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Gelation properties of salt soluble meat protein and soluble wheat protein mixtures
Food Hydrocolloids 17 (2003) 149–159 www.elsevier.com/locate/foodhyd
Gelation properties of salt soluble meat protein and soluble wheat protein mixtures Sarah Comfort, Nazlin K. Howell* School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK Accepted 1 May 2002
Abstract Salt soluble meat proteins (SSMP) and commercially available soluble wheat proteins (SWP) were characterised by SDS-polyacrylamide gel electrophoresis, differential scanning calorimetry (DSC) and small and large deformation testing. DSC scans indicated transitions similar to those of native actomyosin for the salt soluble meat extract whereas SWP did not indicate any transitions between 20 and 120 8C. Small deformation tests on SWP indicated a G0 /G00 crossover gelation temperature of 90 8C and weak gels as judged by frequency sweeps. In contrast, SSMP gelled at 40 8C and formed strong gels on heating to 90 8C. However, on autoclaving at 120 8C, 20% SWP in distilled water produced strong elastic gels with little syneresis, compared with the more brittle gels produced with 20% (w/w) SSMP as indicated by large deformation testing. Mixtures of the two proteins in the ratio SSMP/SWP (15:5) gave strong elastic gels similar to the SWP gels. Even the presence of very small amounts of SWP in the mixture, e.g. SSMP/SWP 20:1 trebled the elastic modulus compared with a SSMP gel and reduced syneresis. This was probably due to the close association of SWP with actomyosin strands as viewed by transmission electron microscopy. However, increased levels of SWP in the mixture, for example SSMP/SWP 10:10 ratio, resulted in the separation of the two protein phases as shown by phase contrast microscopy, and consequently led to lower G0 values in the mixed gels. The addition of 20 mM chloride salts showed that potassium reduced the shear modulus, sodium had no effect and calcium enhanced the shear modulus for SWP gels formed at 120 8C. In contrast, SSMP gels were stronger in the presence of potassium, followed by sodium and calcium. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Soluble wheat proteins; Salt soluble meat proteins; Gelation; Rheology; Phase contrast microscopy; Electron microscopy
1. Introduction Wheat gluten is currently used in meat protein systems to supply proteins and specific amino acids and is a key ingredient in food formulations because of its water and fat binding as well as texturising properties (Maningat, Bassi, & Hesser, 1994). Soluble wheat protein (SWP) is manufactured by non-enzymatic acid deamidation of wheat gluten (Friedli & Howell, 1996). Deamidation generally improves the solubility and the emulsifying properties of proteins by imparting additional negative charges that decreases the isoelectric point of the protein (Hamada, 1994; Howell & Taylor, 1991). Gluten has a considerable number of amino acids containing amide groups (glutamine and asparagine) which are converted into glutamic and aspartic acids by deamidation. Accordingly, the solubility of gluten is * Corresponding author. Tel.: þ 44-1483-876448; fax: þ 44-1483576978. E-mail address: [email protected] (N.K. Howell).
significantly enhanced by deamidation (Howell, Bristow, Copeland, & Friedli, 1998). In contrast to gluten proteins which are very difficult to solubilise, the main muscle proteins myosin and actin are soluble in salt solution (Badii & Howell, 2002a; Howell, 1992). For this reason, 1 –5% salt may be added to comminuted meat during processing. The salt solubilisation process produces a variety of protein aggregates ranging from unassociated individual sarcoplasmic and myofibrillar proteins to intact myofibrils. The salt soluble myofibrillar fraction is largely responsible for the gelation and emulsification properties of comminuted meat products (Acton, Zeigler, & Burge, 1976; Asghar, Samejima, & Yasui, 1983; Badii & Howell, 2002b; Howell, 1978). Thus in this study, myofibrillar proteins were extracted from beef and the composition was characterised prior to gelation studies. Gelation tests were undertaken using large deformation compression testing which are relevant for food gels since the test emulates the action for mastication (Prentice, 1984).
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The properties of interest are the shear modulus of the gel, as given by the initial slope of the stress – strain graph, the force at fracture, Fc (‘break strength’) and deformation at fracture, Dh. While the crude data terms are often quoted it is possible, using the assumption of incompressibility (McEvoy, Ross-Murphy, & Clark, 1985), it is possible to calculate the fracture shear strain (gf) and fracture shear stress (tf). The combination of soluble wheat proteins (SWP) and meat proteins is a system with potential applications in comminuted products. In the pet food industry the combination of wheat gluten and meat proteins has been reported to have been used successfully (Maningat et al., 1994). Pet foods are autoclaved in the canning process and gluten forms its strongest gels at temperatures around 120– 130 8C (Attenburrow, Barnes, Davies, & Ingman, 1990). Thus, the experiments described here were carried out in this temperature range.
2. Materials and methods 2.1. Materials SWP was obtained from Amylum NV, Belgium. Salt soluble meat proteins (SSMP) were extracted from stewing beef purchased from a local supermarket according to a method described later. The ultrafiltration membranes were obtained from Amicon. Electrophoresis reagents: Coomassie Brilliant Blue R250, methanol and glacial acetic acid were obtained from Fisons. All other reagents were Analar grade from Sigma Aldrich, Poole, Dorset. 2.2. Methods 2.2.1. Preparation of the salt soluble meat proteins SSMP were prepared by homogenising stewing steak and salt solution (5% NaCl, 0.02 M NaHCO3, pH 7.6) at a 1:5 ratio at 4 8C. The dispersion was centrifuged at 14,000g for 10 min at 4 8C. The insoluble pellet was discarded and the salt soluble myofibrillar extract was concentrated by filtration using a PM-10 Diaflo ultrafiltration membrane. The effect of different storage conditions was assessed including 4 8C for 16 h; 2 20 8C for 16 h and 2 20 8C for 2 weeks. 2.2.2. Electrophoresis SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out under denaturing conditions as described by Bollag and Edelstein (1994). Samples were prepared by dilution at a 1:1 ratio with a reducing sample buffer and heated for 3 min in a boiling water bath. Any insoluble particulate matter was removed by centrifugation at 10,000g for 10 s. A 5– 20% acrylamide gradient gels were used to resolve the proteins which were stained using a 0.1% (w/v) solution of Coomassie blue R 250 in a 5:5:2 ratio of distilled
water, methanol and glacial acetic acid. Due to difficulty in solubilising large MW gluten proteins, only the SSMP were characterised by SDS-PAGE. 2.2.3. Protein composition by Kjeldahl analysis The protein content of SWP powder and meat extract was determined by Kjeldahl analysis using the standard AOAC (1976) procedure. A factor of N £ 6.25 and N £ 5.8 was used to calculate the protein content of SSMP and SWP, respectively (Egan, Kirk, & Sawyer, 1981). 2.2.4. Differential scanning calorimetry (DSC) DSC measurements were made using a Setaram DSC1 microcalorimeter at a scan rate of 0.1 8C min21 covering the temperature range from 5 to 95 or 130 8C for SSMP and SWP, respectively. Sample pans were filled with approximately 1 g of the protein and a reference pan of distilled water was adjusted to within ^ 0.1 g of the same weight. 2.2.5. Gelation at 120 8C Solutions of 20 and 25% (w/w protein) SSMP, 20 and 21% SWP or mixtures of the two proteins (20:1, 5:15, 10:10, 15:5, 1:20 (SSMP/SWP]) were prepared in distilled water. The protein solutions were poured into 10 mm Visking tubing sacs secured at each end with a knot and wrapped in cling film due to the semi-permeable nature of the tubing. These were autoclaved at 120 8C, 2 atm for 10 min. They were allowed to cool at room temperature for 20 min prior to cutting into 15 mm lengths for testing. The gels were compressed through 12 mm on the Instron Universal Testing Machine at 1 cm min21 using a cylindrical plunger with a diameter of 43 mm (Howell & Lawrie, 1984 ). Shear modulus values (G ) (Pa) of the gel were obtained from the initial slope of the stress – strain graph. In addition, the following equations were used to calculate the fracture shear strain (gf) (strain units) and fracture shear stress (tf) (Pa):
gf ¼ 1:5{ 2 ln½1 2 ðDh=hÞ}
ð1Þ
tf ¼ 0:5Fc =½pR2 =ð1 2 Dh=hÞ
ð2Þ
where h is the undeformed height of the gel cylinder and R the initial cylinder radius. For these equations it is also assumed that the deformed shape is still that of a cylinder not a barrel or hourglass shape. Syneresis in the gels was measured according to Comfort and Howell (2002). The effect of potassium, sodium or calcium chloride (20 mM) on gel formation was investigated for SWP and SSMP by large deformation testing. 2.2.6. Small deformation tests Solutions prepared as described earlier were tested on a Rheometrics controlled stress rheometer using a 40 mm parallel plate geometry with a gap of 0.3 mm. Silicone oil was applied to prevent evaporation during heating. A frequency of 1 rad s21 was used and the applied stress was varied to keep the strain at about 1%. The temperature of the
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peltier plate was programmed to ramp, at a rate of 1 8C min21, from 20 to 90 8C. The sample was held at 90 8C for 1 h until it reached a pseudo-equilibrium. The peltier plate was then cooled to 20 8C at the rate of 2 8C min21. The G0 values were noted at 20 8C before heating, at 90 8C and after cooling at 20 8C. 2.2.7. Phase contrast microscopy The structure of meat proteins, SWP and mixed proteins gels were examined by phase contrast microscopy. Samples, as prepared earlier for gelation studies, were placed on acetone-cleaned microscope slides and viewed under the phase contrast Leitz microscope attached to a Wild MPS 05 system comprising a camera and exposure meter set on camera factor 0.32. 2.2.8. Transmission electron microscopy Identification and disposition of the meat protein and SWP proteins in mixed gels was undertaken by transmission electron microscopy (TEM) according to Comfort and Howell (2002). Samples (1 mm3), cut from gels which were prepared as described earlier, were fixed for 4 h in 5 ml of 4% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 (Howell & Lawrie, 1985). After fixation, the samples were washed several times in the cacodylate buffer before being dehydrated. This was achieved by passing the samples through 25% (w/v) ethanol, 50% (w/v) ethanol, 75% (w/v) ethanol, 90% (w/v) ethanol and finally absolute ethanol. Samples were held for at least 10 min in each solution, and each solution was used twice. After the second incubation in absolute ethanol, the samples were placed in a 1:1 ratio of absolute ethanol and L.R. White resin for 20 min. During this time they were placed on a rotating drum, as the two solutions were immiscible. Finally, the samples were incubated in pure L.R. White resin for 4 h at room temperature in individual capsules (TAAB), and to polymerise the resin samples were incubated for a further 12 h at 60 8C. The resultant embedded samples were sectioned on a microtome and floated onto nickel grids. The samples were stained with uranyl acetate and viewed on a 400T transmission electron microscope.
3. Results 3.1. Characterisation of the proteins
Fig. 1. SDS-PAGE densitometric patterns for (a) meat proteins soluble in salt only (lane 1); water-soluble meat proteins (lane 2) and whole SSMP fraction (lane 3) (b) SSMP after different storage treatments (assignments as for (a)).
SWP powder was found to contain 76% protein and the SSMP fraction contained 31% (w/w) protein. In all the experiments described later, the SWP and SSMP samples were made up w/w protein. Due to the very high molecular weight of the proteins in SWP, characterisation by SDSPAGE electrophoresis was not possible. The electrophoretic pattern and assignments for the separated protein bands are shown for the SSMP was typical for actomyosin (Fig. 1(a)).
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Fig. 2. DSC thermogram of 10% (w/w) SSMP in distilled water, pH 7.0.
The effect of storage is shown in Fig. 1(b). PAGE analysis showed that the myosin heavy chain protein band (200 kDa) was not seen when the SSMP were freeze-dried or exposed to long-term freezing. These processes caused protein denaturation; similar studies are reported for fish muscle proteins which undergo severe protein aggregation (Badii & Howell, 2002a). The myofibrillar proteins stored for a shorter period of 16 h at 4 or 2 20 8C also changed indicating the importance of testing freshly prepared samples. SWP did not indicate conformational transitions by DSC analysis even when heated to 120 8C (not shown), a fact previously reported by Friedli and Howell (1996). This was as expected since the DSC of native wheat gluten does not show any reasonable denaturation peak (Hoseney & Rogers, 1990), although it is clearly changed on heating (Section 3.2.2). For the SSMP the DSC thermogram showed two main endothermic transitions at around 40 – 50 8C for myosin and 65 8C for actin (Fig. 2). The main denaturation transitions of actomyosin fall within this temperature range. Thus, freshly prepared SSMP behaved similarly to native actomyosin and the SWPs may be considered to be in a disordered state. 3.2. Rheological analysis 3.2.1. Large deformation analysis of soluble wheat proteins SWP did not gel at temperatures below 90 8C but formed strong elastic gels at 120 8C which did not undergo syneresis. The minimum concentration required for gelation was similar for both gelation at 90 8C and autoclaved gels (Fig. 3) but the maximum shear modulus (G ) achievable was higher when gels were formed at 120 8C and under pressure. This could be explained by the fact that high temperature favours hydrophobic association up to extreme temperatures when they are disrupted due to the increased kinetic energy of the system. Disulphide – sulphydryl interchange reactions were also implicated in heated gluten protein (Schofield, Bottomley, Timms, & Booth, 1983) together with electrostatic linkages (Attenburrow et al., 1990; Inda & Rha, 1991). In the case of SWP, at concentrations where the number of
Fig. 3. Minimum gelling concentration (C0) of SWP at both 90 and 120 8C.
cross-links formed were not sufficient to form an integral network at 90 8C, the increased temperature did not induce a gel network to form. It is possible that the increased kinetic energy of the system caused as much disruption of the structure as it enhanced cross-link formation. There was therefore no overall effect on the degree of structure formed at higher temperatures. However, at higher concentrations of SWP, when the protein molecules would be in close proximity, any structure disrupted, due to the increased kinetic energy of the system, would be more likely to reform. Along with the enhancement of hydrophobic association the overall effect would be of increased structure formation. 3.2.2. Small deformation analysis of SWP The temperature sweep of 20% (w/w) SWP (with 20 mM calcium) showed an initial fall in the G0 and G00 followed by a small, broad peak at 60 8C (Fig. 4). It was not possible to assign this peak as the denaturation of any of the proteins present in the SWP because the DSC thermogram showed no conformational transitions (Section 3.1). After this peak, the storage modulus continued to fall to a minimum at , 85 8C following which it increased rapidly. The storage modulus became larger than the loss modulus at approximately 90 8C. The frequency sweeps showed that the gel structure formed was weak (Fig. 5(a)). Cooling the structure to 20 8C did not cause any significant increase in the storage modulus but reduced the frequency dependence of the modulus (Fig. 5(b)). 3.2.3. Effect of salts on SWP proteins The effect of chloride salts (20 mM) on gels formed in the autoclave at 120 8C showed that potassium reduced the shear modulus, sodium had no affect and calcium significantly enhanced the gel formation (Fig. 6). This was probably due to the high levels of COOH residues in deamidated SWP contributed mainly by glutamic acid which is the most abundant amino acid present in gluten. Calcium can interact with carboxyl groups to form bridges which strengthen the gel structure (Mulvihill & Kinsella, 1988). The addition of salts also altered the characteristics of the gel produced (Table 1). The addition of both potassium and
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Fig. 4. Temperature sweep (20–90 8C) of 20% (w/w) SWP (20 mM calcium).
sodium salts increased the syneresis seen on compression of the gel. Gels formed in the presence of these monovalent ions did not rupture on compression to 12 mm. The addition of calcium caused a firmer, brittle gel to form which had a high fracture shear stress and strain. It also had a higher capacity to bind water than gels formed in the presence of sodium and potassium.
The storage modulus became greater than the loss modulus almost immediately when the two moduli began to rise to form a peak which reached its maximum at about 50 8C. This corresponded with the main denaturation transition of myosin (Badii & Howell, 2002b; Ziegler & Acton, 1984). At around 70 8C, corresponding to the transition for actin by DSC, the storage modulus began to plateau.
3.2.4. Small deformation analysis of salt soluble meat proteins The temperature sweep of a 20% (w/w) SSMP gelation showed an immediate rise in the storage modulus of the system and the G0 –G00 crossover was at around 40 8C. This indicated that a significant number of cross-links had already been formed at this temperature.
3.2.5. Large deformation analysis of salt soluble meat proteins In contrast to SWP, SSMP gelled readily at about 80 8C but formed brittle gels at 120 8C which underwent syneresis. Gels generally had a higher shear modulus when produced at higher temperatures and pressures. The minimum gelling concentration of SSMP at 90 8C was higher than that when
Fig. 5. Frequency sweep of 20% (w/w) SWP at (a) 90 8C and (b) 20 8C after cooling.
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Fig. 7. Minimum gelling concentration of SSMP gels formed at 90 8C or by an autoclave cycle at 120 8C. Fig. 6. Effect of potassium, sodium or calcium choride (20 mM) on the shear modulus (G ) of 20% (w/w) SWP solutions in distilled water heated to 120 8C.
the gels were formed at 120 8C and under pressure (autoclave treatment) (Fig. 7). Thus, at higher temperatures a more stable network structure was formed. At temperatures above 90 8C any collagen present would be converted into gelatin which, on cooling, may also have contributed to the gel network. Heating meat above 90 8C is known to improve its tenderness because of the breakdown of collagen (Hamm, 1970). Increased pressure is also known to affect the denaturation and gel formation of meat proteins (Hamm, 1970) which may also have contributed to the increase in shear modulus. 3.2.6. Effect of salts on the gelation properties of SSMP The effect of salts on the formation of autoclaved gels was assessed. In general, the addition of salts to the system
made the gels more brittle and less cohesive, i.e. they had a high shear stress and a low shear strain (Table 2). The shear modulus was greatest on the addition of potassium while sodium caused a less marked but significant increase in the shear modulus when compared with that in the meat protein alone (Fig. 8). It has been postulated that an increase in the concentration of Kþ and other monovalent cations lessens the noncovalent interactions required to maintain the structure of myosin and therefore denaturation is enhanced (Goodno & Swenson, 1975). This may explain the increase in shear modulus seen on the addition of sodium and potassium. Gels formed in the presence of calcium also had a higher shear modulus than SSMP gels with no added salt. 3.2.7. Rheological analysis of mixtures of soluble wheat protein and salt soluble protein mixtures The addition of SWP to SSMP gels generally increased the shear modulus of the system (Fig. 9). The replacement of
Table 1 Large deformation analysis of gels made from SWP (20% w/w protein) treated with either 20 mM potassium, sodium or calcium chloride Salt
Fracture shear stress (tf) (kPa) (SD ^ )
None Potassium Sodium Calcium
Did not fracture Did not fracture Did not fracture 44 (12)
Fracture shear strain (gf) (strain units) (SD ^ )
Syneresis on compression% (SD ^ )
1.4 (0.2)
1.1% (0.3) 21.6% (1.4) 16.9% (2.1) 4.2% (0.9)
Table 2 Large deformation analysis of gels made from SSMP (20% w/w protein) treated with either 20 mM potassium, sodium or calcium chloride Salt
Fracture shear stress (tf) (kPa) (SD ^ )
Fracture shear strain (gf) (strain units) (SD ^ )
Syneresis on compression (%) (SD ^ )
None Potassium Sodium Calcium
1.01 (0.5) 35.69 (6.7) 35.62 (3.2) 26.62 (2.0)
1.76 (0.2) 0.80 (0.9) 0.69 (0.2) 0.70 (0.3)
26.2% (1.4) 44.1% (2.8) 37.5% (2.3) 54.7% (2.3)
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Fig. 8. Effect of potassium, sodium or calcium chloride (20 mM) on the shear modulus (G ) (Pa) of 20% (w/w) SSMP solutions heated to 120 8C.
a quarter of the SSMP with SWP produced very strong elastic gels. The gels did not rupture after 12 mm of compression, a characteristic of the SWP gels. Addition of 1% (w/w) SWP to a 20% (w/w) SSMP gel also resulted in substantial increases in gel strength, resulted in a trebling of the shear modulus. The shear modulus was higher than that of a 20% (w/w) SWP gel but was lower than that of a 21% (w/w) SWP gel. The gels also had a high fracture shear stress and shear strain (Table 3) when compared with SSMP gels, which suggested that they were more cohesive and elastic. The addition of small amounts (1% w/w) of SWP to the 20% (w/w) SSMP system caused a dramatic decrease (by 50%) in gel syneresis. The water-loss was reduced still further when a quarter of the SSMP was replaced with SWP. In contrast, large additions of SWP to SSMP resulted in lower gel strength values. For example, at equal ratios of each of the proteins the shear modulus of the gels produced was lower than those seen in gels formed from higher ratios of SWP to SSMP. The gels were also more brittle, with rupture occurring after only 5 mm of compression. The shear modulus was, however, still higher than that of a 20% (w/w) SSMP gel. These gels were most prone to syneresis (Table 3). Similarly, the gels formed from a 3:1 ratio of SWP/SSMP were more brittle than a gel composed of only SWP.
Fig. 9. The shear modulus (G ) (Pa) of SSMP and SWP mixed gels.
However, the addition of only 1% (w/w) SWP to a 20% (w/w) SSMP resulted in a relatively elastic gel similar to that formed from SWP alone. The shear modulus was higher than that of a 20% (w/w) SWP gel but was lower than that of a 21% (w/w) SWP gel. The increased shear modulus appears to be due to the physical disposition of the SWP in the SSMP network as shown in the micrographs later. There was no effect of sodium chloride, used in the preparation of SSMP on the SWP shear modulus as shown in Section 3.2.3 and Fig. 6. 3.3. Analysis of proteins by phase contrast and electron microscopy 3.3.1. Microscopy of soluble wheat protein Phase contrast microscopy. At room temperature 20% (w/w) SWP was observed to contain large protein aggregates (Fig. 10(a)). Heating caused the breakdown of these aggregates (Fig. 10(b)) and this would account for the initial decrease in storage modulus which was seen on heating the protein.
Table 3 Large deformation testing of gels made from mixtures of SWP and SSMP Mixture
Fracture shear stress (tf) (kPa) (SD ^ )
Fracture shear strain (gf) (SD ^ )
Syneresis on compression (SD ^ )
20% SSMP 25% SSMP 20% SSMP 1% SWP 15% SSMP 5% SWP 10% SSMP 10% SWP 5% SSMP 15% SWP 1% SSMP 20% SWP 21% SWP 20% SWP
2.4 (2.2) 37.2 (9.4) 32.5 (6.3) – 13.66 (3.8) 25.48 (2.7) – – –
0.43 (0.08) 0.39 (0.07) 1.01 (0.08) – 0.60 (0.12) 1.37 (0.23) – – –
26.2% (1.4) 19.4% (1.5) 6.7% (2.0) 1.9% (0.7) 8.9% (0.9) 3.6% (1.7) 1.7% (0.4) 0.8% (0.04) 1.1% (0.3)
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Fig. 10. Phase contrast microscopy of 20% (w/w) SWP (20 mM calcium) at (a) 20 8C and (b) 90 8C. Magnification £ 100.
Fig. 11. TEM of a 20% (w/w) SWP (20 mM calcium) gel, (a) magnification £ 8000 and (b) magnification £ 28, 000.
Electron microscopy. SWP gels viewed under the electron microscope (Fig. 11(a)) had a ‘spongy structure’ similar to the wheat gluten gels previously described by Freeman, Shelton, Bjerke, and Skierkowski (1991). At a higher magnification the network strands which made up the gel could be seen (Fig. 11(b)). The strands were between 10 and 30 nm wide and they joined larger aggregates which were approximately 100 nm in diameter. The dimensions of the gluten proteins are not known but the molecular weight of gliadin is in the range 30 –100,000 Da and of glutenin is believed to be in the region of 100,000 Da. Glycinin has a molecular weight of 350,000 Da and dimensions of 11 £ 11 £ 7.5 nm3 (Badley et al., 1975) and so it would be expected that the dimensions of the gluten proteins would be less than this. It was therefore probable that the network strands of the SWP gel were made up of aggregates of the gluten proteins. 3.3.2. Microscopy of salt soluble meat protein Phase contrast microscopy. At room temperature 20% (w/w) SSMP was a transparent dispersion which showed relatively little phase contrast image (Fig. 12(a)). A fibrous network structure formed on heating (Fig. 12(b)). Electron microscopy. Electron micrographs of 20% (w/w) SSMP gels showed cross-sections of its network
fibrous strands which had diameters in the region of 180 nm (Fig. 13(a) and (b)). At high salt concentrations myosin has been seen to associate in an ordered fashion but could be induced to form synthetic filaments (diameter 100 nm) on heating if the ionic strength was lowered (Hermansson, 1994). As expected, at lower concentrations of the protein, a less dense network structure was formed (Fig. 13(b)). The occasional longitudinal section of the fibres were observed and at higher magnification limited transverse associations between the fibres could be seen (Fig. 13(c)). 3.3.3. Microscopy of mixtures of soluble wheat protein and salt soluble proteins Phase contrast microscopy. Phase contrast microscopy of a mixture of SWP and SSMP showed phase separation in the presence of increasing amounts of SWP in the mixture especially at the 1:1 ratio (Fig. 14). Large aggregates of SWP are visible in a continuous network of SSMP fibres. Transmission electron microscopy. When a small amount of the SSMP was replaced by SWP in a mixed gel (17:3 SSMP/SWP), there were large areas of primarily SSMP but much of the SWP was uniformly dispersed throughout the gel (Fig. 15(a) –(c)). The SWP was closely associated with the meat protein fibres which probably led
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Fig. 12. Phase contrast microscopy of SSMP (20% w/w) solution at (a) 20 8C and (b) 90 8C. Magnification £ 100.
Fig. 13. TEM of (a) 20% (w/w) SSMP gel at magnification £ 8000 (b) 13% (w/w) SSMP gel, £ 8000 and (c) 20% (w/w) SSMP gel, £ 28,000.
to the increase in shear modulus values observed by large deformation testing.
4. Conclusions
Fig. 14. Phase contrast micrograph of 10% (w/w) SWP and 10% (w/w) SSMP at 20 8C, £ 100.
SWPs formed weak gels below 90 8C but when autoclaved at 120 8C the proteins interacted to form a strong, elastic gel network. In contrast, SSMP fraction formed stronger gels below 90 8C but on heating at 120 8C SSMP formed a brittle, fibrous gel network with poor water binding capacity.
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Fig. 15. Transmission electron micrographs of mixed gels: (a) 17% (w/w) SSMP and 3% (w/w) SWP, £ 2800 (b) 17% (w/w) SSMP and 3% (w/w) SWP, £ 8000 and (c) 17% (w/w) SSMP and 3% (w/w) SWP, £ 28,000.
SWP can be used to alter the texture properties of mixed gels. With the addition of only 1% SWP a less brittle, smoother texture was noticed which, on the further replacement of meat protein by SWP, became increasingly elastic and cohesive. However, when the ratio of SWP to SSMP approached 1:1 gel strength was reduced. This was probably due to the increased self-association of both proteins causing separate phases to be formed, thus reducing the stabilising effect of the SWP observed between the SSMP strands. These expanses of purely SSMP decreased the shear modulus of the overall gel structure and its waterbinding capacity at 120 8C. Thus the more open, fibrous meat protein gel network appeared to be stabilised by small quantities of the more fine-stranded, ‘sponge-like’ SWP network. The finer stranded nature of the SWP gel gave the structure a higher water-holding capacity and made the mixed gel more cohesive and elastic. The addition of 20 mM chloride salts of potassium, sodium and calcium increased the shear modulus of SSMP gels at 120 8C and only calcium significantly increased the gel strength of SWP gels. Acknowledgments The authors wish to thank MAFF as well as the industrial partners Amylum, Mars plc, Nestle and St Ivel Ltd for
financial support under the Food Processing LINK Programme for project ‘Protein –protein and protein – polysaccharide interactions in food gels’ coordinated by Dr N. Howell. References Acton, J. C., Ziegler, G. R., & Burge, D. L. (1978). Functionality of muscle constituents in the processing of comminuted meat products.CRC Critical Review of Food Science and Nutrition, 18, 99 –121. Asghar, A., Samejima, K., & Yasui, T. (1983). Functionality of muscle proteins in gelation mechanisms of structured meat products. CRC Critical Review of Food Science and Nutrition, 22, 27–106. Attenburrow, G., Barnes, D. J., Davies, A. P., & Ingman, S. J. (1990). Rheological properties of wheat gluten. Journal of Cereal Science, 12, 1 –14. Badii, F., & Howell, N. K. (2002a). A comparison of biochemical changes in cod (Gadus morhua ) and haddock (Melanogrammus aeglefinus ) during frozen storage. Journal of Science and Food Agriculture, 82, 87 –97. Badii, F., & Howell, N. K. (2002b). Changes in the texture and structure of cod and haddock fillets during frozen storage. Food Hydrocolloids, 16, 313 –319. Badley, R. A., Atkinson, R. A., Hauser, H., Oldani, D., Green, J. P., & Stubbs, J. M. (1975). The structure, physical and chemical properties of the soy bean protein glycinin. Biochimica Biophysicica Acta, 412, 214 –228. Bollag, D. M., & Edelstein, S. J. (1994). Protein Methods. New York: Wiley-Liss. Comfort, S. F., & Howell, N. K. (2002). Gelation properties of soya protein isolate with whey protein isolate mixtures. Food Hydrocolloids, 16, 661 –672.
S. Comfort, N.K. Howell / Food Hydrocolloids 17 (2003) 149–159 Egan, H., Kirk, R. S., & Sawyer, R. (1981). Pearsons chemical analysis of foods. London: Longman. Freeman, T. P., Shelton, D. R., Bjerke, J. M., & Skierkowski, K. (1991). The ultrastructure of wheat gluten: Variations related to sample preparation. Cereal Chemistry, 68, 492 –498. Friedli, G. L., & Howell, N. (1996). Gelation properties of deamidated soluble wheat protein. Food Hydrocolloids, 10, 255 –261. Goodno, C. C., & Swenson, C. A. (1975). Thermal transitions of myosin and its helical fragments II. Solvent-induced variations in conformational stability. Biochemistry, 14, 873 –876. Hamada, J. S. (1994). Deamidation of food proteins to improve functionality. CRC Critical Review of Food Science and Nutrition, 34, 283 –292. Hamm, R. (1970). Properties of meat proteins. In R. A. Lawrie (Ed.), Proteins as human food (pp. 167–186). London: Butterworths. Hermansson, A. (1994). Microstructure of protein gels related to functionality. In R. Y. Yada, R. L. Jackman, & J. L. Smith (Eds.), Protein structure–function relationships in foods (pp. 22–43). London: Blackie Academic and Professional. Hoseney, R. C., & Rogers, D. E. (1990). The formation and properties of wheat flour doughs. CRC Critical Review of Food Science and Nutrition, 29, 73–93. Howell, N. (1978). Use of animal proteins in food. Journal of International Flavours and Food Additives, May/June, 119 –126. Howell, N. K. (1992). Protein– protein interactions. In B. J. F. Hudson (Ed.), Biochemistry of food proteins (pp. 35–74). Essex: Elsevier. Howell, N., Bristow, E., Emma Copeland, & Friedli, G. L. (1998). Interaction of deamidated soluble wheat protein with sodium alginate. Food Hydrocolloids, 12, 317 –324.
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Howell, N. K., & Lawrie, R. A. (1984). Functional aspects of blood plasma proteins. 2. Gelling properties. Journal of Food Technology, 19, 289– 295. Howell, N. K., & Lawrie, R. A. (1985). Functional aspects of blood plasma proteins. 4. Elucidation of the mechanism of gelation of plasma and egg albumen proteins. Journal of Food Technology, 20, 489 –504. Howell, N. K., & Taylor, C. (1991). Effect of amidation on the foaming and physicochemical properties of bovine serum albumin. International Journal of Food Science and Technology, 26, 385–395. Inda, A. E., & Rha, C. (1991). Dynamic viscoelastic behaviour of wheat gluten: The effects of hydrogen bonding modification by urea and deuterium oxide. Journal of Textile Studies, 22, 393 –411. Maningat, C. C., Bassi, S., & Hesser, J. M. (1994). Wheat gluten in food and non-food systems. Technical Bulletin—American Institute of Baking Research, 16, 1– 8. McEvoy, H., Ross-Murphy, S. B., & Clark, A. H. (1985). Large deformation and ultimate properties of biopolymer gels: 1. Single biopolymer component systems. Polymer, 26, 1483–1500. Mulvihill, D. M., & Kinsella, J. E. (1988). Gelation of b-lactoglobulin: Effects of sodium chloride and calcium chloride on the rheological and structural properties of gels. Journal of Food Science, 53, 231 –236. Prentice, J. H. (1984). Measurements in the rheology of foodstuffs. New York: Elsevier. Schofield, J. D., Bottomley, R. C., Timms, M. F., & Booth, M. R. (1983). The effect of heat on wheat gluten and the involvement of sulphydryl– disulphide interchange reactions. Journal of Cereal Science, 1, 241– 245. Ziegler, G. R., & Acton, J. C. (1984). Mechanism of gel formation by proteins of muscle tissue. Food Technology, 38, 77–80. See also p. 82.
Gelation properties of salt soluble meat protein and soluble wheat protein mixtures Sarah Comfort, Nazlin K. Howell* School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK Accepted 1 May 2002
Abstract Salt soluble meat proteins (SSMP) and commercially available soluble wheat proteins (SWP) were characterised by SDS-polyacrylamide gel electrophoresis, differential scanning calorimetry (DSC) and small and large deformation testing. DSC scans indicated transitions similar to those of native actomyosin for the salt soluble meat extract whereas SWP did not indicate any transitions between 20 and 120 8C. Small deformation tests on SWP indicated a G0 /G00 crossover gelation temperature of 90 8C and weak gels as judged by frequency sweeps. In contrast, SSMP gelled at 40 8C and formed strong gels on heating to 90 8C. However, on autoclaving at 120 8C, 20% SWP in distilled water produced strong elastic gels with little syneresis, compared with the more brittle gels produced with 20% (w/w) SSMP as indicated by large deformation testing. Mixtures of the two proteins in the ratio SSMP/SWP (15:5) gave strong elastic gels similar to the SWP gels. Even the presence of very small amounts of SWP in the mixture, e.g. SSMP/SWP 20:1 trebled the elastic modulus compared with a SSMP gel and reduced syneresis. This was probably due to the close association of SWP with actomyosin strands as viewed by transmission electron microscopy. However, increased levels of SWP in the mixture, for example SSMP/SWP 10:10 ratio, resulted in the separation of the two protein phases as shown by phase contrast microscopy, and consequently led to lower G0 values in the mixed gels. The addition of 20 mM chloride salts showed that potassium reduced the shear modulus, sodium had no effect and calcium enhanced the shear modulus for SWP gels formed at 120 8C. In contrast, SSMP gels were stronger in the presence of potassium, followed by sodium and calcium. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Soluble wheat proteins; Salt soluble meat proteins; Gelation; Rheology; Phase contrast microscopy; Electron microscopy
1. Introduction Wheat gluten is currently used in meat protein systems to supply proteins and specific amino acids and is a key ingredient in food formulations because of its water and fat binding as well as texturising properties (Maningat, Bassi, & Hesser, 1994). Soluble wheat protein (SWP) is manufactured by non-enzymatic acid deamidation of wheat gluten (Friedli & Howell, 1996). Deamidation generally improves the solubility and the emulsifying properties of proteins by imparting additional negative charges that decreases the isoelectric point of the protein (Hamada, 1994; Howell & Taylor, 1991). Gluten has a considerable number of amino acids containing amide groups (glutamine and asparagine) which are converted into glutamic and aspartic acids by deamidation. Accordingly, the solubility of gluten is * Corresponding author. Tel.: þ 44-1483-876448; fax: þ 44-1483576978. E-mail address: [email protected] (N.K. Howell).
significantly enhanced by deamidation (Howell, Bristow, Copeland, & Friedli, 1998). In contrast to gluten proteins which are very difficult to solubilise, the main muscle proteins myosin and actin are soluble in salt solution (Badii & Howell, 2002a; Howell, 1992). For this reason, 1 –5% salt may be added to comminuted meat during processing. The salt solubilisation process produces a variety of protein aggregates ranging from unassociated individual sarcoplasmic and myofibrillar proteins to intact myofibrils. The salt soluble myofibrillar fraction is largely responsible for the gelation and emulsification properties of comminuted meat products (Acton, Zeigler, & Burge, 1976; Asghar, Samejima, & Yasui, 1983; Badii & Howell, 2002b; Howell, 1978). Thus in this study, myofibrillar proteins were extracted from beef and the composition was characterised prior to gelation studies. Gelation tests were undertaken using large deformation compression testing which are relevant for food gels since the test emulates the action for mastication (Prentice, 1984).
0268-005X/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 8 - 0 0 5 X ( 0 2 ) 0 0 0 4 7 - 4
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The properties of interest are the shear modulus of the gel, as given by the initial slope of the stress – strain graph, the force at fracture, Fc (‘break strength’) and deformation at fracture, Dh. While the crude data terms are often quoted it is possible, using the assumption of incompressibility (McEvoy, Ross-Murphy, & Clark, 1985), it is possible to calculate the fracture shear strain (gf) and fracture shear stress (tf). The combination of soluble wheat proteins (SWP) and meat proteins is a system with potential applications in comminuted products. In the pet food industry the combination of wheat gluten and meat proteins has been reported to have been used successfully (Maningat et al., 1994). Pet foods are autoclaved in the canning process and gluten forms its strongest gels at temperatures around 120– 130 8C (Attenburrow, Barnes, Davies, & Ingman, 1990). Thus, the experiments described here were carried out in this temperature range.
2. Materials and methods 2.1. Materials SWP was obtained from Amylum NV, Belgium. Salt soluble meat proteins (SSMP) were extracted from stewing beef purchased from a local supermarket according to a method described later. The ultrafiltration membranes were obtained from Amicon. Electrophoresis reagents: Coomassie Brilliant Blue R250, methanol and glacial acetic acid were obtained from Fisons. All other reagents were Analar grade from Sigma Aldrich, Poole, Dorset. 2.2. Methods 2.2.1. Preparation of the salt soluble meat proteins SSMP were prepared by homogenising stewing steak and salt solution (5% NaCl, 0.02 M NaHCO3, pH 7.6) at a 1:5 ratio at 4 8C. The dispersion was centrifuged at 14,000g for 10 min at 4 8C. The insoluble pellet was discarded and the salt soluble myofibrillar extract was concentrated by filtration using a PM-10 Diaflo ultrafiltration membrane. The effect of different storage conditions was assessed including 4 8C for 16 h; 2 20 8C for 16 h and 2 20 8C for 2 weeks. 2.2.2. Electrophoresis SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out under denaturing conditions as described by Bollag and Edelstein (1994). Samples were prepared by dilution at a 1:1 ratio with a reducing sample buffer and heated for 3 min in a boiling water bath. Any insoluble particulate matter was removed by centrifugation at 10,000g for 10 s. A 5– 20% acrylamide gradient gels were used to resolve the proteins which were stained using a 0.1% (w/v) solution of Coomassie blue R 250 in a 5:5:2 ratio of distilled
water, methanol and glacial acetic acid. Due to difficulty in solubilising large MW gluten proteins, only the SSMP were characterised by SDS-PAGE. 2.2.3. Protein composition by Kjeldahl analysis The protein content of SWP powder and meat extract was determined by Kjeldahl analysis using the standard AOAC (1976) procedure. A factor of N £ 6.25 and N £ 5.8 was used to calculate the protein content of SSMP and SWP, respectively (Egan, Kirk, & Sawyer, 1981). 2.2.4. Differential scanning calorimetry (DSC) DSC measurements were made using a Setaram DSC1 microcalorimeter at a scan rate of 0.1 8C min21 covering the temperature range from 5 to 95 or 130 8C for SSMP and SWP, respectively. Sample pans were filled with approximately 1 g of the protein and a reference pan of distilled water was adjusted to within ^ 0.1 g of the same weight. 2.2.5. Gelation at 120 8C Solutions of 20 and 25% (w/w protein) SSMP, 20 and 21% SWP or mixtures of the two proteins (20:1, 5:15, 10:10, 15:5, 1:20 (SSMP/SWP]) were prepared in distilled water. The protein solutions were poured into 10 mm Visking tubing sacs secured at each end with a knot and wrapped in cling film due to the semi-permeable nature of the tubing. These were autoclaved at 120 8C, 2 atm for 10 min. They were allowed to cool at room temperature for 20 min prior to cutting into 15 mm lengths for testing. The gels were compressed through 12 mm on the Instron Universal Testing Machine at 1 cm min21 using a cylindrical plunger with a diameter of 43 mm (Howell & Lawrie, 1984 ). Shear modulus values (G ) (Pa) of the gel were obtained from the initial slope of the stress – strain graph. In addition, the following equations were used to calculate the fracture shear strain (gf) (strain units) and fracture shear stress (tf) (Pa):
gf ¼ 1:5{ 2 ln½1 2 ðDh=hÞ}
ð1Þ
tf ¼ 0:5Fc =½pR2 =ð1 2 Dh=hÞ
ð2Þ
where h is the undeformed height of the gel cylinder and R the initial cylinder radius. For these equations it is also assumed that the deformed shape is still that of a cylinder not a barrel or hourglass shape. Syneresis in the gels was measured according to Comfort and Howell (2002). The effect of potassium, sodium or calcium chloride (20 mM) on gel formation was investigated for SWP and SSMP by large deformation testing. 2.2.6. Small deformation tests Solutions prepared as described earlier were tested on a Rheometrics controlled stress rheometer using a 40 mm parallel plate geometry with a gap of 0.3 mm. Silicone oil was applied to prevent evaporation during heating. A frequency of 1 rad s21 was used and the applied stress was varied to keep the strain at about 1%. The temperature of the
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peltier plate was programmed to ramp, at a rate of 1 8C min21, from 20 to 90 8C. The sample was held at 90 8C for 1 h until it reached a pseudo-equilibrium. The peltier plate was then cooled to 20 8C at the rate of 2 8C min21. The G0 values were noted at 20 8C before heating, at 90 8C and after cooling at 20 8C. 2.2.7. Phase contrast microscopy The structure of meat proteins, SWP and mixed proteins gels were examined by phase contrast microscopy. Samples, as prepared earlier for gelation studies, were placed on acetone-cleaned microscope slides and viewed under the phase contrast Leitz microscope attached to a Wild MPS 05 system comprising a camera and exposure meter set on camera factor 0.32. 2.2.8. Transmission electron microscopy Identification and disposition of the meat protein and SWP proteins in mixed gels was undertaken by transmission electron microscopy (TEM) according to Comfort and Howell (2002). Samples (1 mm3), cut from gels which were prepared as described earlier, were fixed for 4 h in 5 ml of 4% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 (Howell & Lawrie, 1985). After fixation, the samples were washed several times in the cacodylate buffer before being dehydrated. This was achieved by passing the samples through 25% (w/v) ethanol, 50% (w/v) ethanol, 75% (w/v) ethanol, 90% (w/v) ethanol and finally absolute ethanol. Samples were held for at least 10 min in each solution, and each solution was used twice. After the second incubation in absolute ethanol, the samples were placed in a 1:1 ratio of absolute ethanol and L.R. White resin for 20 min. During this time they were placed on a rotating drum, as the two solutions were immiscible. Finally, the samples were incubated in pure L.R. White resin for 4 h at room temperature in individual capsules (TAAB), and to polymerise the resin samples were incubated for a further 12 h at 60 8C. The resultant embedded samples were sectioned on a microtome and floated onto nickel grids. The samples were stained with uranyl acetate and viewed on a 400T transmission electron microscope.
3. Results 3.1. Characterisation of the proteins
Fig. 1. SDS-PAGE densitometric patterns for (a) meat proteins soluble in salt only (lane 1); water-soluble meat proteins (lane 2) and whole SSMP fraction (lane 3) (b) SSMP after different storage treatments (assignments as for (a)).
SWP powder was found to contain 76% protein and the SSMP fraction contained 31% (w/w) protein. In all the experiments described later, the SWP and SSMP samples were made up w/w protein. Due to the very high molecular weight of the proteins in SWP, characterisation by SDSPAGE electrophoresis was not possible. The electrophoretic pattern and assignments for the separated protein bands are shown for the SSMP was typical for actomyosin (Fig. 1(a)).
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Fig. 2. DSC thermogram of 10% (w/w) SSMP in distilled water, pH 7.0.
The effect of storage is shown in Fig. 1(b). PAGE analysis showed that the myosin heavy chain protein band (200 kDa) was not seen when the SSMP were freeze-dried or exposed to long-term freezing. These processes caused protein denaturation; similar studies are reported for fish muscle proteins which undergo severe protein aggregation (Badii & Howell, 2002a). The myofibrillar proteins stored for a shorter period of 16 h at 4 or 2 20 8C also changed indicating the importance of testing freshly prepared samples. SWP did not indicate conformational transitions by DSC analysis even when heated to 120 8C (not shown), a fact previously reported by Friedli and Howell (1996). This was as expected since the DSC of native wheat gluten does not show any reasonable denaturation peak (Hoseney & Rogers, 1990), although it is clearly changed on heating (Section 3.2.2). For the SSMP the DSC thermogram showed two main endothermic transitions at around 40 – 50 8C for myosin and 65 8C for actin (Fig. 2). The main denaturation transitions of actomyosin fall within this temperature range. Thus, freshly prepared SSMP behaved similarly to native actomyosin and the SWPs may be considered to be in a disordered state. 3.2. Rheological analysis 3.2.1. Large deformation analysis of soluble wheat proteins SWP did not gel at temperatures below 90 8C but formed strong elastic gels at 120 8C which did not undergo syneresis. The minimum concentration required for gelation was similar for both gelation at 90 8C and autoclaved gels (Fig. 3) but the maximum shear modulus (G ) achievable was higher when gels were formed at 120 8C and under pressure. This could be explained by the fact that high temperature favours hydrophobic association up to extreme temperatures when they are disrupted due to the increased kinetic energy of the system. Disulphide – sulphydryl interchange reactions were also implicated in heated gluten protein (Schofield, Bottomley, Timms, & Booth, 1983) together with electrostatic linkages (Attenburrow et al., 1990; Inda & Rha, 1991). In the case of SWP, at concentrations where the number of
Fig. 3. Minimum gelling concentration (C0) of SWP at both 90 and 120 8C.
cross-links formed were not sufficient to form an integral network at 90 8C, the increased temperature did not induce a gel network to form. It is possible that the increased kinetic energy of the system caused as much disruption of the structure as it enhanced cross-link formation. There was therefore no overall effect on the degree of structure formed at higher temperatures. However, at higher concentrations of SWP, when the protein molecules would be in close proximity, any structure disrupted, due to the increased kinetic energy of the system, would be more likely to reform. Along with the enhancement of hydrophobic association the overall effect would be of increased structure formation. 3.2.2. Small deformation analysis of SWP The temperature sweep of 20% (w/w) SWP (with 20 mM calcium) showed an initial fall in the G0 and G00 followed by a small, broad peak at 60 8C (Fig. 4). It was not possible to assign this peak as the denaturation of any of the proteins present in the SWP because the DSC thermogram showed no conformational transitions (Section 3.1). After this peak, the storage modulus continued to fall to a minimum at , 85 8C following which it increased rapidly. The storage modulus became larger than the loss modulus at approximately 90 8C. The frequency sweeps showed that the gel structure formed was weak (Fig. 5(a)). Cooling the structure to 20 8C did not cause any significant increase in the storage modulus but reduced the frequency dependence of the modulus (Fig. 5(b)). 3.2.3. Effect of salts on SWP proteins The effect of chloride salts (20 mM) on gels formed in the autoclave at 120 8C showed that potassium reduced the shear modulus, sodium had no affect and calcium significantly enhanced the gel formation (Fig. 6). This was probably due to the high levels of COOH residues in deamidated SWP contributed mainly by glutamic acid which is the most abundant amino acid present in gluten. Calcium can interact with carboxyl groups to form bridges which strengthen the gel structure (Mulvihill & Kinsella, 1988). The addition of salts also altered the characteristics of the gel produced (Table 1). The addition of both potassium and
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Fig. 4. Temperature sweep (20–90 8C) of 20% (w/w) SWP (20 mM calcium).
sodium salts increased the syneresis seen on compression of the gel. Gels formed in the presence of these monovalent ions did not rupture on compression to 12 mm. The addition of calcium caused a firmer, brittle gel to form which had a high fracture shear stress and strain. It also had a higher capacity to bind water than gels formed in the presence of sodium and potassium.
The storage modulus became greater than the loss modulus almost immediately when the two moduli began to rise to form a peak which reached its maximum at about 50 8C. This corresponded with the main denaturation transition of myosin (Badii & Howell, 2002b; Ziegler & Acton, 1984). At around 70 8C, corresponding to the transition for actin by DSC, the storage modulus began to plateau.
3.2.4. Small deformation analysis of salt soluble meat proteins The temperature sweep of a 20% (w/w) SSMP gelation showed an immediate rise in the storage modulus of the system and the G0 –G00 crossover was at around 40 8C. This indicated that a significant number of cross-links had already been formed at this temperature.
3.2.5. Large deformation analysis of salt soluble meat proteins In contrast to SWP, SSMP gelled readily at about 80 8C but formed brittle gels at 120 8C which underwent syneresis. Gels generally had a higher shear modulus when produced at higher temperatures and pressures. The minimum gelling concentration of SSMP at 90 8C was higher than that when
Fig. 5. Frequency sweep of 20% (w/w) SWP at (a) 90 8C and (b) 20 8C after cooling.
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Fig. 7. Minimum gelling concentration of SSMP gels formed at 90 8C or by an autoclave cycle at 120 8C. Fig. 6. Effect of potassium, sodium or calcium choride (20 mM) on the shear modulus (G ) of 20% (w/w) SWP solutions in distilled water heated to 120 8C.
the gels were formed at 120 8C and under pressure (autoclave treatment) (Fig. 7). Thus, at higher temperatures a more stable network structure was formed. At temperatures above 90 8C any collagen present would be converted into gelatin which, on cooling, may also have contributed to the gel network. Heating meat above 90 8C is known to improve its tenderness because of the breakdown of collagen (Hamm, 1970). Increased pressure is also known to affect the denaturation and gel formation of meat proteins (Hamm, 1970) which may also have contributed to the increase in shear modulus. 3.2.6. Effect of salts on the gelation properties of SSMP The effect of salts on the formation of autoclaved gels was assessed. In general, the addition of salts to the system
made the gels more brittle and less cohesive, i.e. they had a high shear stress and a low shear strain (Table 2). The shear modulus was greatest on the addition of potassium while sodium caused a less marked but significant increase in the shear modulus when compared with that in the meat protein alone (Fig. 8). It has been postulated that an increase in the concentration of Kþ and other monovalent cations lessens the noncovalent interactions required to maintain the structure of myosin and therefore denaturation is enhanced (Goodno & Swenson, 1975). This may explain the increase in shear modulus seen on the addition of sodium and potassium. Gels formed in the presence of calcium also had a higher shear modulus than SSMP gels with no added salt. 3.2.7. Rheological analysis of mixtures of soluble wheat protein and salt soluble protein mixtures The addition of SWP to SSMP gels generally increased the shear modulus of the system (Fig. 9). The replacement of
Table 1 Large deformation analysis of gels made from SWP (20% w/w protein) treated with either 20 mM potassium, sodium or calcium chloride Salt
Fracture shear stress (tf) (kPa) (SD ^ )
None Potassium Sodium Calcium
Did not fracture Did not fracture Did not fracture 44 (12)
Fracture shear strain (gf) (strain units) (SD ^ )
Syneresis on compression% (SD ^ )
1.4 (0.2)
1.1% (0.3) 21.6% (1.4) 16.9% (2.1) 4.2% (0.9)
Table 2 Large deformation analysis of gels made from SSMP (20% w/w protein) treated with either 20 mM potassium, sodium or calcium chloride Salt
Fracture shear stress (tf) (kPa) (SD ^ )
Fracture shear strain (gf) (strain units) (SD ^ )
Syneresis on compression (%) (SD ^ )
None Potassium Sodium Calcium
1.01 (0.5) 35.69 (6.7) 35.62 (3.2) 26.62 (2.0)
1.76 (0.2) 0.80 (0.9) 0.69 (0.2) 0.70 (0.3)
26.2% (1.4) 44.1% (2.8) 37.5% (2.3) 54.7% (2.3)
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Fig. 8. Effect of potassium, sodium or calcium chloride (20 mM) on the shear modulus (G ) (Pa) of 20% (w/w) SSMP solutions heated to 120 8C.
a quarter of the SSMP with SWP produced very strong elastic gels. The gels did not rupture after 12 mm of compression, a characteristic of the SWP gels. Addition of 1% (w/w) SWP to a 20% (w/w) SSMP gel also resulted in substantial increases in gel strength, resulted in a trebling of the shear modulus. The shear modulus was higher than that of a 20% (w/w) SWP gel but was lower than that of a 21% (w/w) SWP gel. The gels also had a high fracture shear stress and shear strain (Table 3) when compared with SSMP gels, which suggested that they were more cohesive and elastic. The addition of small amounts (1% w/w) of SWP to the 20% (w/w) SSMP system caused a dramatic decrease (by 50%) in gel syneresis. The water-loss was reduced still further when a quarter of the SSMP was replaced with SWP. In contrast, large additions of SWP to SSMP resulted in lower gel strength values. For example, at equal ratios of each of the proteins the shear modulus of the gels produced was lower than those seen in gels formed from higher ratios of SWP to SSMP. The gels were also more brittle, with rupture occurring after only 5 mm of compression. The shear modulus was, however, still higher than that of a 20% (w/w) SSMP gel. These gels were most prone to syneresis (Table 3). Similarly, the gels formed from a 3:1 ratio of SWP/SSMP were more brittle than a gel composed of only SWP.
Fig. 9. The shear modulus (G ) (Pa) of SSMP and SWP mixed gels.
However, the addition of only 1% (w/w) SWP to a 20% (w/w) SSMP resulted in a relatively elastic gel similar to that formed from SWP alone. The shear modulus was higher than that of a 20% (w/w) SWP gel but was lower than that of a 21% (w/w) SWP gel. The increased shear modulus appears to be due to the physical disposition of the SWP in the SSMP network as shown in the micrographs later. There was no effect of sodium chloride, used in the preparation of SSMP on the SWP shear modulus as shown in Section 3.2.3 and Fig. 6. 3.3. Analysis of proteins by phase contrast and electron microscopy 3.3.1. Microscopy of soluble wheat protein Phase contrast microscopy. At room temperature 20% (w/w) SWP was observed to contain large protein aggregates (Fig. 10(a)). Heating caused the breakdown of these aggregates (Fig. 10(b)) and this would account for the initial decrease in storage modulus which was seen on heating the protein.
Table 3 Large deformation testing of gels made from mixtures of SWP and SSMP Mixture
Fracture shear stress (tf) (kPa) (SD ^ )
Fracture shear strain (gf) (SD ^ )
Syneresis on compression (SD ^ )
20% SSMP 25% SSMP 20% SSMP 1% SWP 15% SSMP 5% SWP 10% SSMP 10% SWP 5% SSMP 15% SWP 1% SSMP 20% SWP 21% SWP 20% SWP
2.4 (2.2) 37.2 (9.4) 32.5 (6.3) – 13.66 (3.8) 25.48 (2.7) – – –
0.43 (0.08) 0.39 (0.07) 1.01 (0.08) – 0.60 (0.12) 1.37 (0.23) – – –
26.2% (1.4) 19.4% (1.5) 6.7% (2.0) 1.9% (0.7) 8.9% (0.9) 3.6% (1.7) 1.7% (0.4) 0.8% (0.04) 1.1% (0.3)
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Fig. 10. Phase contrast microscopy of 20% (w/w) SWP (20 mM calcium) at (a) 20 8C and (b) 90 8C. Magnification £ 100.
Fig. 11. TEM of a 20% (w/w) SWP (20 mM calcium) gel, (a) magnification £ 8000 and (b) magnification £ 28, 000.
Electron microscopy. SWP gels viewed under the electron microscope (Fig. 11(a)) had a ‘spongy structure’ similar to the wheat gluten gels previously described by Freeman, Shelton, Bjerke, and Skierkowski (1991). At a higher magnification the network strands which made up the gel could be seen (Fig. 11(b)). The strands were between 10 and 30 nm wide and they joined larger aggregates which were approximately 100 nm in diameter. The dimensions of the gluten proteins are not known but the molecular weight of gliadin is in the range 30 –100,000 Da and of glutenin is believed to be in the region of 100,000 Da. Glycinin has a molecular weight of 350,000 Da and dimensions of 11 £ 11 £ 7.5 nm3 (Badley et al., 1975) and so it would be expected that the dimensions of the gluten proteins would be less than this. It was therefore probable that the network strands of the SWP gel were made up of aggregates of the gluten proteins. 3.3.2. Microscopy of salt soluble meat protein Phase contrast microscopy. At room temperature 20% (w/w) SSMP was a transparent dispersion which showed relatively little phase contrast image (Fig. 12(a)). A fibrous network structure formed on heating (Fig. 12(b)). Electron microscopy. Electron micrographs of 20% (w/w) SSMP gels showed cross-sections of its network
fibrous strands which had diameters in the region of 180 nm (Fig. 13(a) and (b)). At high salt concentrations myosin has been seen to associate in an ordered fashion but could be induced to form synthetic filaments (diameter 100 nm) on heating if the ionic strength was lowered (Hermansson, 1994). As expected, at lower concentrations of the protein, a less dense network structure was formed (Fig. 13(b)). The occasional longitudinal section of the fibres were observed and at higher magnification limited transverse associations between the fibres could be seen (Fig. 13(c)). 3.3.3. Microscopy of mixtures of soluble wheat protein and salt soluble proteins Phase contrast microscopy. Phase contrast microscopy of a mixture of SWP and SSMP showed phase separation in the presence of increasing amounts of SWP in the mixture especially at the 1:1 ratio (Fig. 14). Large aggregates of SWP are visible in a continuous network of SSMP fibres. Transmission electron microscopy. When a small amount of the SSMP was replaced by SWP in a mixed gel (17:3 SSMP/SWP), there were large areas of primarily SSMP but much of the SWP was uniformly dispersed throughout the gel (Fig. 15(a) –(c)). The SWP was closely associated with the meat protein fibres which probably led
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Fig. 12. Phase contrast microscopy of SSMP (20% w/w) solution at (a) 20 8C and (b) 90 8C. Magnification £ 100.
Fig. 13. TEM of (a) 20% (w/w) SSMP gel at magnification £ 8000 (b) 13% (w/w) SSMP gel, £ 8000 and (c) 20% (w/w) SSMP gel, £ 28,000.
to the increase in shear modulus values observed by large deformation testing.
4. Conclusions
Fig. 14. Phase contrast micrograph of 10% (w/w) SWP and 10% (w/w) SSMP at 20 8C, £ 100.
SWPs formed weak gels below 90 8C but when autoclaved at 120 8C the proteins interacted to form a strong, elastic gel network. In contrast, SSMP fraction formed stronger gels below 90 8C but on heating at 120 8C SSMP formed a brittle, fibrous gel network with poor water binding capacity.
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Fig. 15. Transmission electron micrographs of mixed gels: (a) 17% (w/w) SSMP and 3% (w/w) SWP, £ 2800 (b) 17% (w/w) SSMP and 3% (w/w) SWP, £ 8000 and (c) 17% (w/w) SSMP and 3% (w/w) SWP, £ 28,000.
SWP can be used to alter the texture properties of mixed gels. With the addition of only 1% SWP a less brittle, smoother texture was noticed which, on the further replacement of meat protein by SWP, became increasingly elastic and cohesive. However, when the ratio of SWP to SSMP approached 1:1 gel strength was reduced. This was probably due to the increased self-association of both proteins causing separate phases to be formed, thus reducing the stabilising effect of the SWP observed between the SSMP strands. These expanses of purely SSMP decreased the shear modulus of the overall gel structure and its waterbinding capacity at 120 8C. Thus the more open, fibrous meat protein gel network appeared to be stabilised by small quantities of the more fine-stranded, ‘sponge-like’ SWP network. The finer stranded nature of the SWP gel gave the structure a higher water-holding capacity and made the mixed gel more cohesive and elastic. The addition of 20 mM chloride salts of potassium, sodium and calcium increased the shear modulus of SSMP gels at 120 8C and only calcium significantly increased the gel strength of SWP gels. Acknowledgments The authors wish to thank MAFF as well as the industrial partners Amylum, Mars plc, Nestle and St Ivel Ltd for
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