at all temperatures,thus indicating very minor participation of ionic bondsin gel formation.With regard to hydrogen bonds, in general proteinsolubility in SC (0.6 mol/L NaCl + 1.5 mol/L urea) alsoshowed low values. Gels with egg white were sig-nificantly less solubilized than the others at 60–90 °C,which suggests a predominance of stronger interactionsin these gels. The two-step heat treatment (35 °C/90 °C)

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produced a significant (P ≤ 0.05) reduction in thepresence of hydrogen bonds in gels with sodiumcaseinate or gluten, giving similar values to thoseregistered for the gels with egg white or soy protein.The highest values of protein solubility were obtainedwith SD (0.6 mol/L NaCl + 8 mol/L urea) as a measureof hydrophobic interactions, and were exhibited by gelsheated at 50–60 °C, which are typical modori tem-peratures. The modori phenomenon, which is apprecia-ble rheologically as deterioration of texture, wasconsiderably more pronounced in gels containing soyprotein, casein or gluten where folding test scoresdropped sharply (22). The mechanism causing this isgenerally poorly understood. Proteolytic activity ofalkaline proteases in the muscle has been suggested asthe most probable cause of gel weakening in thistemperature range (32, 33). On the other hand Niwa(34) suggested that modori could come about throughexcessive formation of hydrophobic interactions, thiswould mean that the network built up at settingtemperature undergoes a thermal shrinkage, releasingwater and causing more uneven dispersal of thenetwork. Direct cooking at 90 °C caused a significantdecline in hydrophobic interactions, with the exception

of the gel with added egg white where solubility wasstill low at 60 °C. According to Miyajima (35) tem-peratures beyond 58 °C could weaken hydrophobicinteractions by destabilizing the hydrogen bonds link-ing water molecules, which would interfere with hydro-phobic hydration. Another and perhaps more likelyexplanation could be that at this cooking temperaturethere is a more extensive formation of stronger bonds,such as disulfide or other covalent bonds, which are notsusceptible to cleavage in the extracting solution (36).Disulfide bonds in a relatively high quantity weredetected in gels with added egg white at 60 and 90 °C(Table 1). This could be related to the fact that eggwhite tends to form thermostable gels with extensivecross-linking by disulfide bonds (24, 37). In contrast,less than 1 g/L of soluble protein in SE was obtained forthe other gels at 90 °C. These low values could meanthat the experimental conditions employed for measur-ing disulfide bonds were not sensitive enough and onlyregistered appreciable data when these bonds werepresent in a large quantity. Gels containing soy protein,sodium caseinate or gluten generally exhibited morehydrophobic 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 (nonspecificassociations), 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 60 °C 90 °C 35/90 °C

Nonspecific associationsEgg white 5.37 a/x 4.57 a/y 2.67 a/z 1.28 a/v 1.34 a/vSoy 3.26 b/x 1.74 b/y 1.80 b/yz 1.95 b/z 1.43 a/zCasein 5.71 a/x 5.44 c/x 5.61 c/x 4.90 c/y 3.22 b/zGluten 3.00 b/x 2.76 d/x 2.52 d/y 2.23 d/z 1.59 a/v

Ionic bondsEgg white 0.60 a/x 0.31 ab/y 0.57 a/xy 0.50 a/xy 0.46 a/ySoy 0.27 b/x 0.45 a/y 0.32 a/x 0.48 a/y 0.57 b/yCasein 0.12 c/x 0.23 c/y 0.16 b/x 0.15 b/x 0.10 c/xGluten 0.89 d/x 0.08 b/y 0.39 a/z 0.26 c/z 0.41 d/yz

Hydrogen bondsEgg white 2.20 a/x 0.56 a/y 0.56 a/y 0.90 a/y 0.56 a/ySoy 2.72 a/x 2.85 b/x 1.59 b/y 1.27 b/z 0.75 b/zCasein 4.16 b/x 2.60 b/yz 2.97 c/z 2.27 c/z 1.07 c/yvGluten 2.62 a/x 0.47 a/x 2.41 c/x 2.85 d/y 0.73 b/z

Hydrophobic interactionsEgg white 4.01 a/x 12.60 a/y 5.80 a/z 3.47 a/xz 2.74 a/vSoy 6.40 b/x 13.88 b/y 15.21 b/z 9.71 b/v 7.24 b/xCasein 6.06 c/x 11.49 a/y 11.31 c/y 8.06 c/z 4.68 c/vGluten 6.41 b/x 15.80 c/y 14.37 b/y 8.55 c/z 4.72 c/v

Disulfide bondsEgg white 0.50 a/x 0.24 a/y 2.66 a/v 3.29 a/u 0.52 a/xSoy 0.89 b/x 0.54 b/y 0.32 b/y 0.58 b/y 0.47 a/yCasein 0.26 c/x 0.70 c/y 0.51 c/z 0.95 c/y 0.30 b/xGluten 0.92 b/x 0.13 d/y 0.28 b/y 0.70 b/x 0.18 c/y

Different letters a, b, c etc. indicate significant differences among gels containing the variousnonmuscle proteins for each type of bond.Different letters x, y, z etc. indicate significant differences among heating treatments for eachtype of bond.

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aggregate via hydrophobic interactions (4, 38–40), butas is shown further below, they also interfere to someextent in the cross-linking of the myofibrillar proteins.Hydrophobic interactions were especially low both inthe suwari (35 °C) and in the heat-set (35 °C/90 °C)gels. Since very little formation of disulfide bonds wasdetected (due in part to low sensitivity of the experi-mental technique), it could be assumed that other typesof covalent bonds predominate. At setting tempera-tures such covalent bonds have been attributed to theaction of transglutaminase (TGase) enzymes (12, 13).In gels made with the two-step treatment therefore, thebonds formed at setting temperatures apparently stillremained, and increased only slightly after cooking.

Electrophoretic studyElectrophoretic profiles of the fraction soluble in SA(0.05 mol/L NaCl) and in SD (0.6 mol/L NaCl + 8mol/L urea) of gels with added egg white made at 35, 90and 35/90 °C are shown in Fig. 1. In the SA solubilizedfraction from the suwari gel (35 °C) (Fig. 1a) the bandscorresponding to actin, tropomyosin, troponins andother low molecular weight (MW) proteins were clearlyapparent, as well as two new bands of 42 kDa and 76kDa, which in previous assays were found to corre-spond to ovalbumin and conalbumin, respectively. Ma

and Holme (41) reported two similar bands in electro-phoretic profiles of native egg white protein. Theconsiderable presence of egg white proteins in thisfraction confirmed that these were chiefly responsiblefor the high level of nonspecific associations referred toabove (Table 1). This suggests that at the giventemperature the egg white was weakly linked to the gelmatrix. On solubilizing with SD (Fig. 1b) a new bandappeared in the sample application zone which corre-sponded to an aggregate of proteins (Ag) polymerizedby bonds stronger than hydrophobic interactions. Thisaggregate was not seen in the SA fraction because itremained entirely in the precipitate. The small numberof hydrophobic interactions detected at this tem-perature (Table 1) and the absence of the MHC band inthese conditions of solubilization indicate that theMHC was completely polymerized by disulfide or othercovalent bonds. In gels directly cooked at 90 °C, themain visible band appearing in the SA fraction (Fig. 1c)presented similar MW to that of tropomyosin, suggest-ing that tropomyosin is not directly involved in gelformation. These results are consistent with the findingsof Samejima et al. (42) and Jimenez-Colmenero et al.(43). There were also minor traces of MHC and apolypeptide of 89 kDa which could be a product ofproteolysis. In the SD fraction (Fig. 1d) both the actinband and the MHC band were prominent, whichsuggests that they became partially involved in the gelnetwork by means of both hydrogen and hydrophobicbonds. Nevertheless, the presence of a large aggregatein the sample application zone (Ag) as well as in theinterphase between the stacking and resolving gels,together with the absence of albumin and conalbuminbands, suggests that the egg white was completelyaggregated and may have interacted to some extentwith the bulk of the MHC through disulfide bonds orother covalent bonds. Foegeding et al. (24) found thatmyosin and albumin in a model system interacted toform a gel matrix at temperatures beyond 80 °C, thisbeing the temperature at which albumin undergoessufficient heat alteration. The affinity of egg white forMHC at high temperature may be the reason why aproportion of the MHC continued to interact by meansof 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, thussuggesting that the bulk of the MHC together with theegg white proteins was considerably polymerized bydisulfide or other covalent bonds still remaining in theinsoluble fraction.The electrophoretic profiles of gels made with additionof soy protein are shown in Fig. 2. In the SA solublefraction of the suwari gel (Fig. 2a) some bandsappeared which preliminary results showed to corre-spond to different protein fractions of the soy isolate.This would indicate that at the given temperature aconsiderable part of the added nonmuscle protein wasnonspecifically associated. On solubilizing with SD(Fig. 2b), however, a new fraction of soy isolateappeared, and the MHC band was also faintly visible.This would explain the higher protein solubility in SD

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/Lurea) 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)

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(as a measure of hydrophobic interactions) (Table 1)recorded in the suwari gel with soy protein as comparedto the gel with egg white. In the SD solubilized fractionof gels cooked directly at 90 °C (Fig. 2d) the MHCband, the actin band and the soy isolate fractionsappeared more strongly marked than in the suwari gel(Fig. 2b), indicating considerable participation of allthese proteins in the formation of the definitive gel viahydrogen bonds or hydrophobic interactions.In the heat-set gel (35/90 °C) (Fig. 2e) the MHC wascompletely polymerized, and the aggregate (Ag) andactin band were less visible than in directly cooked gels,indicating greater involvement of these myofibrillarproteins in gel formation by means of bonds strongerthan hydrophobic interactions. A considerable part ofthe soy proteins remained in the SD soluble fractionand was largely responsible for the fact that proteinsolubility in SD (as a measure of hydrophobic inter-actions) was higher in the gel with soy protein than inthe others.In the gel with casein made at 35 °C the bandscorresponding to MHC and casein were not clearlyvisible in the electrophoretic profiles of the SA and SDfractions (Fig. 3a,b). Thus, MHC was found to becompletely polymerized by bonds stronger than hydro-phobic interactions, such as disulfide bonds or covalentbonds, the latter possibly resulting from TGase activity,

which could be responsible for the covalent linkagebetween the MHC and the casein (25). In gels directlycooked at 90 °C, two proteins of 30 kDa and 25 kDa,which are the molecular weights of α- and â-casein,appeared in the electrophoretic profile of the SAsoluble fraction (Fig. 3c). A considerable part of thecasein, then, seems not to have interacted with themyofibrillar proteins in the gel matrix. No bondcorresponding to MHC was observed in either the SAor SD solubilized fractions (Fig. 3c,d), indicating thatpolymerization was complete apart from casein. Thereason for the low capacity of casein to interact withmuscle proteins at high temperature may be that thecomponent molecules of casein are highly amphipatic,with a strong tendency to associate in the form ofmicelles by means of hydrophobic interactions andhydrogen bonds. Moreover, casein has very few sulphy-dryl groups and hence little capacity to form disulfidebonds with myofibrillar proteins (40, 44). In the gelmade with the two-step treatment (Fig. 3e), the caseinbands appeared less intense than in the directly cookedgel, from which it was assumed that following thesetting phase part of the casein remained in associationwith the MHC, linked by bonds stronger than hydro-phobic interactions.The electrophoretic profiles of gels made with addedgluten (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/Lurea) 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/Lurea) 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 ofproteins of high molecular weight and is characterizedby its high degree of insolubility; it was thereforeassumed that it formed large aggregates, which wouldbe retained in the precipitate along with the bulk of theMHC. As with the other ingredients, tropomyosin wasscarcely involved in formation of the gel network andactin appeared to interact chiefly via hydrophobicinteractions or hydrogen bonds in the gel directlycooked at 90 °C.

Conclusions

Gels made with addition of egg white presented anetwork characterized by the predominance of strongerbonding than in the gels with added soy protein, caseinor gluten. The modori phenomenon at 50–60 °C wasapparent in all gels and largely related to an increase ofhydrophobic interactions. In cooked gels (90 °C) and inpre-set gels (35/90 °C) the egg white proteins werecompletely polymerized by stronger bonds than hydro-phobic interactions, whereas these interactions werethose mainly responsible for the aggregation of soyproteins, casein and gluten. On the other hand, aconsiderable part of sodium caseinate remained non-specifically associated to the gel network.

Acknowledgements

This research was financed by the Comision Inter-ministerial de Ciencia y Tecnologia (CICyT) underproject ALI-910899-CO3-01 (1991/1994).

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