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Rearrangements in Acid-Induced Casein Gels during and after Gel Formation
Rearrangements in Acid-Induced Casein Gels during and after Gel Formation By Ton van Vliet, John A. Lucey', Katja Grolle, and Pieter Walstra DEPARTMENT O F FOOD SCIENCE, WAGENINGEN AGRICULTURAL UNIVERSITY, P.O. BOX 8129, 6700 EV WAGENINGEN, T H E NETHERLANDS DEPARTMENT OF FOOD TECHNOLOGY, MASSEY UNIVERSITY, PALMERSTON NORTH, NEW ZEALAND
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1 Introduction In rennet-induced milk gels, extensive rearrangements of the network structure occur after gel formation which are related to the strong tendency of this type of gel to exhibit syneresis. These rearrangements are more extensive if temperature is higher and/or pH is lower (for pH 2 5.15). The extent of rearranging can be related to the dynamics (average life-time) of the proteinprotein bonds as expressed in terms of the loss tangent, and to the yielding/ fracture force of the casein strands. At pH < 5, rearrangement and, accordingly, endogenous syneresis was considered to be virtually absent.I4 A quality defect in the production of set yoghurts, which usually have a pH below 4.5, is that some serum separates on top of the product. Moreover, it has been reported' that acid gels formed of non-pre-heated milk show extensive syneresis when incubated at temperatures above 40 "C. Probably, these phenomena are also related to (some) rearrangements of the casein particles and strands during and after gel formation. To investigate this, the permeability, the dynamic moduli and the fracture properties of glucono-dlactone (GDL) induced casein gels were determined as a function of the ageing time and of the measuring and ageing temperatures. In this paper some results are presented for gels with pH between 4.6 and 4.9. It has been ~ h o w nthat ~ . ~during aggregation of casein particles, for instance caused by a pH decrease, clusters are formed which o n average have a fractal structure. The number of primary casein particles, N , , in such a cluster scales with the radius R as 335
Rearrangements in Acid-Induced Casein Gels
336
$ = ($)”. N
where D is the fractal dimensionality (D < 3 ) , aeffis the radius of the effective building blocks forming the fractal cluster, and Nu is the number of primary casein particles forming such a building block. A gel will be formed when the average of the volume fraction of particles in the fractal clusters becomes equal to the overall particle volume fraction I$ in the system:’
(R,) is a measure of the average aggregate radius at the point that a gel is formed. In fact, it gives an upper cut-off length, the largest length scale at which the fractal regime exists; at longer distances, a homogeneous scaling of particle density with distance is observed. For a full characterization of the fractal character of a gel one needs three parameters, viz. D, aeffand (R,). Systems with the same D but a different aeffwill have a different structure at a length scale larger than a,ff.8In the case of an attractive interparticle force, there will be a tendency for phase separation during the aggregation stage. This tendency will be counteracted by the incorporation of the (aggregates of) particles in the gel network. Depending on the actual conditions this may lead to more or less clustering of particles, These clusters may then be considered as the building blocks of the
2 Materials and Methods Standard 2.5 wt% sodium caseinate dispersions were made by dissolving 3 g of a commercial sodium caseinate powder (DMV International, casein content 86.2%) in 100 g demineralized water containing 100 ppm thiomersal (BDH Chemicals). To allow for equilibrium, the dispersions were stirred at 25 “C for 16-20 h before use. Gel formation was induced by adding glucono-6-lactone (GDL). Dynamic moduli of the gels were determined at small deformations with a Bohlin VOR rheometer, equipped with a cup and bob system (inner and outer radii 14.00 and 15.25 mm, respectively). Gel formation was followed at a frequency of 0.1 Hz and a maximum shear strain of y 5 0.01. At this strain the samples showed linear behaviour. To prevent evaporation, samples were covered with a thin layer of vegetable oil. Frequency sweeps were done between 0.001 and 1.0 Hz at similar maximum strains. Temperature was controlled within a 0.1 “C. Large deformation and fracture properties were determined by applying a constant shear-rate of 0.00185 s - l . A more extensive description is given by Lucey et aZ.” The permeability of the gels was determined using the ‘tube’ method as described by van Dijk et aZ.” Measuring and gelation temperatures were the same, unless stated otherwise. Syneresis experiments were performed on one-
T. van Vliet, J. A . Lucey, K . Grolle, and P. Walstra
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dimensional slabs (height -6 mm) as described by van Dijk et al." Syneresis was initiated by wetting the surface of the gel with demineralized water. For confocal scanning laser microscopy (CSLM), gels were prepared as usual, except that one drop of diluted Rhodamine dye was added to 100 ml of the Na caseinate dispersion. Gelation occurred in a special object slide; temperature was controlled within f 0.1 "C. Gels were examined by a BIORAD MCR600 system.
3 Results and Discussion Some results of syneresis experiments on GDL-induced 2.51 wt% sodium caseinate gels at various temperatures are shown in Figure 1. At pH = 4.9 the extent of syneresis is much stronger at a higher temperature. At 30 "C the tendency to exhibit syneresis was clearly less at a pH of 4.7 than at 4.92. relative height ("h) 105
loo
95
90
85
80
75
Figure 1
0
I
I
I
1000
2OOo
3000
time (s)
4000
so00
ReIative height of sodium caseinate gels as a function of time after start of syneresis. Measuring and gel formation temperature: 0, 20 "C; A, 30 "C; 0, 40 "C,all p H == 4.92; a, 30 "C, p H 4.7
Rearrangements in Acid-Induced Casein Gels
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According to Darcy's law, the overall liquid flux v in one direction through a gel is" v =-(B/qc) APIAx,
(3)
where B is the permeability coefficient, qc is the viscosity of the liquid, and APIAx is the pressure gradient. The quantity B is a measure of the number and size of the largest 'capillaries' (pores) present in a gel. Factors favouring a large liquid flux, and thereby fast syneresis, are a high syneresis pressure, A P , a small flow distance, A x , and a high permeability coefficient. The permeability coefficients observed were 0.9,4.1 and 25 X m2, for 'young' gels formed and tested at 20, 30 and 40 "C, respectively (Table 1 ) . The presence of larger pores in the gels made at higher temperature is also clear from confocal scanning laser microscopy pictures (Figure 2). At 20 and 30 "C the size of the largest pores is ca. 5 and 20 pm, respectively. For a gel consisting of aggregated clusters the size of these clusters will be about the same as that of the largest pores, provided that no extensive rearrangements of the gel structure has occurred after gel formation. Assuming that this is not the case, it is possible to calculate the size of the building blocks of these clusters using equation (2), if the volume fraction of casein is known. Taking the voluminosity values at 20 and 30 "C as 3.1 and 2.7 ml g-I, respectively, leads to volume fractions of q5 = 0.078 and @ = 0.068, respectively.'2 The fractal dimensionality of acid casein gels formed by GDL addition is about 2.35.'This results in a e f fvalues of ca. 50 and 160 nm for the gels formed at 20 and 30 "C, respectively. The radius of the primary casein particles will be around 50 nm under the prevalent conditions.I3 This is in agreement with the value calculated for acffat 20 "C, but not with that calculated for the gels formed at 30 "C. This then implies that there is no extensive rearrangement ( e . g . , complete fusion of particles or the formation of dense aggregates) during gel formation at 20 "C. Table 1
The permeability coefficient B of GDL-induced acid sodium caseinate gels f o r three formation and measuring temperatures. The effect of a temporary (30 min) higher temperature is also shown. The value of B was determined about 1-2 h after visible gel formation
Temperaiure of formaiion and measurementI"C 20 20 30 30 40
Additional temporary temperaturePC
BIpm2
0.9 1.6 4.1 5.4 25
T. van Vliet, J . A . Lucey, K . Grolle, and P . Walstra
Figure 2
339
Effect of gelation temperature on confocal scanning laser micrographs of sodium caseinate gels formed at pH 4.6: (a) 20 "C; (6) 30 "C. Gels were analysed about 17 h after G D L addition. Scale bar 25 ,urn
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Rearrangements in Acid-Induced Casein Gels
At 30 "C the situation is completely different, i.e. ueff is much larger than the radius of the primary particles. Moreover, the value of ueff is likely to be an underestimation, because in reality the effective building blocks formed due to the rearrangement processes will not consist of completely fused primary particles, but of dense aggregates with a volume fraction of particles below 1. Taking the arbitrary value of 0.74 for this volume fraction would result in an aeff value of cu. 250 nm.'* The conclusion is that, during gel formation at 30 "C, extensive rearrangements must occur at the scale of the size of the primary particles. Rearrangement processes take longer for increasing aggregate size because the distances involved are longer. Above a certain size the rearrangement processes are too slow to keep up with the flocculation process and fractal clusters are formed and ultimately a gel. The gel formed then has a much more inhomogeneous structure than in the absence of rearrangements. The permeability data and the confocal scanning laser microscopy pictures indicate that the clear difference in syneresis behaviour will at least be partly due to the presence of larger capillaries in the gels formed at the higher temperature. The fact that at 30 "Csyneresis is less for a gel of pH 4.7 than 4.92 indicates that other factors play a part. Moreover, the analysis given above leaves open the question whether rearrangements occur also after gel formation. To investigate the latter question first, gel permeability was also determined after applying a temperature cycle. Some results are given in Table 1. It is seen that a temporary higher temperature results in a somewhat larger permeability, indicating that some rearrangements of the gel can occur at 40 "C. Preliminary results have indicated that a temperature cycle 2 b 3 b 2 0 "C does not significantly affect B (results not shown). The rate of gel formation is slower at a lower temperature for a given amount of GDL added (Figure 3). After a gel is formed, G' initially increases very quickly, but this increase tends to level off faster to a semi-plateau value for a higher ageing temperature, in agreement with what has been observed before for skim milk gels.I4 The effect of a temporary higher temperature on the G' versus ageing time curve is shown in Figure 4. A higher temperature for 30 minutes clearly resulted in a higher value of G'. A temporary lowering of temperature did not induce an irreversible change in G' (results not shown). Combination of the results obtained for a temperature cycle for B and G' shows that, for gels formed at 20 or 30 "C, a temporary temperature of 40 "C induces or enhances rearrangements in the gels at two levels, i . e . (i) within and between the casein particles, and (ii) at the level of the casein strands making up the network. Presumably, the first type of rearrangement mainly leads to fusion of the casein particles. It will occur at both temperatures investigated,: but faster at the higher temperature. The second type of rearrangement will lead to fracture of strands, and so to a higher B value and a lower G'. However, this latter effect is overshadowed by the increase in G' due to the fusion of casein particles. As mentioned above, syneresis is stimulated by fracture and/or yielding of the casein strands which in turn is favoured by the following circumstances: (i) thin strands, so that the number of protein-protein bonds per cross section is
34 1
T. van Vliet, J . A . Lucey, K . Grolle, and P . Walstra G' (Nrn2) 600
-I;
c c
400
20°C
--
I : ,. o o o A A
0 0
100000
D A D A
a AOA
200000 time after addition of GDL fs)
Figure 3
Storage modulus G' as a function of ageing time for sodium caseinate gels acidified with 0.376 % G D L for variour ageing temperatures. The final pH was ca. 4.6
small (small fracture stress), and (ii) a short average life-time of the proteinprotein bonds, as is expressed in a high tan 6 (= G" / G').3*4If tan 6 is high, a casein strand may yield due to a tensile force resulting in an elongational flow in the strand causing it to become thinner and thinner. If the strand becomes too thin, even a small tensile force will be larger than its fracture force, and it will fracture. Some results obtained for the fracture properties of sodium caseinate gels are shown in Table 2. Both results obtained for gels at ca. 10 minutes after gel formation and for aged gels (pH 4.6) are given. In all cases the casein networks fracture but the system as a whole remains continuous; so it is said to have yielded. As can be seen, the fracture stress of the casein network strongly decreases with increasing gel formation and measuring temperature. The fracture stress increases and the fracture strain decreases with ageing of the gel, probably due to ongoing fusion of the casein particles.
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Rearrangements in Acid-Induced Casein Gels
G' (Nm-2)
400
m
m
I
0
I
-1
I
I
1 4 m
IeOOOO
time (s) Figure 4
Storage modulus G' as a function of ageing time for sodium caseinate gels acidified with 0.3 % G D L . The gels were formed and aged at 20 "C. During ageing, the temperature was temporarily (for 30 minutes) increased to 30 "C (broken line) or 40 "C (full curve)
Table 2
Fracture stress or. and fracture strain yfr of the casein network f o r 2.51 wt% sodium caseinate gels formed by the addition of 0.376 wt% GDL. Ageing and measuring temperatures, Tgerand T,,,,,,, are indicated. Fresh gels were tested ca. I 0 min after gelation; old gels were tested when p H was 4.6
20 20 30 30 40
20 30 30 40 40
102
2.1
-
-
28 -
9
1.5 -
1.5
326 207 164 62 22
0.75 0.85 1.o 0.67 1.2
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The temperature effect on ufrand yfr is not only due to the measuring temperature. Increasing the latter clearly leads to a reduction in uf,, but still there is a large effect of gelation and ageing temperature (Table 2). Probably the latter effect has its origin primarily in the more inhomogeneous structure of the gels formed at higher temperature. The effect of measuring temperature implies that a higher temperature leads to weaker interactions between the casein particles and probably to a lower fracture force of the casein strands. This lower fracture force is the reason that strands may break, as is clear from the increase in B, if gels formed at 20 or 30 "C are temporarily brought to 40 "C (Table 1). The relaxation behaviour (average life-time) is independent of ageing time and measuring temperature in the range 20-40 "C,and tan 6 is ca. 0.22 at all temperatures." This implies that slow yielding of the strands probably does not play an important role, in contrast to what is the case in casein gels with a pH above 5.15, where it is an important factor determining gel structure as a function of ageing t i ~ n e . ~ . ~ As shown in Figure 1, GDL-induced casein gels may exhibit syneresis at a higher temperature. This will partly be due to the larger value of B. The question is: what causes APlAx? In principle, APlAx may be due to an endogenous syneresis pressure caused by ongoing fusion of the casein particles or due to the pressure gradient caused by the weight of the casein network. An estimate of the former is hardly possible. The latter can be estimated by using APlAx = &,Apg, where &,is the net volume fraction of protein (ca. 0.017), Ap is the density difference between the protein and the solution (taken as 500 kg m-3), and g is the acceleration due to g r a ~ i t y . 'This ~ results in a pressure gradient of about 100 Pa m-'. According to equation (l),and using the values 4 of B given in Table 1, this would result in an overall liquid flux of about x low8or 25 x lo-' m s-l for temperatures of 20,30 and 40 "C, respectively (Table 3). The actual rate of decrease in height of the gels due to 'syneresis' given in Figure 1can be estimated from the initial slope, and is found to be ca. 4 x 4X and 1 X lop6m s-l, respectively. For all temperatures the actual rate by which the height of the gels is reduced is larger than the overall liquid flux due to the weight of the gel (Table l), implying the presence of a (small) endogenous syneresis pressure, which then would be larger at a higher Table 3
The rate of decrease in height due to syneresis of GDL-induced sodium caseinate gels and the calculated liquid flux due to the weight of the gels. (Sodium caseinate concentration 2.51 wt%; p H = 4.92)
TemperatureI "C
4 4 4 1
20 30 30" 40
'In this case pH
Rate of decrease in heightlm s-'
= 4.7.
x 10-8 x
x 10-8 x 10-6
Liquid flux (vlm s-I) calculated from eqn ( I ) 1 x 10-8 4 x 10-8 4 x 10-8 2.5 x 10-7
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Rearrangements in Acid-Induced Casein Gels
temperature. During the ongoing decrease in pH and/or ageing of the gel, this endogenous syneresis pressure presumably decreases,' resulting in a smaller tendency of the gels to exhibit syneresis. At pH 4.7 and 30 "C the rate of decrease in height of the gel was found to be similar to the calculated liquid flux v (Table 3 ) , and so there was virtually no endogenous syneresis pressure. The observed, mostly small, syneresis pressures are presumably due to the process of fusion of the casein particles. This would also explain why syneresis in acid gels rapidly decreases in rate, as observed by van Dijk.16This in contrast to the syneresis in rennet casein gels, which proceeds for a far longer time, and which is due to rearrangement of the particle strands in the
4 Conclusions In GDL-induced gel formation, extensive rearrangements at the particle level occur, leading to dense particle clusters during the aggregation stage at a temperature of 30 or 40 "C. These rearrangements are virtually absent if the gel is formed at 20 "C. After the gel is formed, an increase in temperature may induce minor rearrangements. The observed small endogenous syneresis pressures are presumably due to t h e process of fusion of the casein particles, in contrast to what is the case for rennet casein gels.
Acknowledgement The authors thank Annemarie Schoonman for performing some of the experiments.
References 1. P. Walstra, H. J . M. van Dijk, and T. Geurts, Neth. Milk Dairy J., 1985, 39,209. 2 . S. P. F. M. Roefs, T. van Vliet, H . C. J. M. van den Bijgaart, A. E. A . de GrootMostert, and P. Walstra, Neth. Milk Dairy J., 1990, 44, 159. 3. T. van Vliet, H. J . M. van Dijk, P. Zoon, and P. Walstra, Colloid Polym. Sci., 1991, 269, 620. 4. T. van Vliet and P. Walstra, J . Food E n g . , 1994, 22, 75. 5. V. R. Harwalkar and M. Kalab, Food Microstruct., 1986, 5 , 287. 6. L. G. B. Bremcr, B. H. Bijsterbosch, R. Schrijvers, T. van Vliet, and P. Walstra, Colloids Surf., 1990, 51, 159. 7. L. G. B. Brerner, T. van Vliet, and P. Walstra, J . Chem. Soc., Furuduy Trans. 1, 1989,85, 3359. 8. B. H. Bijsterbosch, M . T. A . Bos, E. Dickinson, J . H. J. van Opheusden. and P. Walstra, Furuduy Discuss., 1995, 101, 51. 9. E. Dickinson, in 'Food Macromolecules and Colloids', ed. E. Dickinson and D. Lorient, Royal Society of Chemistry, Cambridge, 1995, p. 1 . 10. J. A. Lucey, K. Grolle, T. van Vliet, T. Geurts, and P. Walstra, submitted for publication.
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11. H. J. M. van Dijk and P. Walstra, Neth. Milk Dairy J . , 1986, 40,3. 12. J. A. Lucey, T. van W e t , T. Geurts, and P.Walstra, submitted for publication. 13. P.Walstra and R. Jenness, ‘Dairy Chemistry and Physics’, Wiley, New York, 1984. 14. M. Arshad, M. Paulsson, and P. Dejmek, J . Dairy Sci., 1993,76,3310. 15. T. van Vliet and P. Walstra, in ‘Food Colloids’, ed. R. D. Bee, P. Richmond and J. Mingins, Royal Society of Chemistry, Cambridge, 1989, p. 206. 16. H. J. M. van Dijk, PhD Thesis, Wageningen Agricultural University, Netherlands, 1982.
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1 Introduction In rennet-induced milk gels, extensive rearrangements of the network structure occur after gel formation which are related to the strong tendency of this type of gel to exhibit syneresis. These rearrangements are more extensive if temperature is higher and/or pH is lower (for pH 2 5.15). The extent of rearranging can be related to the dynamics (average life-time) of the proteinprotein bonds as expressed in terms of the loss tangent, and to the yielding/ fracture force of the casein strands. At pH < 5, rearrangement and, accordingly, endogenous syneresis was considered to be virtually absent.I4 A quality defect in the production of set yoghurts, which usually have a pH below 4.5, is that some serum separates on top of the product. Moreover, it has been reported' that acid gels formed of non-pre-heated milk show extensive syneresis when incubated at temperatures above 40 "C. Probably, these phenomena are also related to (some) rearrangements of the casein particles and strands during and after gel formation. To investigate this, the permeability, the dynamic moduli and the fracture properties of glucono-dlactone (GDL) induced casein gels were determined as a function of the ageing time and of the measuring and ageing temperatures. In this paper some results are presented for gels with pH between 4.6 and 4.9. It has been ~ h o w nthat ~ . ~during aggregation of casein particles, for instance caused by a pH decrease, clusters are formed which o n average have a fractal structure. The number of primary casein particles, N , , in such a cluster scales with the radius R as 335
Rearrangements in Acid-Induced Casein Gels
336
$ = ($)”. N
where D is the fractal dimensionality (D < 3 ) , aeffis the radius of the effective building blocks forming the fractal cluster, and Nu is the number of primary casein particles forming such a building block. A gel will be formed when the average of the volume fraction of particles in the fractal clusters becomes equal to the overall particle volume fraction I$ in the system:’
(R,) is a measure of the average aggregate radius at the point that a gel is formed. In fact, it gives an upper cut-off length, the largest length scale at which the fractal regime exists; at longer distances, a homogeneous scaling of particle density with distance is observed. For a full characterization of the fractal character of a gel one needs three parameters, viz. D, aeffand (R,). Systems with the same D but a different aeffwill have a different structure at a length scale larger than a,ff.8In the case of an attractive interparticle force, there will be a tendency for phase separation during the aggregation stage. This tendency will be counteracted by the incorporation of the (aggregates of) particles in the gel network. Depending on the actual conditions this may lead to more or less clustering of particles, These clusters may then be considered as the building blocks of the
2 Materials and Methods Standard 2.5 wt% sodium caseinate dispersions were made by dissolving 3 g of a commercial sodium caseinate powder (DMV International, casein content 86.2%) in 100 g demineralized water containing 100 ppm thiomersal (BDH Chemicals). To allow for equilibrium, the dispersions were stirred at 25 “C for 16-20 h before use. Gel formation was induced by adding glucono-6-lactone (GDL). Dynamic moduli of the gels were determined at small deformations with a Bohlin VOR rheometer, equipped with a cup and bob system (inner and outer radii 14.00 and 15.25 mm, respectively). Gel formation was followed at a frequency of 0.1 Hz and a maximum shear strain of y 5 0.01. At this strain the samples showed linear behaviour. To prevent evaporation, samples were covered with a thin layer of vegetable oil. Frequency sweeps were done between 0.001 and 1.0 Hz at similar maximum strains. Temperature was controlled within a 0.1 “C. Large deformation and fracture properties were determined by applying a constant shear-rate of 0.00185 s - l . A more extensive description is given by Lucey et aZ.” The permeability of the gels was determined using the ‘tube’ method as described by van Dijk et aZ.” Measuring and gelation temperatures were the same, unless stated otherwise. Syneresis experiments were performed on one-
T. van Vliet, J. A . Lucey, K . Grolle, and P. Walstra
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dimensional slabs (height -6 mm) as described by van Dijk et al." Syneresis was initiated by wetting the surface of the gel with demineralized water. For confocal scanning laser microscopy (CSLM), gels were prepared as usual, except that one drop of diluted Rhodamine dye was added to 100 ml of the Na caseinate dispersion. Gelation occurred in a special object slide; temperature was controlled within f 0.1 "C. Gels were examined by a BIORAD MCR600 system.
3 Results and Discussion Some results of syneresis experiments on GDL-induced 2.51 wt% sodium caseinate gels at various temperatures are shown in Figure 1. At pH = 4.9 the extent of syneresis is much stronger at a higher temperature. At 30 "C the tendency to exhibit syneresis was clearly less at a pH of 4.7 than at 4.92. relative height ("h) 105
loo
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90
85
80
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Figure 1
0
I
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I
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so00
ReIative height of sodium caseinate gels as a function of time after start of syneresis. Measuring and gel formation temperature: 0, 20 "C; A, 30 "C; 0, 40 "C,all p H == 4.92; a, 30 "C, p H 4.7
Rearrangements in Acid-Induced Casein Gels
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According to Darcy's law, the overall liquid flux v in one direction through a gel is" v =-(B/qc) APIAx,
(3)
where B is the permeability coefficient, qc is the viscosity of the liquid, and APIAx is the pressure gradient. The quantity B is a measure of the number and size of the largest 'capillaries' (pores) present in a gel. Factors favouring a large liquid flux, and thereby fast syneresis, are a high syneresis pressure, A P , a small flow distance, A x , and a high permeability coefficient. The permeability coefficients observed were 0.9,4.1 and 25 X m2, for 'young' gels formed and tested at 20, 30 and 40 "C, respectively (Table 1 ) . The presence of larger pores in the gels made at higher temperature is also clear from confocal scanning laser microscopy pictures (Figure 2). At 20 and 30 "C the size of the largest pores is ca. 5 and 20 pm, respectively. For a gel consisting of aggregated clusters the size of these clusters will be about the same as that of the largest pores, provided that no extensive rearrangements of the gel structure has occurred after gel formation. Assuming that this is not the case, it is possible to calculate the size of the building blocks of these clusters using equation (2), if the volume fraction of casein is known. Taking the voluminosity values at 20 and 30 "C as 3.1 and 2.7 ml g-I, respectively, leads to volume fractions of q5 = 0.078 and @ = 0.068, respectively.'2 The fractal dimensionality of acid casein gels formed by GDL addition is about 2.35.'This results in a e f fvalues of ca. 50 and 160 nm for the gels formed at 20 and 30 "C, respectively. The radius of the primary casein particles will be around 50 nm under the prevalent conditions.I3 This is in agreement with the value calculated for acffat 20 "C, but not with that calculated for the gels formed at 30 "C. This then implies that there is no extensive rearrangement ( e . g . , complete fusion of particles or the formation of dense aggregates) during gel formation at 20 "C. Table 1
The permeability coefficient B of GDL-induced acid sodium caseinate gels f o r three formation and measuring temperatures. The effect of a temporary (30 min) higher temperature is also shown. The value of B was determined about 1-2 h after visible gel formation
Temperaiure of formaiion and measurementI"C 20 20 30 30 40
Additional temporary temperaturePC
BIpm2
0.9 1.6 4.1 5.4 25
T. van Vliet, J . A . Lucey, K . Grolle, and P . Walstra
Figure 2
339
Effect of gelation temperature on confocal scanning laser micrographs of sodium caseinate gels formed at pH 4.6: (a) 20 "C; (6) 30 "C. Gels were analysed about 17 h after G D L addition. Scale bar 25 ,urn
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At 30 "C the situation is completely different, i.e. ueff is much larger than the radius of the primary particles. Moreover, the value of ueff is likely to be an underestimation, because in reality the effective building blocks formed due to the rearrangement processes will not consist of completely fused primary particles, but of dense aggregates with a volume fraction of particles below 1. Taking the arbitrary value of 0.74 for this volume fraction would result in an aeff value of cu. 250 nm.'* The conclusion is that, during gel formation at 30 "C, extensive rearrangements must occur at the scale of the size of the primary particles. Rearrangement processes take longer for increasing aggregate size because the distances involved are longer. Above a certain size the rearrangement processes are too slow to keep up with the flocculation process and fractal clusters are formed and ultimately a gel. The gel formed then has a much more inhomogeneous structure than in the absence of rearrangements. The permeability data and the confocal scanning laser microscopy pictures indicate that the clear difference in syneresis behaviour will at least be partly due to the presence of larger capillaries in the gels formed at the higher temperature. The fact that at 30 "Csyneresis is less for a gel of pH 4.7 than 4.92 indicates that other factors play a part. Moreover, the analysis given above leaves open the question whether rearrangements occur also after gel formation. To investigate the latter question first, gel permeability was also determined after applying a temperature cycle. Some results are given in Table 1. It is seen that a temporary higher temperature results in a somewhat larger permeability, indicating that some rearrangements of the gel can occur at 40 "C. Preliminary results have indicated that a temperature cycle 2 b 3 b 2 0 "C does not significantly affect B (results not shown). The rate of gel formation is slower at a lower temperature for a given amount of GDL added (Figure 3). After a gel is formed, G' initially increases very quickly, but this increase tends to level off faster to a semi-plateau value for a higher ageing temperature, in agreement with what has been observed before for skim milk gels.I4 The effect of a temporary higher temperature on the G' versus ageing time curve is shown in Figure 4. A higher temperature for 30 minutes clearly resulted in a higher value of G'. A temporary lowering of temperature did not induce an irreversible change in G' (results not shown). Combination of the results obtained for a temperature cycle for B and G' shows that, for gels formed at 20 or 30 "C, a temporary temperature of 40 "C induces or enhances rearrangements in the gels at two levels, i . e . (i) within and between the casein particles, and (ii) at the level of the casein strands making up the network. Presumably, the first type of rearrangement mainly leads to fusion of the casein particles. It will occur at both temperatures investigated,: but faster at the higher temperature. The second type of rearrangement will lead to fracture of strands, and so to a higher B value and a lower G'. However, this latter effect is overshadowed by the increase in G' due to the fusion of casein particles. As mentioned above, syneresis is stimulated by fracture and/or yielding of the casein strands which in turn is favoured by the following circumstances: (i) thin strands, so that the number of protein-protein bonds per cross section is
34 1
T. van Vliet, J . A . Lucey, K . Grolle, and P . Walstra G' (Nrn2) 600
-I;
c c
400
20°C
--
I : ,. o o o A A
0 0
100000
D A D A
a AOA
200000 time after addition of GDL fs)
Figure 3
Storage modulus G' as a function of ageing time for sodium caseinate gels acidified with 0.376 % G D L for variour ageing temperatures. The final pH was ca. 4.6
small (small fracture stress), and (ii) a short average life-time of the proteinprotein bonds, as is expressed in a high tan 6 (= G" / G').3*4If tan 6 is high, a casein strand may yield due to a tensile force resulting in an elongational flow in the strand causing it to become thinner and thinner. If the strand becomes too thin, even a small tensile force will be larger than its fracture force, and it will fracture. Some results obtained for the fracture properties of sodium caseinate gels are shown in Table 2. Both results obtained for gels at ca. 10 minutes after gel formation and for aged gels (pH 4.6) are given. In all cases the casein networks fracture but the system as a whole remains continuous; so it is said to have yielded. As can be seen, the fracture stress of the casein network strongly decreases with increasing gel formation and measuring temperature. The fracture stress increases and the fracture strain decreases with ageing of the gel, probably due to ongoing fusion of the casein particles.
342
Rearrangements in Acid-Induced Casein Gels
G' (Nm-2)
400
m
m
I
0
I
-1
I
I
1 4 m
IeOOOO
time (s) Figure 4
Storage modulus G' as a function of ageing time for sodium caseinate gels acidified with 0.3 % G D L . The gels were formed and aged at 20 "C. During ageing, the temperature was temporarily (for 30 minutes) increased to 30 "C (broken line) or 40 "C (full curve)
Table 2
Fracture stress or. and fracture strain yfr of the casein network f o r 2.51 wt% sodium caseinate gels formed by the addition of 0.376 wt% GDL. Ageing and measuring temperatures, Tgerand T,,,,,,, are indicated. Fresh gels were tested ca. I 0 min after gelation; old gels were tested when p H was 4.6
20 20 30 30 40
20 30 30 40 40
102
2.1
-
-
28 -
9
1.5 -
1.5
326 207 164 62 22
0.75 0.85 1.o 0.67 1.2
T. van Vliet, J . A. Lucey, K . Grolle, and P. Walstra
343
The temperature effect on ufrand yfr is not only due to the measuring temperature. Increasing the latter clearly leads to a reduction in uf,, but still there is a large effect of gelation and ageing temperature (Table 2). Probably the latter effect has its origin primarily in the more inhomogeneous structure of the gels formed at higher temperature. The effect of measuring temperature implies that a higher temperature leads to weaker interactions between the casein particles and probably to a lower fracture force of the casein strands. This lower fracture force is the reason that strands may break, as is clear from the increase in B, if gels formed at 20 or 30 "C are temporarily brought to 40 "C (Table 1). The relaxation behaviour (average life-time) is independent of ageing time and measuring temperature in the range 20-40 "C,and tan 6 is ca. 0.22 at all temperatures." This implies that slow yielding of the strands probably does not play an important role, in contrast to what is the case in casein gels with a pH above 5.15, where it is an important factor determining gel structure as a function of ageing t i ~ n e . ~ . ~ As shown in Figure 1, GDL-induced casein gels may exhibit syneresis at a higher temperature. This will partly be due to the larger value of B. The question is: what causes APlAx? In principle, APlAx may be due to an endogenous syneresis pressure caused by ongoing fusion of the casein particles or due to the pressure gradient caused by the weight of the casein network. An estimate of the former is hardly possible. The latter can be estimated by using APlAx = &,Apg, where &,is the net volume fraction of protein (ca. 0.017), Ap is the density difference between the protein and the solution (taken as 500 kg m-3), and g is the acceleration due to g r a ~ i t y . 'This ~ results in a pressure gradient of about 100 Pa m-'. According to equation (l),and using the values 4 of B given in Table 1, this would result in an overall liquid flux of about x low8or 25 x lo-' m s-l for temperatures of 20,30 and 40 "C, respectively (Table 3). The actual rate of decrease in height of the gels due to 'syneresis' given in Figure 1can be estimated from the initial slope, and is found to be ca. 4 x 4X and 1 X lop6m s-l, respectively. For all temperatures the actual rate by which the height of the gels is reduced is larger than the overall liquid flux due to the weight of the gel (Table l), implying the presence of a (small) endogenous syneresis pressure, which then would be larger at a higher Table 3
The rate of decrease in height due to syneresis of GDL-induced sodium caseinate gels and the calculated liquid flux due to the weight of the gels. (Sodium caseinate concentration 2.51 wt%; p H = 4.92)
TemperatureI "C
4 4 4 1
20 30 30" 40
'In this case pH
Rate of decrease in heightlm s-'
= 4.7.
x 10-8 x
x 10-8 x 10-6
Liquid flux (vlm s-I) calculated from eqn ( I ) 1 x 10-8 4 x 10-8 4 x 10-8 2.5 x 10-7
344
Rearrangements in Acid-Induced Casein Gels
temperature. During the ongoing decrease in pH and/or ageing of the gel, this endogenous syneresis pressure presumably decreases,' resulting in a smaller tendency of the gels to exhibit syneresis. At pH 4.7 and 30 "C the rate of decrease in height of the gel was found to be similar to the calculated liquid flux v (Table 3 ) , and so there was virtually no endogenous syneresis pressure. The observed, mostly small, syneresis pressures are presumably due to the process of fusion of the casein particles. This would also explain why syneresis in acid gels rapidly decreases in rate, as observed by van Dijk.16This in contrast to the syneresis in rennet casein gels, which proceeds for a far longer time, and which is due to rearrangement of the particle strands in the
4 Conclusions In GDL-induced gel formation, extensive rearrangements at the particle level occur, leading to dense particle clusters during the aggregation stage at a temperature of 30 or 40 "C. These rearrangements are virtually absent if the gel is formed at 20 "C. After the gel is formed, an increase in temperature may induce minor rearrangements. The observed small endogenous syneresis pressures are presumably due to t h e process of fusion of the casein particles, in contrast to what is the case for rennet casein gels.
Acknowledgement The authors thank Annemarie Schoonman for performing some of the experiments.
References 1. P. Walstra, H. J . M. van Dijk, and T. Geurts, Neth. Milk Dairy J., 1985, 39,209. 2 . S. P. F. M. Roefs, T. van Vliet, H . C. J. M. van den Bijgaart, A. E. A . de GrootMostert, and P. Walstra, Neth. Milk Dairy J., 1990, 44, 159. 3. T. van Vliet, H. J . M. van Dijk, P. Zoon, and P. Walstra, Colloid Polym. Sci., 1991, 269, 620. 4. T. van Vliet and P. Walstra, J . Food E n g . , 1994, 22, 75. 5. V. R. Harwalkar and M. Kalab, Food Microstruct., 1986, 5 , 287. 6. L. G. B. Bremcr, B. H. Bijsterbosch, R. Schrijvers, T. van Vliet, and P. Walstra, Colloids Surf., 1990, 51, 159. 7. L. G. B. Brerner, T. van Vliet, and P. Walstra, J . Chem. Soc., Furuduy Trans. 1, 1989,85, 3359. 8. B. H. Bijsterbosch, M . T. A . Bos, E. Dickinson, J . H. J. van Opheusden. and P. Walstra, Furuduy Discuss., 1995, 101, 51. 9. E. Dickinson, in 'Food Macromolecules and Colloids', ed. E. Dickinson and D. Lorient, Royal Society of Chemistry, Cambridge, 1995, p. 1 . 10. J. A. Lucey, K. Grolle, T. van Vliet, T. Geurts, and P. Walstra, submitted for publication.
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11. H. J. M. van Dijk and P. Walstra, Neth. Milk Dairy J . , 1986, 40,3. 12. J. A. Lucey, T. van W e t , T. Geurts, and P.Walstra, submitted for publication. 13. P.Walstra and R. Jenness, ‘Dairy Chemistry and Physics’, Wiley, New York, 1984. 14. M. Arshad, M. Paulsson, and P. Dejmek, J . Dairy Sci., 1993,76,3310. 15. T. van Vliet and P. Walstra, in ‘Food Colloids’, ed. R. D. Bee, P. Richmond and J. Mingins, Royal Society of Chemistry, Cambridge, 1989, p. 206. 16. H. J. M. van Dijk, PhD Thesis, Wageningen Agricultural University, Netherlands, 1982.