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Rheological investigations of alkaline-induced gelation of skimmed milk and reconstituted skimmed milk concentrates
Food Hydrocolloids 14 (2000) 197–201 www.elsevier.com/locate/foodhyd
Rheological investigations of alkaline-induced gelation of skimmed milk and reconstituted skimmed milk concentrates Y. Hemar 1, A.J.R. Law, D.S. Horne, J. Leaver* Hannah Research Institute, Ayr, KA6 5HL, Scotland, UK Received 14 June 1999; accepted 13 September 1999
Abstract The gelation of milk and of reconstituted milk at various concentrations of solids, as induced by alkaline pH, has been investigated using dynamic rheological methods. The gels formed at pH 12 were relatively weak and exhibited an exponential relationship between the elastic modulus G 0 and the protein concentration f G 0 , f3:9 : Gelation time decreased with both increasing temperature and protein concentration. Chromatographic analysis of the proteins showed that storage at pH 12 resulted in changes in their chemical structure. q 2000 Elsevier Science Ltd. All rights reserved. Keywords: Milk; Gelation; Rheology; Alkaline-induced
1. Introduction Under physiological conditions approximately 80% of the protein molecules present in bovine milk are packaged into submicron-sized colloidal particles, the casein micelles. These micelles consist of four species of phosphoproteins that are relatively lacking in secondary structure. An average micelle contains approximately 10,000 protein molecules and 3000 amorphous calcium phosphate microgranules. The as1-, as2- and b-casein molecules interact with the microgranules via clusters of phosphoserine residues (Holt, 1992). The remaining k-casein species consists of singly or doubly phosphorylated protein molecules that do not interact to any great extent with the microgranules. Instead, most of the k-casein molecules are believed to coat the exterior of the micelles. Their highly electronegatively charged C-terminal regions projecting from the surface of the micelle form a “hairy layer” that, through a combination of steric and electrostatic repulsion, prevents individual micelles from aggregating and maintains the colloidal structure of milk. The non-micellar proteins have globular structures in which intramolecular disulphide
* Corresponding author. Tel.: 144-01292-674091; fax: 144-01292674008. E-mail address: [email protected] (J. Leaver). 1 Current address: Institute for Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand.
bridges play an important role. These are collectively termed the milk serum, or whey, proteins. Gelation of milk has been of great interest for thousands of years. Rennet-gels (cheese) and acid-gels (yoghurt) are of major commercial interest and have therefore been studied in great detail. Rennet gels are formed by the action of enzymes that cleave the k-casein molecules, removing the stabilising layer of caseinomacropeptide, resulting in aggregation of the casein micelles. Since the isoelectric point of the casein is in the region of pH 5 and the natural pH of milk is around 6.7, acid gels are formed by reducing the net charge on the micelles, which again causes individual particles to aggregate. Four decades ago, Beeby and Kumetat (1959a,b) showed that when the pH of milk was brought to 11.7 by the addition of calcium hydroxide at temperatures below 108C, the viscosity of the system increased within a few minutes and then subsequently decreased to reach a constant value. After 10 days the systems gelled. Their study concentrated mainly on the early stage of the viscosity change and their explanation was that, as a result of electrostatic repulsion at high pH, the casein micelles expand and their volume increases, as does the viscosity of the system. After reaching a maximum viscosity, the micelles undergo dissociation causing a decrease in viscosity. Some proteins can form gels at alkaline pH, e.g. b-lactogloblin at pH 8 (Mulvihill, Rector & Kinsella, 1991). Recently, Sanchez and Burgos (1997) showed that at high pH, a hydrolysate of sunflower globulin produced by the
0268-005X/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved. PII: S0268-005 X( 99)00 049-1
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Y. Hemar et al. / Food Hydrocolloids 14 (2000) 197–201
which has the same salt content as the serum phase but which is effectively protein free, was prepared by filtering milk through a membrane with a nominal molecular weight cut-off of 10,000 da. Samples (30 ml), were adjusted to pH 12 at 208C by dropwise addition of NaOH (5 M). 2.2. Rheological measurements
Fig. 1. Elastic modulus G 0 (B), loss modulus G 00 (A) and strain (V) as a function of time, for a 10% (w/v) reconstituted milk at pH 12.
action of the proteolytic enzyme trypsin, also formed a gel at 208C. Gelation time increased with pH but decreased with concentration. The storage modulus at pH 8 was also found to increase exponentially with protein concentration. To our knowledge, there have been no reports in the literature of detailed studies of the gelation of milk at high alkaline pH. The aims of this work were therefore to investigate, using dynamic rheological methods, the gelation of milk at pH 12 and to study the influence of protein concentration and temperature on gel elasticity and gelation time.
2. Material and methods 2.1. Materials Raw bulk milk was collected from the Hannah Research Institute’s herd of Friesian cows and fat was removed by centrifuging at 1000g for 40 min at 48C. Low heat skim milk powder (SMP) was prepared at the Institute. The total protein content of the SMP was 32.5% and the water content was less than 4%. Reconstituted skim milk was prepared by dissolving SMP in distilled water. Solutions were stirred and stored at 48C for 16 h. Sodium caseinate, a calcium phosphate-free isoelectric precipitate of casein, was prepared by adjusting the pH of skimmed milk to 4.6. The insoluble casein was washed and redissolved by adjusting the pH to 7.0 with NaOH prior to freeze drying. Whey proteins were prepared by extensively dialysing the pH 4.6 filtrate against water prior to freeze drying. b-Lactoglobulin (BLG) was purchased from Sigma Chemical Co. (Poole, Dorset, UK). Micellar casein was prepared by high speed centrifugation of milk at 50,000 g for 1 h, the serum, which contains the noncolloidal proteins, being discarded. Milk ultrafiltrate,
Rheological measurements were performed on a controlled stress Bohlin CVO rheometer (Bohlin Instruments, Gloucestershire, UK). The double gap concentric cylinder geometry, which requires 30 ml of sample, was used. Measurements were performed maintaining applied stress constant throughout the run. In order to minimise disturbance of the gelation process and avoid potential shear-induced retardation of the gelation, a discrete oscillation protocol was adopted. This performed one measurement requiring three oscillation cycles every 10 min. Unless specified in the text, the measurements were performed at 0.1 Hz at temperatures of 10, 20, 30 and 408C with a precision of 0.18C. Gelation time was defined as the time at which the elastic modulus G 0 equals the loss modulus G 00 (Djabourov, Leblond & Papon, 1988). Although there are other methods for determining gelation time, this practice is now widely adopted. 2.3. Protein analysis To examine changes in the proteins on prolonged storage, milk was adjusted to pH 12 at 208C and samples taken at 0, 4 and 20 h. Caseins, together with denatured whey proteins, were precipitated by adjusting the samples to pH 4.6. The protein composition of the precipitate was analysed using anion-exchange fast protein liquid chromatography (FPLC) on a Mono Q HR5/5 column (Davies & Law, 1987) and cation-exchange FPLC on a Mono S HR 5/5 column (Hollar, Law, Dalgleish & Brown, 1991). Columns were purchased from Pharmacia Biotech (St Albans, UK). 3. Results and discussion As the pH of the milk increases, so too does the net negative charge on the proteins. Electrostatic repulsive forces induce expansion of the casein micelle and eventually this results in a decrease in turbidity due to dissociation of the micelles as postulated by Beeby and Kumetat (1959a). In protein systems, gelation can be induced by changing a variety of physical parameters such as pH, temperature and ionic strength (see Oakenfull, Pearce & Burley, 1997 and references therein). As a result of the formation of one or more types of crosslinks, e.g. disulfide bridges, hydrophobic interactions, and hydrogen bonds, a three-dimensional network is achieved. The kinetics of gelation of a 10% (w/w) reconstituted milk at pH 12 and 208C are shown in Fig. 1. The initial part of the curve shows that
Y. Hemar et al. / Food Hydrocolloids 14 (2000) 197–201
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at all of the concentrations investigated, both G 0 and G 00 showed a very small increase with frequency. The elastic modulus, G 0 at 0.1 Hz was plotted as a function of milk concentration (Fig. 3a). Using a power law fit, we found that G 0 , f 3:9 : Similar exponents have been found in gelling solutions (Nakamura, Harada & Tanaka, 1993). Though typical of milk gel systems, the value of the elastic modulus remained small (,1000 Pa) even at relatively high concentrations. Gelation time decreased with increasing protein concentration (Fig. 3b). Samples lost their gel structure if subjected to weak shear (by simple hand shaking for example), but the gel structure reformed if the shaken sample was allowed to stand for a few hours at room temperature. This indicates that the bonds (crosslinking) involved in the gelled system were weak and to some extent reversible. Gelation time decreased with increasing temperature.
Fig. 2. Elastic modulus G 0 (solid symbols) and loss modulus G 00 (open symbols) as a function of frequency for 10% (B), 15% (X) and 20% (O) total solids reconstituted milks. Measurements were performed 15 h after adjusting the pH of the sample.
the character of the sample was purely viscous, with a loss modulus, G 00 , very close to that of milk and a very low elastic modulus, G 0 . After 1 h both G 0 and G 00 began to increase. G 0 increased more rapidly than G 00 and passed beyond it. Since the gelation point is defined as the crossing point G 0 G 00 ; the gelation time (tg) in this example, was 1.34 h. Above the gelation point, the values of G 0 and G 00 tend towards a plateau value with G 00 being approximately one decade lower than G 0 . Through this period of change in the shear moduli, as also plotted in Fig. 1, the developed strain falls smoothly from an initial value of approximately 30 to a final value of 0.001, dropping most precipitously in the region of the defined gel point, where it has value ,0.2. Similar sharp falls in strain were recorded in the regions of gel points determined under the various reaction conditions studied (data not shown). The excessive initial strains could disturb the gelation and retard the gelation point measurement but the rapid and large changes in both moduli and strain suggest such effects should be negligible. Thus the changes in gelation time observed are indicative of direct reflections of the varying reaction. Any systematic errors incurred are likely to be no greater than would be encountered by selecting another measuring frequency or adopting another definition of the gel point. Similar measurements were performed on milks of different concentrations of solids at pH 12. In each case, after 15 h, when the system reached its plateau value, the viscoelastic behaviour of the system was measured as a function of frequency. These frequency spectra show the typical behaviour expected of a gelled system (Fig. 2). As expected,
Fig. 3. Dependence of (a) the elastic modulus G 0 and (b) the gelation time on milk concentration (expressed as total solids). Solid lines are power law fits to the data.
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Fig. 4. Gelation time as a function of temperature for skimmed milk (A), 10% total solids (B), and 15% (X) total solids reconstituted milks. The lines are drawn as a guide only.
This effect was enhanced by increasing the milk concentration. Gelation times for skimmed raw milk, and reconstituted milk at 10 and 15 wt% are shown in Fig. 4. At the highest temperature, the gelation times of the different milks were very close to each other, with gelation effectively being instantaneous. These measurements were not extended to higher milk concentration since the transition to the gel occurred before the sample could be transferred to the rheometer cell (i.e. G 0 . G 00 from the beginning). In an attempt to determine which of the milk components were responsible for the observed gelation, sodium caseinate, whey proteins, purified b-lactoglobulin and whey protein-free casein micelles were resuspended in either ultrafiltrate or water and adjusted to pH 12 at 208C (Table 1). Apart from pH-adjusted milk, the only system, which gelled was where casein micelles were resuspended in ultrafiltrate. Replacing ultrafiltrate with water prevented gelation, as did replacing micelles with caseinate. The total calcium content of bovine milk is approximately 30 mM of which 20 mM is micellar calcium. Calcium binding Table 1 Gelation of various systems adjusted to pH 12 and stored at 208C Component type
Concentration (wt%)
Solvent
Gel formation
Sodium caseinate Sodium caseinate b-Lactoglobulin Whey proteins Casein pellet Casein pellet
10 10 1 1 10 10
Water Ultra filtrate Water Water Water Ultra filtrate
No No No No No Yes
Fig. 5. Anion-exchange FPLC on a Mono Q column of the proteins precipitated at pH 4.6 from milk stored at pH 12 for 0, 4 and 20 h at room temperature. Identities of the individual proteins in the original milk are marked.
therefore probably also contributed to the protein–protein crosslinks in the gel but a Ca 21 concentration greater than the 10 mM present in ultrafiltrate is required for these bonds to form. Anion and cation exchange FPLC analysis of the proteins precipitated at pH 4.6 from milk samples kept for 4 and 20 h at pH 12 showed considerable changes had occurred in the chemical structure of the proteins. The profiles of the samples on the anion exchange column are shown in Fig. 5. In general, peaks became more diffuse, and comparison of the same samples separated by these techniques indicated that considerable changes had occurred after 4 h and very little of the original protein was unmodified after 24 h. It should be noted that by the time gelation occurred in milk samples, considerable covalent modification of the proteins had taken place as shown in the FPLC analyses, and the altered proteins were forming the gel. The colour of the sample changed from green to brown but only in samples containing lactose suggesting that Maillard-type reactions may be occurring at this high pH. Whether these are the causes of the modification of the proteins is not known but other reactions such as dephosphorylation, deamination and cleavage of disulphide bridges would also be expected to occur under these conditions.
Acknowledgements Core funding for the Hannah Research Institute is provided by The Scottish Executive Rural Affairs Department. Y.H. is funded by the EU Framework IV sponsored
Y. Hemar et al. / Food Hydrocolloids 14 (2000) 197–201
project “Structure, rheology and physical stability of aggregated particle systems containing proteins and lipids”. References Beeby, R., & Kumetat, K. (1959). Viscosity changes in concentrated skimmilk treated with alkali, urea and calcium-complexing agents. I. The importance of casein micelle. Journal of Dairy Research, 26, 248–257. Beeby, R., & Kumetat, K. (1959). Viscosity changes in concentrated skimmilk treated with alkali, urea and calcium-complexing agents. II. The influence of concentration, temperature and rate of shear. Journal of Dairy Research, 26, 258–263. Davies, D. T., & Law, A. J. R. (1987). Quantitative fractionation of casein mixtures by fast protein liquid chromatography. Journal of Dairy Research, 54, 369–376. Djabourov, M., Leblond, J., & Papon, P. (1988). Gelation of aqueous gelatin solutions. II. Rheology of the sol–gel transition. Journal de Physique France, 49, 333–343. Hollar, C. M., Law, A. J. R., Dalgleish, D. G., & Brown, R. J. (1991).
201
Separation and quantification of major casein fractions using cationexchange fast protein liquid chromatography. Journal of Dairy Science, 74, 2403–2409. Holt, C. (1992). Structure and stability of bovine casein micelles. In J. J. Anfinsen & J. T. Edsall & F. M. Richards & D. S. Eisenberg (Eds.), (pp. 63–151). Advances in protein chemistry, 43. New York: Academic Press. Mulvihill, D. M., Rector, D., & Kinsella, J. E. (1991). Mercaptoethanol, N-ethylmaleimide, propylene glycol and urea effects on rheological properties of thermally induced [b]-lactoglobulin gels at alkaline pH. Journal of Food Science, 56, 1338–1341. Nakamura, K., Harada, K., & Tanaka, Y. (1993). Viscoelastic properties of aqueous gellan solutions: the effects of concentration on gelation. Food Hydrocolloids, 7, 435–447. Oakenfull, D., Pearce, J., & Burley, R. W. (1997). Protein gelation. In S. Damodaran & A. Paraf (Eds.), Food proteins and their applications, (pp. 111–142). New York: Marcel Dekker. Sanchez, A. C., & Burgos, J. (1997). Gelation of sunflower globulin hydrolysates: rheological and calorimetric studies. Journal of Agricultural and Food Chemistry, 45, 2407–2412.
Rheological investigations of alkaline-induced gelation of skimmed milk and reconstituted skimmed milk concentrates Y. Hemar 1, A.J.R. Law, D.S. Horne, J. Leaver* Hannah Research Institute, Ayr, KA6 5HL, Scotland, UK Received 14 June 1999; accepted 13 September 1999
Abstract The gelation of milk and of reconstituted milk at various concentrations of solids, as induced by alkaline pH, has been investigated using dynamic rheological methods. The gels formed at pH 12 were relatively weak and exhibited an exponential relationship between the elastic modulus G 0 and the protein concentration f G 0 , f3:9 : Gelation time decreased with both increasing temperature and protein concentration. Chromatographic analysis of the proteins showed that storage at pH 12 resulted in changes in their chemical structure. q 2000 Elsevier Science Ltd. All rights reserved. Keywords: Milk; Gelation; Rheology; Alkaline-induced
1. Introduction Under physiological conditions approximately 80% of the protein molecules present in bovine milk are packaged into submicron-sized colloidal particles, the casein micelles. These micelles consist of four species of phosphoproteins that are relatively lacking in secondary structure. An average micelle contains approximately 10,000 protein molecules and 3000 amorphous calcium phosphate microgranules. The as1-, as2- and b-casein molecules interact with the microgranules via clusters of phosphoserine residues (Holt, 1992). The remaining k-casein species consists of singly or doubly phosphorylated protein molecules that do not interact to any great extent with the microgranules. Instead, most of the k-casein molecules are believed to coat the exterior of the micelles. Their highly electronegatively charged C-terminal regions projecting from the surface of the micelle form a “hairy layer” that, through a combination of steric and electrostatic repulsion, prevents individual micelles from aggregating and maintains the colloidal structure of milk. The non-micellar proteins have globular structures in which intramolecular disulphide
* Corresponding author. Tel.: 144-01292-674091; fax: 144-01292674008. E-mail address: [email protected] (J. Leaver). 1 Current address: Institute for Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand.
bridges play an important role. These are collectively termed the milk serum, or whey, proteins. Gelation of milk has been of great interest for thousands of years. Rennet-gels (cheese) and acid-gels (yoghurt) are of major commercial interest and have therefore been studied in great detail. Rennet gels are formed by the action of enzymes that cleave the k-casein molecules, removing the stabilising layer of caseinomacropeptide, resulting in aggregation of the casein micelles. Since the isoelectric point of the casein is in the region of pH 5 and the natural pH of milk is around 6.7, acid gels are formed by reducing the net charge on the micelles, which again causes individual particles to aggregate. Four decades ago, Beeby and Kumetat (1959a,b) showed that when the pH of milk was brought to 11.7 by the addition of calcium hydroxide at temperatures below 108C, the viscosity of the system increased within a few minutes and then subsequently decreased to reach a constant value. After 10 days the systems gelled. Their study concentrated mainly on the early stage of the viscosity change and their explanation was that, as a result of electrostatic repulsion at high pH, the casein micelles expand and their volume increases, as does the viscosity of the system. After reaching a maximum viscosity, the micelles undergo dissociation causing a decrease in viscosity. Some proteins can form gels at alkaline pH, e.g. b-lactogloblin at pH 8 (Mulvihill, Rector & Kinsella, 1991). Recently, Sanchez and Burgos (1997) showed that at high pH, a hydrolysate of sunflower globulin produced by the
0268-005X/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved. PII: S0268-005 X( 99)00 049-1
198
Y. Hemar et al. / Food Hydrocolloids 14 (2000) 197–201
which has the same salt content as the serum phase but which is effectively protein free, was prepared by filtering milk through a membrane with a nominal molecular weight cut-off of 10,000 da. Samples (30 ml), were adjusted to pH 12 at 208C by dropwise addition of NaOH (5 M). 2.2. Rheological measurements
Fig. 1. Elastic modulus G 0 (B), loss modulus G 00 (A) and strain (V) as a function of time, for a 10% (w/v) reconstituted milk at pH 12.
action of the proteolytic enzyme trypsin, also formed a gel at 208C. Gelation time increased with pH but decreased with concentration. The storage modulus at pH 8 was also found to increase exponentially with protein concentration. To our knowledge, there have been no reports in the literature of detailed studies of the gelation of milk at high alkaline pH. The aims of this work were therefore to investigate, using dynamic rheological methods, the gelation of milk at pH 12 and to study the influence of protein concentration and temperature on gel elasticity and gelation time.
2. Material and methods 2.1. Materials Raw bulk milk was collected from the Hannah Research Institute’s herd of Friesian cows and fat was removed by centrifuging at 1000g for 40 min at 48C. Low heat skim milk powder (SMP) was prepared at the Institute. The total protein content of the SMP was 32.5% and the water content was less than 4%. Reconstituted skim milk was prepared by dissolving SMP in distilled water. Solutions were stirred and stored at 48C for 16 h. Sodium caseinate, a calcium phosphate-free isoelectric precipitate of casein, was prepared by adjusting the pH of skimmed milk to 4.6. The insoluble casein was washed and redissolved by adjusting the pH to 7.0 with NaOH prior to freeze drying. Whey proteins were prepared by extensively dialysing the pH 4.6 filtrate against water prior to freeze drying. b-Lactoglobulin (BLG) was purchased from Sigma Chemical Co. (Poole, Dorset, UK). Micellar casein was prepared by high speed centrifugation of milk at 50,000 g for 1 h, the serum, which contains the noncolloidal proteins, being discarded. Milk ultrafiltrate,
Rheological measurements were performed on a controlled stress Bohlin CVO rheometer (Bohlin Instruments, Gloucestershire, UK). The double gap concentric cylinder geometry, which requires 30 ml of sample, was used. Measurements were performed maintaining applied stress constant throughout the run. In order to minimise disturbance of the gelation process and avoid potential shear-induced retardation of the gelation, a discrete oscillation protocol was adopted. This performed one measurement requiring three oscillation cycles every 10 min. Unless specified in the text, the measurements were performed at 0.1 Hz at temperatures of 10, 20, 30 and 408C with a precision of 0.18C. Gelation time was defined as the time at which the elastic modulus G 0 equals the loss modulus G 00 (Djabourov, Leblond & Papon, 1988). Although there are other methods for determining gelation time, this practice is now widely adopted. 2.3. Protein analysis To examine changes in the proteins on prolonged storage, milk was adjusted to pH 12 at 208C and samples taken at 0, 4 and 20 h. Caseins, together with denatured whey proteins, were precipitated by adjusting the samples to pH 4.6. The protein composition of the precipitate was analysed using anion-exchange fast protein liquid chromatography (FPLC) on a Mono Q HR5/5 column (Davies & Law, 1987) and cation-exchange FPLC on a Mono S HR 5/5 column (Hollar, Law, Dalgleish & Brown, 1991). Columns were purchased from Pharmacia Biotech (St Albans, UK). 3. Results and discussion As the pH of the milk increases, so too does the net negative charge on the proteins. Electrostatic repulsive forces induce expansion of the casein micelle and eventually this results in a decrease in turbidity due to dissociation of the micelles as postulated by Beeby and Kumetat (1959a). In protein systems, gelation can be induced by changing a variety of physical parameters such as pH, temperature and ionic strength (see Oakenfull, Pearce & Burley, 1997 and references therein). As a result of the formation of one or more types of crosslinks, e.g. disulfide bridges, hydrophobic interactions, and hydrogen bonds, a three-dimensional network is achieved. The kinetics of gelation of a 10% (w/w) reconstituted milk at pH 12 and 208C are shown in Fig. 1. The initial part of the curve shows that
Y. Hemar et al. / Food Hydrocolloids 14 (2000) 197–201
199
at all of the concentrations investigated, both G 0 and G 00 showed a very small increase with frequency. The elastic modulus, G 0 at 0.1 Hz was plotted as a function of milk concentration (Fig. 3a). Using a power law fit, we found that G 0 , f 3:9 : Similar exponents have been found in gelling solutions (Nakamura, Harada & Tanaka, 1993). Though typical of milk gel systems, the value of the elastic modulus remained small (,1000 Pa) even at relatively high concentrations. Gelation time decreased with increasing protein concentration (Fig. 3b). Samples lost their gel structure if subjected to weak shear (by simple hand shaking for example), but the gel structure reformed if the shaken sample was allowed to stand for a few hours at room temperature. This indicates that the bonds (crosslinking) involved in the gelled system were weak and to some extent reversible. Gelation time decreased with increasing temperature.
Fig. 2. Elastic modulus G 0 (solid symbols) and loss modulus G 00 (open symbols) as a function of frequency for 10% (B), 15% (X) and 20% (O) total solids reconstituted milks. Measurements were performed 15 h after adjusting the pH of the sample.
the character of the sample was purely viscous, with a loss modulus, G 00 , very close to that of milk and a very low elastic modulus, G 0 . After 1 h both G 0 and G 00 began to increase. G 0 increased more rapidly than G 00 and passed beyond it. Since the gelation point is defined as the crossing point G 0 G 00 ; the gelation time (tg) in this example, was 1.34 h. Above the gelation point, the values of G 0 and G 00 tend towards a plateau value with G 00 being approximately one decade lower than G 0 . Through this period of change in the shear moduli, as also plotted in Fig. 1, the developed strain falls smoothly from an initial value of approximately 30 to a final value of 0.001, dropping most precipitously in the region of the defined gel point, where it has value ,0.2. Similar sharp falls in strain were recorded in the regions of gel points determined under the various reaction conditions studied (data not shown). The excessive initial strains could disturb the gelation and retard the gelation point measurement but the rapid and large changes in both moduli and strain suggest such effects should be negligible. Thus the changes in gelation time observed are indicative of direct reflections of the varying reaction. Any systematic errors incurred are likely to be no greater than would be encountered by selecting another measuring frequency or adopting another definition of the gel point. Similar measurements were performed on milks of different concentrations of solids at pH 12. In each case, after 15 h, when the system reached its plateau value, the viscoelastic behaviour of the system was measured as a function of frequency. These frequency spectra show the typical behaviour expected of a gelled system (Fig. 2). As expected,
Fig. 3. Dependence of (a) the elastic modulus G 0 and (b) the gelation time on milk concentration (expressed as total solids). Solid lines are power law fits to the data.
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Y. Hemar et al. / Food Hydrocolloids 14 (2000) 197–201
Fig. 4. Gelation time as a function of temperature for skimmed milk (A), 10% total solids (B), and 15% (X) total solids reconstituted milks. The lines are drawn as a guide only.
This effect was enhanced by increasing the milk concentration. Gelation times for skimmed raw milk, and reconstituted milk at 10 and 15 wt% are shown in Fig. 4. At the highest temperature, the gelation times of the different milks were very close to each other, with gelation effectively being instantaneous. These measurements were not extended to higher milk concentration since the transition to the gel occurred before the sample could be transferred to the rheometer cell (i.e. G 0 . G 00 from the beginning). In an attempt to determine which of the milk components were responsible for the observed gelation, sodium caseinate, whey proteins, purified b-lactoglobulin and whey protein-free casein micelles were resuspended in either ultrafiltrate or water and adjusted to pH 12 at 208C (Table 1). Apart from pH-adjusted milk, the only system, which gelled was where casein micelles were resuspended in ultrafiltrate. Replacing ultrafiltrate with water prevented gelation, as did replacing micelles with caseinate. The total calcium content of bovine milk is approximately 30 mM of which 20 mM is micellar calcium. Calcium binding Table 1 Gelation of various systems adjusted to pH 12 and stored at 208C Component type
Concentration (wt%)
Solvent
Gel formation
Sodium caseinate Sodium caseinate b-Lactoglobulin Whey proteins Casein pellet Casein pellet
10 10 1 1 10 10
Water Ultra filtrate Water Water Water Ultra filtrate
No No No No No Yes
Fig. 5. Anion-exchange FPLC on a Mono Q column of the proteins precipitated at pH 4.6 from milk stored at pH 12 for 0, 4 and 20 h at room temperature. Identities of the individual proteins in the original milk are marked.
therefore probably also contributed to the protein–protein crosslinks in the gel but a Ca 21 concentration greater than the 10 mM present in ultrafiltrate is required for these bonds to form. Anion and cation exchange FPLC analysis of the proteins precipitated at pH 4.6 from milk samples kept for 4 and 20 h at pH 12 showed considerable changes had occurred in the chemical structure of the proteins. The profiles of the samples on the anion exchange column are shown in Fig. 5. In general, peaks became more diffuse, and comparison of the same samples separated by these techniques indicated that considerable changes had occurred after 4 h and very little of the original protein was unmodified after 24 h. It should be noted that by the time gelation occurred in milk samples, considerable covalent modification of the proteins had taken place as shown in the FPLC analyses, and the altered proteins were forming the gel. The colour of the sample changed from green to brown but only in samples containing lactose suggesting that Maillard-type reactions may be occurring at this high pH. Whether these are the causes of the modification of the proteins is not known but other reactions such as dephosphorylation, deamination and cleavage of disulphide bridges would also be expected to occur under these conditions.
Acknowledgements Core funding for the Hannah Research Institute is provided by The Scottish Executive Rural Affairs Department. Y.H. is funded by the EU Framework IV sponsored
Y. Hemar et al. / Food Hydrocolloids 14 (2000) 197–201
project “Structure, rheology and physical stability of aggregated particle systems containing proteins and lipids”. References Beeby, R., & Kumetat, K. (1959). Viscosity changes in concentrated skimmilk treated with alkali, urea and calcium-complexing agents. I. The importance of casein micelle. Journal of Dairy Research, 26, 248–257. Beeby, R., & Kumetat, K. (1959). Viscosity changes in concentrated skimmilk treated with alkali, urea and calcium-complexing agents. II. The influence of concentration, temperature and rate of shear. Journal of Dairy Research, 26, 258–263. Davies, D. T., & Law, A. J. R. (1987). Quantitative fractionation of casein mixtures by fast protein liquid chromatography. Journal of Dairy Research, 54, 369–376. Djabourov, M., Leblond, J., & Papon, P. (1988). Gelation of aqueous gelatin solutions. II. Rheology of the sol–gel transition. Journal de Physique France, 49, 333–343. Hollar, C. M., Law, A. J. R., Dalgleish, D. G., & Brown, R. J. (1991).
201
Separation and quantification of major casein fractions using cationexchange fast protein liquid chromatography. Journal of Dairy Science, 74, 2403–2409. Holt, C. (1992). Structure and stability of bovine casein micelles. In J. J. Anfinsen & J. T. Edsall & F. M. Richards & D. S. Eisenberg (Eds.), (pp. 63–151). Advances in protein chemistry, 43. New York: Academic Press. Mulvihill, D. M., Rector, D., & Kinsella, J. E. (1991). Mercaptoethanol, N-ethylmaleimide, propylene glycol and urea effects on rheological properties of thermally induced [b]-lactoglobulin gels at alkaline pH. Journal of Food Science, 56, 1338–1341. Nakamura, K., Harada, K., & Tanaka, Y. (1993). Viscoelastic properties of aqueous gellan solutions: the effects of concentration on gelation. Food Hydrocolloids, 7, 435–447. Oakenfull, D., Pearce, J., & Burley, R. W. (1997). Protein gelation. In S. Damodaran & A. Paraf (Eds.), Food proteins and their applications, (pp. 111–142). New York: Marcel Dekker. Sanchez, A. C., & Burgos, J. (1997). Gelation of sunflower globulin hydrolysates: rheological and calorimetric studies. Journal of Agricultural and Food Chemistry, 45, 2407–2412.