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Influence of milk proteins on κ-carrageenan gelation
International Dairy Journal 9 (1999) 359}364
In#uence of milk proteins on i-carrageenan gelation Athina Tziboula*, David S. Horne Hannah Research Institute, Ayr, KA6 5HL, UK
Abstract The in#uence of milk proteins on the gelation of i-carrageenan during cooling from 60}53C was investigated at constant ionic strength, using small deformation rheology. When carrageenan was dispersed in skim milk ultra"ltrate (SMUF), gel development proceeded in two distinct steps: the "rst at the onset of gelation was a coil-to-helix transition of carrageenan followed by a second stage of aggregation and gelation. In serum, the initial growth of the complex modulus (GH) was followed by a temporary decrease in GH, then recovery of structure and further gelation. We believe that this anomaly in gel development was the result of aggregation and subsequent re-stabilisation of the aggregates in an encompassing network. In the presence of casein micelles gelation took place in a single step, suggesting an interaction between i-carrageenan and the casein micelles which has to be satis"ed "rst and reduces the availability of carrageenan for the gelation ro( le. Thus, the minimum concentration of polymer required for gelation was greater in milk than in SMUF or serum. It is also demonstrated in this paper that the gelation pro"les show scaling behaviour and can be described by two independent parameters: the gel strength at in"nite time and the gelation time. 1999 Elsevier Science Ltd. All rights reserved. Keywords: i-Carrageenan; Caseins; Milk
1. Introduction Carrageenans are a generic class of linear polymers of alternating a-1,4 and b-1,3 anhydrogalactose residues with varying amounts of sulphate half ester groups. Their functionality ranges from thickeners to gellants and stabilising agents depending on their ability to form thermoreversible gels or not. For water-thickening applications non-gelling carrageeanans (such as jcarrageenan) are preferred, whilst for gelling applications, i- or ι-carrageenans are most commonly used. The carrageenan functionality in milk has been attributed to a speci"c interaction between carrageenan and i-casein to form a complex which aggregates into a three-dimensional gel network (Snoeren, 1976). This interaction has been ascribed to electrostatic attraction between the negatively charged sulphate groups of the carrageenan and a predominately positively charged region in the peptide chain of i-casein (Snoeren, 1976; Payens, 1972; Snoeren, Payens, Jeurnink & Both, 1975; Grindrod and Nickerson, 1968). In milk caseins exist associated with each other and with calcium phosphate * Corresponding author. Tel.: #44-1292-674088; fax: #44-1292674008. E-mail address: [email protected] (A. Tziboula)
to form aggregates, the casein micelles. It is accepted that i-casein is located on the surface of the micelles (Schmidt, 1982; Horne, 1984; Dalgleish, Horne & Law, 1989), therefore a similar interaction between carrageenan and icasein is possible. Nevertheless, cations such as K> or Ca> which promote carrageenan gelation are also present in milk and can also in#uence carrageenan gelation. Previous studies on i-carrageenan gelation in milk showed that gel formation and properties were governed by the total ionic content and the carrageenan concentration (Drohan, Tziboula, McNulty & Horne, 1997). Although milk proteins did not in#uence gelation at concentrations of carrageenan greater than 200 ppm, they became important at low polymer levels. For example doubling the concentration of casein micelles in the carrageenan}milk mixtures prevented gel formation. Subsequently, it was shown that the rate of cooling of the milk}carrageenan mixtures in#uenced the gelation temperature and gel strength. Fast cooling rates resulted in delayed gelation (i.e. lower gelation temperatures) and weaker gels. This e!ect was more pronounced at low carrageenan concentrations and when the polysaccharide was dispersed in skim milk ultra"ltrate (SMUF) rather than in milk serum or milk (Tziboula & Horne, 1998). This behaviour demonstrated that for meaningful comparisons and interpretation of the data carrageenan
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gelation studies should be carried out at slow cooling rates. This paper deals with the e!ect of carrageenan concentration (4200 ppm) on the gelation temperature and the mechanical properties of the gels in the presence of di!erent milk protein fractions. i-Carrageenan was dispersed in SMUF, skim milk or skim milk serum. SMUF was the milk fraction obtained after the removal of the milk proteins and consisted mainly of lactose, ions and the proteose-peptone fraction in milk. Skim milk serum was the fraction of milk obtained after the removal of the casein fraction and consisted of SMUF plus the whey proteins. The ionic concentration in the mixtures was kept constant equivalent to that of skim milk. This approach allowed us to evaluate the relative importance of the di!erent milk proteins and ionic composition of milk on i-carrageenan gelation. A simple scaling approach, previously applied in rennet gels, was used to examine the parameters in#uencing i-carrageenan gelation in the presence of milk proteins (Horne, 1995).
2. Materials and methods 2.1. Milk and milk fractions Bulk milk from the Institute herd was skimmed by centrifugation at 1200 g for 40 min at 43C (Mistral 6000 centrifuge; Sanyo Gallenkamp plc., Leicester, UK). From the skimmed milk the following fractions were prepared: (i) Skim milk ultra"ltrate (SMUF): was prepared using a pressure "lter equipped with a membrane of molecular weight cut-o! 10 kda (Amicon Ltd, Danvers, MA). (ii) Serum fraction was obtained by centrifugation of skim milk at 43 000 g for 120 min. The supernatant obtained consisted of whey proteins, low molecular weight material and soluble caseins; this fraction was designated the serum fraction. The soluble caseins were removed from the serum fraction by isoelectric precipitation at pH 4.6 followed by centrifugation (1000 g for 5 min; Mistral 6000 centrifuge; Sanyo Gallenkamp plc., Leicester, UK) and "ltration through Whatman No. 1 "lter paper. The ionic equilibria of the serum fraction was re-established to that of skim milk by exhaustive dialysis against skimmed milk over 48 h, three changes of dialysate, serum to milk ratio 1 : 250). The puri"ed serum fraction was then rapidly frozen by immersion in liquid nitrogen and stored at !203C. 2.2. i-carrageenan i-Carrageenan from Euchema cottonoii (Sigma Chemicals, Poole, UK) was converted to the sodium form using
cation exchange resin (Amberlite IR120, BDH Ltd, Poole, UK). The polysaccharide was recovered by "ltration and freeze dried. The extent of conversion of the carrageenan to the sodium form was evaluated using atomic absorption spectroscopy. The sodium content was estimated to be 5.67% w/w. 2.3. Milk protein}carrageenan mixtures i-Carrageenan (80}200 ppm) was added to the appropriate milk fraction while stirring at room temperature. The mixture was heated to 703C for 20 min to hydrate the carrageenan and then transferred to the rheometer held at 603C. 2.4. Rheological study Low-frequency oscillation (0.08 Hz) was performed on a controlled stress rheometer (CVO, Bohlin Instruments, Gloucestershire, UK) using the double gap measuring geometry. The geometry consists of a hollow cylinder (diameter 45 mm) which is lowered into a cylindrical groove in an outer cylinder (diameter 50 mm). An oscillating stress (5 mPa) was applied to the sample (30 ml) as it was cooled from 60}53C at a rate of 13C/15 min. The instrument calculates the phase angle at which the strain response lags behind the applied stress and hence the storage modulus (G ), viscous modulus (G ) complex modulus (GH) and complex viscosity (gH) can be extracted. The gelation temperature of the mixtures was taken as the point where tan d"G /G "1. The applied stress of 5 mPa was just above the minimum permissible stress with the double-gap geometry whilst operating within the linear viscoelastic region of the structures formed. The strain response varied with the viscoelastic properties of the milk}carrageenan mixtures during the cooling cycle. The initial strain response (at 593C) was constant at 1.42$0.01 whilst at 53C it ranged from 0.4 for the weakest structures to 0.002 for stronger gels. Evaporation was prevented by "tting the solvent trap on the double-gap geometry.
3. Results and discussion 3.1. Inyuence of milk proteins on i-carrageenan gelation Typical gelation pro"les of SMUF}, milk} and serum}carrageenan mixtures during cooling from 60 to 203C, are shown in Fig. 1a}c. As the temperature decreased from 60 to 403C there was a lag period where the elastic contributions to the mechanical properties of the mixtures was very small. The phase lag (d) of the strain response to the applied stress was greater than 453. At these temperatures, i-carrageenan existed in the random coil conformation. The onset of structure formation was
A. Tziboula, D.S. Horne / International Dairy Journal 9 (1999) 359 } 364
Fig. 1. Plots of the shear modulus (GH) development during cooling of (a) skim milk ultra"ltrate-i-carrageenan mixtures; (b) skim milk-icarrageenan mixtures; (c) skim milk serum-i-carrageenan mixtures. Data obtained from low-frequency oscillation at frequency 0.08 Hz and applied stress 5 mPa. i-Carrageenan concentration: - - - - - - 200 ppm; } } } } 170 pmm; *** 140 ppm; } - } - 80 ppm. GT, gelation temperature; ¹ , Temperature of coil-to-helix transition of carrageenan. 20
signalled at around 383C by a reduction in d and a rapid increase in the complex modulus (GH). This point corresponded to the conformational coil-to-helix transition of i-carrageenan (¹ ) and it was not in#uenced by the 20 concentration of carrageenan or the composition of the dispersing medium. It is well established that the gelation temperature of carrageenan is in#uenced by the total ionic concentration and can be predicted by calculating the cation activity contributions from the various components in the solution (Rees, 1969; Rochas and Rinaudo, 1980). For the carrageenan concentrations studied here, the total cation concentration was "xed at levels equivalent to that of skim milk, hence the coil}helix transition temperature was also una!ected. As the cooling continued there came a point at which d"453. At this point the contributions to structure from the viscous and elastic components becomes equal. This point is de"ned as the rheological gelation temperature (GT). Unlike ¹ , 20 GT was a function of the polymer concentration and di!ered with the nature of the milk protein present in the mixtures. In SMUF (Fig. 1a) gelation was a two stage process: the conformation transition of carrageenan at 383C was
361
marked by a measurable increase in GH and it was followed by a second discreet rise in GH at slightly lower temperatures. The second step on the GH development corresponded to the rheological GT. Both gel strength and GT were a function of the concentration of carrageenan. Decreasing the polymer content lowered GT and resulted in weaker gels. When i-carrageenan was dispersed in milk (Fig. 1b) the initial sharp rise in GH at ¹ was followed by immediate aggregation and gela20 tion. Although the rheological GT was 2}33C lower than ¹ the whole process proceeded smoothly in one step. 20 As before the gel strength was governed by the concentration of the gelling agent in the system albeit for the same carrageenan concentration the skim milk}carrageenan gels were stronger than the SMUF}carrageenan gels. It is also noteworthy that gelation was not observed at i-carrageenan concentrations of 140 and 80 ppm. The path of gel formation was also di!erent in the presence of whey proteins (Fig. 1c). Immediately after the "rst sharp increase in GH at ¹ there was a noticeable 20 decrease in the development of the shear modulus (Fig. 1c). In a separate experiment we monitored the path of i-carrageenan gelation in the presence of whey protein and observed the formation of visible aggregates in the vicinity ¹ . On further cooling of the samples, i-car20 rageenan gelation proceeded faster than the aggregation of the whey protein}carrageenan complex hence the recovery in the development of the complex modulus (Fig. 1c). Therefore, it seemed likely that two antagonistic events took place; on the one hand interaction between i-carrageenan and whey proteins and aggregate formation which had a structure damaging e!ect and on the other hand i-carrageenan gelation which was structure forming. This &interference' of milk proteins with gel formation has been reported previously (Tziboula & Horne, 1998). It was found that at low i-carrageenan concentrations an increase in the micellar casein/or whey protein content whilst maintaining the ionic strength equivalent to that of skim milk, prevented gel formation. In all cases, the rheological GT showed carrageenan concentration dependence (Table 1). Lowering the polymer concentration resulted in a sharp decrease in GT. Furthermore, there was a critical carrageenan concentration below which gelation did not take place. This critical polymer concentration was lower for the serum}carrageenan and SMUF}carrageenan rather than the milk} carrageenan mixtures. Again at such low carrageenan levels there was domination of protein}carrageenan interactions which interfered with gel formation. The e!ect of carrageenan concentration on GT was a manifestation of the time dependency of the gelation process and is discussed in detail in the following section. Finally, the milk serum}carrageenan gels were stronger than the milk} or SMUF}carrageenan gels (Fig. 2).
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Table 1 Gelation temperature of SMUF}, skim milk}, and serum}carrageenan mixtures as a function of i-carrageenan concentration i-Carrageenan conc. (ppm)
80 140 170 200
Gelation temperature (GT) (3C) SMUF
Skim milk
Serum
17.3 30.3 32.3 33.7
No gelation No gelation 36.6 36.5
13.5 33.8 32.2 33.4
Fig. 2. In#uence of i-carrageenan concentration on gel strength at 53C. 䊏 skim milk ultra"ltrate-i-carrageenan gels; 䊐 skim milk-i-carrageenan gels; skim milk serum-i-carrageenan gels.
These results suggested that in a complex system such as milk, there were many often antagonistic events that can in#uence the carrageenan gelation. The data presented above suggested that i-carrageenan was capable of forming complexes with both casein micelles and whey proteins. With the casein micelles there was a strong interaction which had to be satis"ed "rst, reducing the availability of carrageenan for the gelation ro( le; this explains why the minimum carrageenan concentration required for gelation was higher in milk than in SMUF or serum. At su$ciently high polymer concentrations i-carrageenan gelation e!ects dominated the system entrapping casein micelles and casein micelle}carrageenan complexes. Similarly in serum, the initial rise in GH at ¹ was hindered by the formation of aggregates, possibly 20 between carrageenan and whey proteins. At su$ciently high polymer concentrations gelation overcomes aggregation entrapping aggregates in the carrageenan network. The resultant whey protein}carrageenan gels were much stronger than the equivalent SMUF- or milk-gels. 3.2. Scaling behaviour of the shear moduli during i-carrageenan gelation In rennet gelation studies Horne (1995,1996) has demonstrated that GH development follows a simple scaling
Fig. 3. The data of Fig. 1a replotted as a function of (a) time; (b) reduced time (t/t ) where t is the gelation time of the individual run; (c) GH for %2 %2 each curve normalised to the value of the modulus attained at reduced time t/t "1.8 on that curve. %2
behaviour governed by two independent parameters: the gelation time and the ultimate gel strength. Although the mechanism by which the aggregating particles are produced is di!erent in rennet gels, we applied the same mathematical approach in this work. Thus the gelation pro"les shown in Fig. 1a were as a function of time (t) and then as a function of the reduced time t/t , with t %2 %2 the gelation time varying for each individual curve (Fig. 3a}b). A series of curves were produced ordered according to their "nal gel strength, which in turn was a function of the polymer concentration. This suggested that the shear modulus should be normalised against the shear modulus at in"nite time. As the latter is rather impractical, we normalised the reduced plots to the values of the shear moduli at an arbitrarily chosen value of reduced time (i.e. at tH"t/t "1.8) (Fig. 3c). The %2 di!erent curves pro"ling the development of gel strength at di!erent i-carrageenan concentrations were reduced to a common curve which demonstrates a simple scaling behaviour. For the concentrations of carrageenan used in this work, similar scaling behaviour was also found in the
A. Tziboula, D.S. Horne / International Dairy Journal 9 (1999) 359 } 364
Fig. 4. The data of Fig. 1b replotted as a function of (a) time; (b) reduced time (t/t ) where t is the gelation time of the individual run; (c) %2 %2 GH for each curve normalised to the value of the modulus attained at reduced time t/t "1.7 on that curve. %2
milk} and serum}carrageenan mixtures (Figs. 4 and 5). The di!erent shapes of the reduced curves in the di!erent media illustrates the signi"cance of the milk proteins on i-carrageenan gelation. The presence of scaling behaviour in i-carrageenan gels implies that the e!ect of polymer concentration on the rheological GT is simply a manifestation of the timedependent growth of the gel. At low carrageenan concentrations it will take longer to form interhelical cross links between carrageenan molecules and initiate the aggregation process which leads to gel formation. As with the rennet gels, the presence of scaling behaviour implies that the mathematical expression describing the shear modulus development (GH) can be factorised into the produce of two functions: G*"G* f (t/t ) (1) %2 Therefore, measured G* is a function only of the asymptotic value of the shear modulus G* , and the reduced time variable t/t . De"ning the value of t/t %2 %2 "xes the value of f (t/t ). Thus, the value of the elastic %2
363
Fig. 5. The data of Fig. 1c replotted as a function of (a) time; (b) reduced time (t/t ) where t is the gelation time of the individual run; %2 %2 (c) GH for each curve normalised to the value of the modulus attained at reduced time t/t "1.8 on that curve. %2
modulus at some speci"ed value of the reduced time will always be the same fraction of G* . Therefore, compari sons of the values of GH at a reaction time of 1.8t will %2 provide the same information as would be obtained at in"nite reaction time. This gel strength at in"nite time, can be regarded as an expression of the static components in the gelation process and it is a function of the polymer concentration. The gelation time controls the dynamics of gel-"rming when the static components are invariant between experiments. The existence of scaling behaviour in the presence of milk proteins implies that carrageenan gel formation in the presence of milk proteins involves mainly carrageenan}carrageenan crosslinkages and not carrageenan}protein interactions.
4. Conclusions In this work we studied the kinetics of i-carrageenan gelation in the presence of milk proteins. It was shown that the path of gel formation di!ered according to the
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A. Tziboula, D.S. Horne / International Dairy Journal 9 (1999) 359 } 364
composition of the carrageenan dispersing medium. In SMUF (i.e. in the absence of milk proteins) gelation proceeded in two discreet steps, the "rst corresponding to the conformational transition of the polymer which was then followed by aggregation and gelation. On the contrary in the presence casein micelles gelation proceeded fast in a single step following the conformational transition of carrageenan. In the presence of serum proteins gelation proceeded in a single step but the initial rise of GH was disrupted by aggregate formation. Higher carrageenan concentrations were required for gelation in milk rather than milk serum or skim milk ultra"ltrate. Our results suggested that in milk} carrageenan mixtures two largely independent and antagonistic events take place: on the one hand complex formation between i-carrageenan and the milk proteins which interferes with the path of gel formation and on the other hand carrageenan network formation and gelation. The milk protein}carrageenan complexes become entrapped in this network. The rheological characteristics of the mixtures are governed by factors that in#uence either of these two events. In i-carrageenan gels, GH development followed a simple scaling behaviour governed by two independent parameters: the gelation time and the ultimate gel strength. The e!ect of polymer concentration on GT was simply a consequence of the time dependency of the gel growth. The existence of scaling behaviour implied that gel formation is the result of carrageenan}carrageenan cross-linkages and not carrageenan}protein linkages.
Acknowledgements This research was funded by The Scottish O$ce Agriculture, Environment and Fisheries Department.
References Dalgleish, D. G., Horne, D. S., & Law, A. J. R. (1989). Size related di!erences in bovine casein micelles. Biochimica et Biophysica Acta, 991, 383}387. Drohan, D. D., Tziboula, A., McNulty, D., & Horne, D. S. (1997). Milk protein}carrageenan interactions. Food Hydrocolloids, 11, 101}107. Grindrod, J., & Nickerson, T. A. (1968). E!ect of various gums on skim milk and puri"ed milk proteins. Journal of Dairy Science, 51, 834}841. Horne, D. S. (1984). Steric e!ects in the coagulation of casein micelles by ethanol. Biopolymers, 23, 989}993. Horne, D. S. (1995). Scaling behaviour of shear moduli during the formation of Rennet milk gels. In E. Dickinson, & D. Lorient, Food macromolecules and colloids (pp. 456}461). The Royal Society of Chemistry. Horne, D. S. (1996). Aspects of scaling behaviour kinetics of particle gel formation. Journal de Chimie Physique et de Physico Chimie Biologique, 93, 977}986. Payens, T. A. J. (1972). Light scattering of protein reactivity of polysaccharides, especially of carrageenans. Journal of Dairy Science, 55, 141}150. Rees, D. A. (1969). Structure, conformation and mechanism in the formation of polysaccharide gels and networks. Advances in Carbohydrate Chemistry and Biochemistry, 24, 267}332. Rochas, C., & Rinaudo, M. (1980). Activity coe$cients of counterions and conformation in i-carrageenan systems. Biopolymers, 19, 2165}2175. Snoeren, Th. M. H., Payens, T. A., Jeurnink, J., & Both, P. (1975). Electrostatic interaction between i-carrageenan and i-casein. Milchwissenschaft, 30, 393}396. Snoeren, Th. H. M. (1976). i-Carrageenan. A study on its physicochemical properties, sol}gel transition and interaction with milk proteins. Thesis, Nederlands Instituut voor Zuivelonderzoek, Ede, The Netherlands. Schmidt, D. G. (1982). Association of caseins and casein micelle structure. In P. F. Fox, Developments in dairy chemistry (pp. 61}86). London: Applied Science Publishers. Tziboula, A., & Horne, D. S. (1998). In#uence of milk proteins on the gel transition temperature and the mechanical properties of weak i-carrageenan gels. In G. O. Phillips, P. A. Williams, & D. J. Wedlock, Gums and stabilisers for the food industry, vol. 9 (pp. 202}211). Oxford: IRL Press.
In#uence of milk proteins on i-carrageenan gelation Athina Tziboula*, David S. Horne Hannah Research Institute, Ayr, KA6 5HL, UK
Abstract The in#uence of milk proteins on the gelation of i-carrageenan during cooling from 60}53C was investigated at constant ionic strength, using small deformation rheology. When carrageenan was dispersed in skim milk ultra"ltrate (SMUF), gel development proceeded in two distinct steps: the "rst at the onset of gelation was a coil-to-helix transition of carrageenan followed by a second stage of aggregation and gelation. In serum, the initial growth of the complex modulus (GH) was followed by a temporary decrease in GH, then recovery of structure and further gelation. We believe that this anomaly in gel development was the result of aggregation and subsequent re-stabilisation of the aggregates in an encompassing network. In the presence of casein micelles gelation took place in a single step, suggesting an interaction between i-carrageenan and the casein micelles which has to be satis"ed "rst and reduces the availability of carrageenan for the gelation ro( le. Thus, the minimum concentration of polymer required for gelation was greater in milk than in SMUF or serum. It is also demonstrated in this paper that the gelation pro"les show scaling behaviour and can be described by two independent parameters: the gel strength at in"nite time and the gelation time. 1999 Elsevier Science Ltd. All rights reserved. Keywords: i-Carrageenan; Caseins; Milk
1. Introduction Carrageenans are a generic class of linear polymers of alternating a-1,4 and b-1,3 anhydrogalactose residues with varying amounts of sulphate half ester groups. Their functionality ranges from thickeners to gellants and stabilising agents depending on their ability to form thermoreversible gels or not. For water-thickening applications non-gelling carrageeanans (such as jcarrageenan) are preferred, whilst for gelling applications, i- or ι-carrageenans are most commonly used. The carrageenan functionality in milk has been attributed to a speci"c interaction between carrageenan and i-casein to form a complex which aggregates into a three-dimensional gel network (Snoeren, 1976). This interaction has been ascribed to electrostatic attraction between the negatively charged sulphate groups of the carrageenan and a predominately positively charged region in the peptide chain of i-casein (Snoeren, 1976; Payens, 1972; Snoeren, Payens, Jeurnink & Both, 1975; Grindrod and Nickerson, 1968). In milk caseins exist associated with each other and with calcium phosphate * Corresponding author. Tel.: #44-1292-674088; fax: #44-1292674008. E-mail address: [email protected] (A. Tziboula)
to form aggregates, the casein micelles. It is accepted that i-casein is located on the surface of the micelles (Schmidt, 1982; Horne, 1984; Dalgleish, Horne & Law, 1989), therefore a similar interaction between carrageenan and icasein is possible. Nevertheless, cations such as K> or Ca> which promote carrageenan gelation are also present in milk and can also in#uence carrageenan gelation. Previous studies on i-carrageenan gelation in milk showed that gel formation and properties were governed by the total ionic content and the carrageenan concentration (Drohan, Tziboula, McNulty & Horne, 1997). Although milk proteins did not in#uence gelation at concentrations of carrageenan greater than 200 ppm, they became important at low polymer levels. For example doubling the concentration of casein micelles in the carrageenan}milk mixtures prevented gel formation. Subsequently, it was shown that the rate of cooling of the milk}carrageenan mixtures in#uenced the gelation temperature and gel strength. Fast cooling rates resulted in delayed gelation (i.e. lower gelation temperatures) and weaker gels. This e!ect was more pronounced at low carrageenan concentrations and when the polysaccharide was dispersed in skim milk ultra"ltrate (SMUF) rather than in milk serum or milk (Tziboula & Horne, 1998). This behaviour demonstrated that for meaningful comparisons and interpretation of the data carrageenan
0958-6946/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 0 8 8 - 6
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gelation studies should be carried out at slow cooling rates. This paper deals with the e!ect of carrageenan concentration (4200 ppm) on the gelation temperature and the mechanical properties of the gels in the presence of di!erent milk protein fractions. i-Carrageenan was dispersed in SMUF, skim milk or skim milk serum. SMUF was the milk fraction obtained after the removal of the milk proteins and consisted mainly of lactose, ions and the proteose-peptone fraction in milk. Skim milk serum was the fraction of milk obtained after the removal of the casein fraction and consisted of SMUF plus the whey proteins. The ionic concentration in the mixtures was kept constant equivalent to that of skim milk. This approach allowed us to evaluate the relative importance of the di!erent milk proteins and ionic composition of milk on i-carrageenan gelation. A simple scaling approach, previously applied in rennet gels, was used to examine the parameters in#uencing i-carrageenan gelation in the presence of milk proteins (Horne, 1995).
2. Materials and methods 2.1. Milk and milk fractions Bulk milk from the Institute herd was skimmed by centrifugation at 1200 g for 40 min at 43C (Mistral 6000 centrifuge; Sanyo Gallenkamp plc., Leicester, UK). From the skimmed milk the following fractions were prepared: (i) Skim milk ultra"ltrate (SMUF): was prepared using a pressure "lter equipped with a membrane of molecular weight cut-o! 10 kda (Amicon Ltd, Danvers, MA). (ii) Serum fraction was obtained by centrifugation of skim milk at 43 000 g for 120 min. The supernatant obtained consisted of whey proteins, low molecular weight material and soluble caseins; this fraction was designated the serum fraction. The soluble caseins were removed from the serum fraction by isoelectric precipitation at pH 4.6 followed by centrifugation (1000 g for 5 min; Mistral 6000 centrifuge; Sanyo Gallenkamp plc., Leicester, UK) and "ltration through Whatman No. 1 "lter paper. The ionic equilibria of the serum fraction was re-established to that of skim milk by exhaustive dialysis against skimmed milk over 48 h, three changes of dialysate, serum to milk ratio 1 : 250). The puri"ed serum fraction was then rapidly frozen by immersion in liquid nitrogen and stored at !203C. 2.2. i-carrageenan i-Carrageenan from Euchema cottonoii (Sigma Chemicals, Poole, UK) was converted to the sodium form using
cation exchange resin (Amberlite IR120, BDH Ltd, Poole, UK). The polysaccharide was recovered by "ltration and freeze dried. The extent of conversion of the carrageenan to the sodium form was evaluated using atomic absorption spectroscopy. The sodium content was estimated to be 5.67% w/w. 2.3. Milk protein}carrageenan mixtures i-Carrageenan (80}200 ppm) was added to the appropriate milk fraction while stirring at room temperature. The mixture was heated to 703C for 20 min to hydrate the carrageenan and then transferred to the rheometer held at 603C. 2.4. Rheological study Low-frequency oscillation (0.08 Hz) was performed on a controlled stress rheometer (CVO, Bohlin Instruments, Gloucestershire, UK) using the double gap measuring geometry. The geometry consists of a hollow cylinder (diameter 45 mm) which is lowered into a cylindrical groove in an outer cylinder (diameter 50 mm). An oscillating stress (5 mPa) was applied to the sample (30 ml) as it was cooled from 60}53C at a rate of 13C/15 min. The instrument calculates the phase angle at which the strain response lags behind the applied stress and hence the storage modulus (G ), viscous modulus (G ) complex modulus (GH) and complex viscosity (gH) can be extracted. The gelation temperature of the mixtures was taken as the point where tan d"G /G "1. The applied stress of 5 mPa was just above the minimum permissible stress with the double-gap geometry whilst operating within the linear viscoelastic region of the structures formed. The strain response varied with the viscoelastic properties of the milk}carrageenan mixtures during the cooling cycle. The initial strain response (at 593C) was constant at 1.42$0.01 whilst at 53C it ranged from 0.4 for the weakest structures to 0.002 for stronger gels. Evaporation was prevented by "tting the solvent trap on the double-gap geometry.
3. Results and discussion 3.1. Inyuence of milk proteins on i-carrageenan gelation Typical gelation pro"les of SMUF}, milk} and serum}carrageenan mixtures during cooling from 60 to 203C, are shown in Fig. 1a}c. As the temperature decreased from 60 to 403C there was a lag period where the elastic contributions to the mechanical properties of the mixtures was very small. The phase lag (d) of the strain response to the applied stress was greater than 453. At these temperatures, i-carrageenan existed in the random coil conformation. The onset of structure formation was
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Fig. 1. Plots of the shear modulus (GH) development during cooling of (a) skim milk ultra"ltrate-i-carrageenan mixtures; (b) skim milk-icarrageenan mixtures; (c) skim milk serum-i-carrageenan mixtures. Data obtained from low-frequency oscillation at frequency 0.08 Hz and applied stress 5 mPa. i-Carrageenan concentration: - - - - - - 200 ppm; } } } } 170 pmm; *** 140 ppm; } - } - 80 ppm. GT, gelation temperature; ¹ , Temperature of coil-to-helix transition of carrageenan. 20
signalled at around 383C by a reduction in d and a rapid increase in the complex modulus (GH). This point corresponded to the conformational coil-to-helix transition of i-carrageenan (¹ ) and it was not in#uenced by the 20 concentration of carrageenan or the composition of the dispersing medium. It is well established that the gelation temperature of carrageenan is in#uenced by the total ionic concentration and can be predicted by calculating the cation activity contributions from the various components in the solution (Rees, 1969; Rochas and Rinaudo, 1980). For the carrageenan concentrations studied here, the total cation concentration was "xed at levels equivalent to that of skim milk, hence the coil}helix transition temperature was also una!ected. As the cooling continued there came a point at which d"453. At this point the contributions to structure from the viscous and elastic components becomes equal. This point is de"ned as the rheological gelation temperature (GT). Unlike ¹ , 20 GT was a function of the polymer concentration and di!ered with the nature of the milk protein present in the mixtures. In SMUF (Fig. 1a) gelation was a two stage process: the conformation transition of carrageenan at 383C was
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marked by a measurable increase in GH and it was followed by a second discreet rise in GH at slightly lower temperatures. The second step on the GH development corresponded to the rheological GT. Both gel strength and GT were a function of the concentration of carrageenan. Decreasing the polymer content lowered GT and resulted in weaker gels. When i-carrageenan was dispersed in milk (Fig. 1b) the initial sharp rise in GH at ¹ was followed by immediate aggregation and gela20 tion. Although the rheological GT was 2}33C lower than ¹ the whole process proceeded smoothly in one step. 20 As before the gel strength was governed by the concentration of the gelling agent in the system albeit for the same carrageenan concentration the skim milk}carrageenan gels were stronger than the SMUF}carrageenan gels. It is also noteworthy that gelation was not observed at i-carrageenan concentrations of 140 and 80 ppm. The path of gel formation was also di!erent in the presence of whey proteins (Fig. 1c). Immediately after the "rst sharp increase in GH at ¹ there was a noticeable 20 decrease in the development of the shear modulus (Fig. 1c). In a separate experiment we monitored the path of i-carrageenan gelation in the presence of whey protein and observed the formation of visible aggregates in the vicinity ¹ . On further cooling of the samples, i-car20 rageenan gelation proceeded faster than the aggregation of the whey protein}carrageenan complex hence the recovery in the development of the complex modulus (Fig. 1c). Therefore, it seemed likely that two antagonistic events took place; on the one hand interaction between i-carrageenan and whey proteins and aggregate formation which had a structure damaging e!ect and on the other hand i-carrageenan gelation which was structure forming. This &interference' of milk proteins with gel formation has been reported previously (Tziboula & Horne, 1998). It was found that at low i-carrageenan concentrations an increase in the micellar casein/or whey protein content whilst maintaining the ionic strength equivalent to that of skim milk, prevented gel formation. In all cases, the rheological GT showed carrageenan concentration dependence (Table 1). Lowering the polymer concentration resulted in a sharp decrease in GT. Furthermore, there was a critical carrageenan concentration below which gelation did not take place. This critical polymer concentration was lower for the serum}carrageenan and SMUF}carrageenan rather than the milk} carrageenan mixtures. Again at such low carrageenan levels there was domination of protein}carrageenan interactions which interfered with gel formation. The e!ect of carrageenan concentration on GT was a manifestation of the time dependency of the gelation process and is discussed in detail in the following section. Finally, the milk serum}carrageenan gels were stronger than the milk} or SMUF}carrageenan gels (Fig. 2).
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Table 1 Gelation temperature of SMUF}, skim milk}, and serum}carrageenan mixtures as a function of i-carrageenan concentration i-Carrageenan conc. (ppm)
80 140 170 200
Gelation temperature (GT) (3C) SMUF
Skim milk
Serum
17.3 30.3 32.3 33.7
No gelation No gelation 36.6 36.5
13.5 33.8 32.2 33.4
Fig. 2. In#uence of i-carrageenan concentration on gel strength at 53C. 䊏 skim milk ultra"ltrate-i-carrageenan gels; 䊐 skim milk-i-carrageenan gels; skim milk serum-i-carrageenan gels.
These results suggested that in a complex system such as milk, there were many often antagonistic events that can in#uence the carrageenan gelation. The data presented above suggested that i-carrageenan was capable of forming complexes with both casein micelles and whey proteins. With the casein micelles there was a strong interaction which had to be satis"ed "rst, reducing the availability of carrageenan for the gelation ro( le; this explains why the minimum carrageenan concentration required for gelation was higher in milk than in SMUF or serum. At su$ciently high polymer concentrations i-carrageenan gelation e!ects dominated the system entrapping casein micelles and casein micelle}carrageenan complexes. Similarly in serum, the initial rise in GH at ¹ was hindered by the formation of aggregates, possibly 20 between carrageenan and whey proteins. At su$ciently high polymer concentrations gelation overcomes aggregation entrapping aggregates in the carrageenan network. The resultant whey protein}carrageenan gels were much stronger than the equivalent SMUF- or milk-gels. 3.2. Scaling behaviour of the shear moduli during i-carrageenan gelation In rennet gelation studies Horne (1995,1996) has demonstrated that GH development follows a simple scaling
Fig. 3. The data of Fig. 1a replotted as a function of (a) time; (b) reduced time (t/t ) where t is the gelation time of the individual run; (c) GH for %2 %2 each curve normalised to the value of the modulus attained at reduced time t/t "1.8 on that curve. %2
behaviour governed by two independent parameters: the gelation time and the ultimate gel strength. Although the mechanism by which the aggregating particles are produced is di!erent in rennet gels, we applied the same mathematical approach in this work. Thus the gelation pro"les shown in Fig. 1a were as a function of time (t) and then as a function of the reduced time t/t , with t %2 %2 the gelation time varying for each individual curve (Fig. 3a}b). A series of curves were produced ordered according to their "nal gel strength, which in turn was a function of the polymer concentration. This suggested that the shear modulus should be normalised against the shear modulus at in"nite time. As the latter is rather impractical, we normalised the reduced plots to the values of the shear moduli at an arbitrarily chosen value of reduced time (i.e. at tH"t/t "1.8) (Fig. 3c). The %2 di!erent curves pro"ling the development of gel strength at di!erent i-carrageenan concentrations were reduced to a common curve which demonstrates a simple scaling behaviour. For the concentrations of carrageenan used in this work, similar scaling behaviour was also found in the
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Fig. 4. The data of Fig. 1b replotted as a function of (a) time; (b) reduced time (t/t ) where t is the gelation time of the individual run; (c) %2 %2 GH for each curve normalised to the value of the modulus attained at reduced time t/t "1.7 on that curve. %2
milk} and serum}carrageenan mixtures (Figs. 4 and 5). The di!erent shapes of the reduced curves in the di!erent media illustrates the signi"cance of the milk proteins on i-carrageenan gelation. The presence of scaling behaviour in i-carrageenan gels implies that the e!ect of polymer concentration on the rheological GT is simply a manifestation of the timedependent growth of the gel. At low carrageenan concentrations it will take longer to form interhelical cross links between carrageenan molecules and initiate the aggregation process which leads to gel formation. As with the rennet gels, the presence of scaling behaviour implies that the mathematical expression describing the shear modulus development (GH) can be factorised into the produce of two functions: G*"G* f (t/t ) (1) %2 Therefore, measured G* is a function only of the asymptotic value of the shear modulus G* , and the reduced time variable t/t . De"ning the value of t/t %2 %2 "xes the value of f (t/t ). Thus, the value of the elastic %2
363
Fig. 5. The data of Fig. 1c replotted as a function of (a) time; (b) reduced time (t/t ) where t is the gelation time of the individual run; %2 %2 (c) GH for each curve normalised to the value of the modulus attained at reduced time t/t "1.8 on that curve. %2
modulus at some speci"ed value of the reduced time will always be the same fraction of G* . Therefore, compari sons of the values of GH at a reaction time of 1.8t will %2 provide the same information as would be obtained at in"nite reaction time. This gel strength at in"nite time, can be regarded as an expression of the static components in the gelation process and it is a function of the polymer concentration. The gelation time controls the dynamics of gel-"rming when the static components are invariant between experiments. The existence of scaling behaviour in the presence of milk proteins implies that carrageenan gel formation in the presence of milk proteins involves mainly carrageenan}carrageenan crosslinkages and not carrageenan}protein interactions.
4. Conclusions In this work we studied the kinetics of i-carrageenan gelation in the presence of milk proteins. It was shown that the path of gel formation di!ered according to the
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composition of the carrageenan dispersing medium. In SMUF (i.e. in the absence of milk proteins) gelation proceeded in two discreet steps, the "rst corresponding to the conformational transition of the polymer which was then followed by aggregation and gelation. On the contrary in the presence casein micelles gelation proceeded fast in a single step following the conformational transition of carrageenan. In the presence of serum proteins gelation proceeded in a single step but the initial rise of GH was disrupted by aggregate formation. Higher carrageenan concentrations were required for gelation in milk rather than milk serum or skim milk ultra"ltrate. Our results suggested that in milk} carrageenan mixtures two largely independent and antagonistic events take place: on the one hand complex formation between i-carrageenan and the milk proteins which interferes with the path of gel formation and on the other hand carrageenan network formation and gelation. The milk protein}carrageenan complexes become entrapped in this network. The rheological characteristics of the mixtures are governed by factors that in#uence either of these two events. In i-carrageenan gels, GH development followed a simple scaling behaviour governed by two independent parameters: the gelation time and the ultimate gel strength. The e!ect of polymer concentration on GT was simply a consequence of the time dependency of the gel growth. The existence of scaling behaviour implied that gel formation is the result of carrageenan}carrageenan cross-linkages and not carrageenan}protein linkages.
Acknowledgements This research was funded by The Scottish O$ce Agriculture, Environment and Fisheries Department.
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