Journal of Food Science. Vol. 47. 1982. pp. 1241-44

Conditions for the Formation of Heat-Induced Gels of Some Globular Food Proteins

Per Olof Hegg

Author Hegg is affiliated with the Dept. of Food Technology, Univ. of Lund, Box. 740, S-220 07 Lund, Sweden.


The quality of thermally induced aggregates of the globular proteins conalbumin, serum albumin, beta-lactoglobulin and lysozyme has been examined at various salt concentrations and pH values. The properties of the aggregates were characterized by their dry matter content. The results are given as simple phase diagrams, The following areas of dry matter content were found: solubility; transparent and opaque gels (dry matter content of 5-9%); precipitates (dry matter content above 9). Gels were formed only close to conditions of solubility. Only serum albumin was found to be a protein with good gelling properties. A small gelling area was registered for beta-lactoglobulin, while no gelling was observed for conalbumin or lysozyme. under the conditions examined. No common simple physical characteristic of the proteins used could be correlated to good gelling behavior.


the importance of gel formation in food is well known in, for instance, many meat, egg and milk .product. In these products proteins are considered to be the main texture building component. The mechanism of protein gelling is, however, poorly understood and theories on gel formation are instead to be found in carbohydrate chemistry (c.f. Rees, 1972). It is conceivable, however, that some of these theories could be applied also to protein chemistry.

It has been reported earlier that the egg white protein ovalbumin forms excellent heat induced gels under certain conditions. These conditions, however, must be exactly defined in terms of, for instance, salt concentration and pH. In the case of ovalbumin transparent gets are only found between pH 10 and 11 at physiological salt concentration. At another salt concentration transparent gels are formed within a different pH-interval (Hegg et al., 1979). Other proteins seem either not to form gels by heating (Hegg, 1978) or the conditions required for gel formation are different when compared to ovalbumin.

This investigation was initiated to map the conditions required for globular proteins to form thermally induced gels. It would be of considerable value in food technology if a common environmental condition or certain protein characteristic could be correlated with the ability to form heat induced gels. To this end the following common food proteins, which have different qualities in many respects, were selected: conalbumin and lysozyme from egg white, serum albumin from bovine blood, and beta-lactoglobulin from milk. . It should be stressed that the protein preparations used were extremely pure and well characterized.


Protein preparations

Bovine serum albumin (lot no. 16C-7201), conalbumin (lot no. 34C-8200) and lysozyme (lot no. 74C-8041) were all obtained from Sigma Chemical Co. Serum albumin was free of bound fatty acids and conalbumin from bound iron, as determined by differential scanning calorimetry (Gumpen et al., 1979, Donovan and Rose, 1975). Both preparations were saltfree. Lysozyme was desalted prior to use according to Hegg et al. (1979). beta-lactoglobulin was obtained saltfree by preparation from flesh whole raw milk (Hegg, 1980). None of the used preparations were contaminated by other proteins as determined by SDS-polyacrylamide gel electrophoresis.

Determination of water-holding capacity

The experiments were performed at the fixed protein concentration of 44 mg/ml. Protein concentrations were determined according to the following: beta-lactoglobulin A 1%/1cm=9.6 (Townend et al., 1960), serum albumin A 1%/1cm=6.67 Reynolds et al.. 1967), conalbumin A 1%/1cm=11.3 (Glazer and McKenzie, 1963) and lysozyme A 1%/1cm,=26.9 (Ogasahara and Hamaguchi, 1967). All values are given at the wavelength of 290 nm.

Protein solutions were prepared as earlier described (Hegg et al. 1979). They were heated from 25-95° with a rate of 10°C/min. Heat treatments were carried out in glass tubes (7 x 75 mm) containing 1 ml of protein solution (Hegg et al. 1979). After heating to 95 C the tubes were immediately cooled in ice water and centrifuged at 30,000 x g for 45 min. The percentage of thermally aggregated protein was calculated from the decrease in absorbance of the supernatants at 280 nm. In most cases aggregation was complete (> 90% insoluble protein) at 95°C

The water-holding capacity of the aggregates formed was characterized by their dry matter (d.m.) content. The dry matter content (%) was calculated from (C0 . V0 a)/(Wa) where C0 is the initial protein concentration (mg/ml); V0. the initial solution volume (ml); a, percentage aggregation; and Wa, the weight of the aggregate (mg). A discussion of the d.m. content is given elsewhere (Hegg et al., 1979), The reproducibility was ±3.5 % calculated on the measured d.m. contents. In the few cases when complete aggregation was not reached at 95°C the figure for d.m. content is marked with an asterisk. Complete solubility was defined as no observed phase separation after centrifugation.




Fig. 1-The dry matter content (%) of conalbumin aggregates formed through hearing to 95°C in different concentrations of NaCl between, pH 3 and 11. Heating rate was 10°C/min. Dry matter contents are given with figures at each pH for 0, 85 mM, 170 mM and 340 mM added NaCl. S indicates solubility, The lines differentiate areas of different dry matter content, /// 5-9% and=above 9%, The unhatched area indicates that thermal aggregation does not occur. Dry matter contents when aggregation was incomplete have been marked with an asterisk.

When no sodium chloride was added to the conalbumin solution, thermally induced aggregates were only formed within the pH-interval from around 6 to pH 85 (Fig. 1). Consequently, aggregation in a saltfree protein solution only occurred around the isoelectric point of the protein (approximately pH 6 for conalbumin). The aggregates obtained were characterized by their dry matter content and these values were all high in the pH-interval described above. High dry matter values indicate that the aggregates visibly appear as "white loose precipitates" which are effectively packed together by centrifugation. No transparent gels, which are characterized by dry matter values of around 5% or opaque gels with dry matter contents between 5 and 9% were observed.

The limits between solubility and aggregation gradually moved towards lower and higher pH-values. respectively, when the salt concentration was increased. As can be seen from the incomplete aggregation near the boundary (dry matter figures marked with an asterisk) there was a gradual transition from solubility to complete aggregation. With the exception of a few points. the dry matter values in Fig, 1 are all above 13, i.e. the aggregates visibly appeared as precipitates. No systematic tendency to a decrease or increase in dry matter was found with changing conditions. The quality of heat induced aggregates of conalbumin thus seem to be largely independent of salt concentration and pH.

The very steep course of the solubility/aggregation line observed for conalbumin at acidic pH-values was not observed earlier for ovalbumin. The boundary between solubility and aggregation seems to be closely related to the titration curve of the protein (Hegg, 1919). Conalbumin has a pH-induced transition between pH 3.5 and 5, and thus a profound alteration in the amount of titratable groups in this pH interval (Wisnia et al., 1961).



Fig. 2 . The dry matter content (%) of serum albumin aggregates formed through heating to 95°C in n different concentrations of NaCl between pH 3 and 11, For details, see legend to Fig. 1.

Heat induced aggregation in saltfree solution of serumalbumin occurred within a more narrow pH-interval than for conalbumin. The isoelectric point is located around the middle of this interval (Fig 2).

Serum albumin has a similar pH-induced transition at acidic pH values as conalbumin. Thus, the course of the boundary between solubility and aggregation at acidic pH values is the same for these two proteins. At alkaline pH-values they differ, however, and serum albumin is soluble under conditions ranging front pH 6.5 in saltfree solution to 11 in NaCl. This difference compared to conalbumin is mainly due to the fact that serum albumin undergoes structural transitions also at alkaline pH-values (Steinhardt and Reynolds, 1969).

The most important difference between the two proteins is, however, the extremely large gelling area for serum albumin at neutral and weak alkaline pH-values (Fig. 2). At physiological salt concentration for instance, gels are formed in the wide pH-interval 6.-9.5. Both transparent and opaque gels are, found within this interval. Transparent gels are characterized by a dry matter content around 5% and these gels are nearly always found close to the boundary between solubility and aggregation. This is s probably due to the higher net charge repulsion between the protein molecules required for the formation of a gel with transparency (Hegg, 1978).

Precipitates of serum albumin were always observed near the isoelectric point regardless of the salt concentration used. This is in agreement with the results on conalbumin Progressively lower dry matter contents were obtained towards the solubility/aggregation limit on the acidic side and even at these acidic pH-values a narrow gelling area could be distinguished. A gradual decrease in dry matter content was also registered from the isoelectric point towards alkaline pH-values. This tendency was, however, casually broken at pH 8, where lower values were found at all salt concentrations. The reason for this is probably due to the structural transition mentioned above.



Fig. 3. The dry, matter content (%) of beta-lactoglobulin aggregates formed through heating to 95°C at different concentrations of NaCl between pH 3 and 11. For details, see legend to Fig. 1.

beta-Lactoglobulin is the most abundant whey protein and has an isoelectric point of around 5.2 (McKenzie, 1971). The protein has a pH-induced transition above pH 7 (McKenzie et al., 1967), which accounts for the solubility characteristics (Fig. 3) similar to those registered for serum albumin The solubility/aggregation boundary at acidic pH-values more reflects the shape of a normal titration behavior (Hegg, 1978).

Gel formation occurred close to the aggregation/solubility boundary both in on the acidic and alkaline side of the isoelectric point. Both these areas of gel formation were however, very narrow. Furthermore, only opaque gels were obtained. The behavior earlier found for ovalbumin (Hegg, et al., 1979) that at any salt concentration the dry matter contents were at highest around the isoelectric point and then gradually decreased towards lower and higher pH values was observed also for beta-lactoglobulin. Compared to the other proteins in this investigation, the dry matter contents were generally lower for beta-lactoglobulin and dry matter values above 20% were found only in saltfree solotion.



Fig. 4. The dry matter content (%) of lysozyme aggregates formed through heating to 95°C In different concentrations of NaCl between pH 3 and 11. For details, see legend to Fig. 1.

Lysozyme is the smallest globular protein used in this investigation with a molecular weight of 14,500 (Dayhoff, 1972). The protein has a small solubility area, and is soluble only on the acidic side of the isoelectric point (Fig. 4). This is due to the high isoelectric point (pH 10.5-11.0) of the protein (Gilbert, 1971).

The dry matter contents found in the, aggregates were all very high, i.e. only precipitates were formed. No gel formation, either of transparent or opaque gels, could be detected. Furthermore, there was an extremely slow transformation from solubility to complete precipitation for lysozyme. Several of the dry matter values in Fig. 4 are marked with an asterisk which indicates incomplete aggregation,


The solubility areas for the proteins given in Fig. 1-4 show the same general appearance. From the isoelectric point, these areas expand with a decreasing or increasing pH and shrink with an increasing salt concentration. Thus, thermal non-aggregating conditions are largely determined by the state of ionization of the titratable groups of the proteins, and the differences found in extension of the boundaries between solubility and aggregation mainly reflect different net charges of the proteins under various conditions (Hegg, et al., 1979). The, effect of salt and pH on the ,, attractive forces is small in comparison with the effect on the repulsive net charge. Consequently, it would be possible to predict the boundaries between solubility and aggregation for any globular protein by knowing titration curves, amount of salt present in the sample and simple physical data, as the, isoelectric point, pH-induced transitions etc.

Ovalbumin has earlier been reported to be a protein with good gelling properties, i.e. gels were formed at a wide pH- and salt concentration range (Hegg et al., 1979). Serum albumin (Fig. 2) has gelling properties comparable to those of ovalbumin. The gelling area for serum albumen is, however, displaced towards lower pH values and higher salt concentrations and the area where transparent gels were formed was also more narrow.

beta-Lactoglobulin has an isoelectric point close to that of serum albumin and a similar pH-induced transition at alkaline pH values. For beta-lactoglobulin, however, only a very small gelling area was detected (Fig. 3), Obviously these simple physical characteristics could not be used as a correlation to good getting property of a protein.

Areas of gel formation could not be distinguished for conalbumin (Fig. 1) or lysozyme (Fig. 4). Our data do not indicate a simple physical characteristic in common to account for their lack of ability to form gels thermally. It must be pointed out however, that only a fixed protein concentration of 4.4% has been used in this investigation. A higher concentration would possibly facilitate the formation of a three-dimensional protein network and thus the formation of a gel structure. It cannot be excluded that a critical concentration for gelling exists for every protein and that this concentration in the case of conalbumin and lysozyme is very high. The kinetics of heating is another factor that affect the gelling behaviour A low rate of heating generally has a positive effect on gel formation (Hegg, 1978). From these points of view the experimental conditions selected in this investigation are quite unfavorable for gel formation since they were designed to sort out a protein with good gelling properties from a poor one.

Disulphide bridges and sulphydryl groups have been suggested to be important for crosslinking of Proteins. The proteins used in this investigation differ widely in their content of these groups and thus no correlation between disulphide or sulphydryl content and the ability to form thermally induced gels was found. Some other proteins apart from those reported on here have also been investigated. Notably, myoglobin containing no disulphide bridge or free sulphydryl group (Mailer and Cordes, 1966), was found to be an extremely potent gelling protein, further indicating that these parameters are unimportant in gel formation of these proteins. The forces which keep the framework of the gel together must instead be found in the hydrophobic and hydrogen bonds, which become available during thermal denaturation of the globular proteins. These forces counteract the repulsive net charge of the proteins, and a delicate balance between attractive and repulsive forces seems to be a prerequisite for the formation of a gel framework (Hegg et al., 1979). The differences in the ability to form gels might reflect different types of intermolecular interactions in the aggregates of the proteins examined. It has been proposed that ß-sheet hydrogen bonding might be important in aggregate formation (Clark et al., 1981). The ß-sheet content in the native state of serum albumin, for instance, is reported as low and that of ß -lactoglobulin as high (Wasylewski, 1979). There is therefore no indication that a high content of intramolecular ß -sheet structure in the native state would facilitate the formation of intermolecular ß-sheet formation in the denatured state. Instead the potent gelling proteins serum albumin, ovalbumin and myoglobin seem to have a high helical content in the native state (Joly, 1965; Chen et al., 1974). Since the poor gelling protein lysozyme also has a high helix content (Chen et al., 1974) this seems not to be decisive factor in gel formation either.

The contribution from the hydrophobic forces to the formation of the gel framework is difficult to assess since hydrophobicity is not easy either to calculate or to measure. None of the proteins used are known to have an extreme content of hydrophobic amino acids. Furthermore, the location of the hydrophobic amino acids within the molecule is probably more important for the development of intermolecular interactions than the total content. Further speculation in this matter is not meaningful, since except for lysozyme, the protein structures for the model proteins examined are unknown.

In conclusion, it seems possible to predict the conditions required for aggregation and solubility of a globular protein but hard to identify a special protein characteristic which is crucial for gel formation. If gel formation occurs, however, this is found close to the boundary between aggregation and solubility.

Studies at various protein concentrations might possibly add further clues to the mechanism of protein gelling. As no characteristic property of the proteins used in this investigation could be correlated with a good gelling behaviour it could not be excluded that most proteins might form thermally induced gels. If so, the question of gelling might be reduced to find the right condition for the actual protein


Chen, Y.H., Yang, J.T., and Chau. K.H. 1974. Determination of the helix and ß-form of proteins in aqueous solution by circular dichroism. Biochemistry 13: 3350.

Clark, A.H., Saunderson, D.H.P., and Suggett, A. 1981. infrared and laser-Raman spectroscopic studies of thermally-induced globular protein gels. Int. J. Peptide Protein Res. 17: 353.

Dayhoff, M.D. (Ed.) 1972. "Atlas of Protein Sequence and Structure." Vol.. 5. The National Biomedical Research Foundation Washington, DC.

Donovan. J.W. and Rose, K.D. 1975. Iron binding to conalbumin Calorimetric evidence for two distinct species with one bound iron atom. J. Biol. Chem. 260: 6026.

Gilbert, A.B. 1971. Egg albumen and its formation. Physiol. Biochem. Dom. Fowl 3: 1291.

Glazer, A.N, and McKenzie, H.A. 1963. The denaturation of proteins. 4. Conalbumin and iron (III)-conalbumin in urea solution. Biochim. Biophys, Acta. 71: 109.

Gumpen, S., Hegg, P.O., and Martens, H. 1979. Thermal stability at fatty acid-serum albumin complexes studied by differential' scanning calorimetry. Biochim. Biophys. Acta 574: 189.

Hegg P.O. 1978. Thermal aggregation and denaturation of egg white proteins. A model study of food protein behaviour, Ph.D. thesis, Univ. of Lund, Lund, Sweden.

Hegg, P.O. 1980. Thermal stability of beta-lactoglobulin as a function of pH and the relative concentration of sodium dodecylsulphate. Acta Agric. Scand. 30: 401.

Hegg, P.O., Martens, H, and Löfquist,, B. 1979. Effects of pH and neutral salts on the formation and quality of thermal aggregates of ovalbumin. A study on thermal aggregation and denaturation. J. Sci. Food Agric. 30: 981.

Joly, M. 1966. A physico-chemical approach to the denaturation of proteins", p. 279. Academic Press, New York.

Mahler, H.R. and Cordes, E.H. 1966. In "Biological Chemistry," p. 104. Harper and Row, New York.

McKenzie, H.A, 1971, In "Milk Proteins. Chemistry and Molecular Biology," Vol. 2, Academic Press, New York.

McKenzie, H.A., Sawyer, W.H., and Smith, M.B. 1967. Optical rotatory dispersion and sedimentation in the study of association-dissociation: bovine beta-lactoglobulin near pH 5. Biochim. Biophys. Acta. 147: 73.

Ogasahara. K. and Hamaguchi, K. 1967. Structure of lysozyme. 1. Effect of pH on the stability of lysozyme. J. Biochem. 61: 199.

Rees, D.A. 1972. Polysaccharide gels. A molecular view. Chemistry & Industry 19: 630.

Reynolds, J.A., Herbert, S., Polar, H., and Steinhardt, J. 1994 The binding of divers detergent anions to bovine serum albumin. Biochemistry 6: 937.

Steinhardt, J. and Reynolds, J.A. 1969. In "Multiple Equilibria in Proteins." Academic Press, New York.

Townsend, R., Weinberger, L., and Timasheff, S.N. 1960. Molecular interactions in beta-lactoglobulin. 4. The dissociation of beta-lactoglobulin below pH 3.5. J. Am. Chem. Soc. 82: 3175.

Wasylewski, Z. 1979. Protein-cationic detergent interaction. Fourier transform infrared and laser raman spectroscopic studies on the interaction between proteins and dodecyl pyridinium bromide. Act. Biochim. Polonica 26: 205.

Wishnia, A., Weber, 1., and Warner, R.C. 1961. The hydro[illegible] equilibria of conalbumin. J. Am. Chem. Soc. 83: 2071.

Ms received 11/27/81; revised 2/24/82; accepted 3/3/82.

I thank Gunnel Lundh and Sigrid Häggström for technical assistance. This investigation was supported by grant No. 78-3756 from the. Swedish Board of Technical Development.