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Acidified Gel Interface
PII : S0958-6946(98)00125-3
Int. Dairy Journal 8 (1998) 801—806 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/99/$ — see front matter
Formation of a Protein Aggregate Layer at a Milk/Acidified Gel Interface Franyoise Warina*, Alexandre Voirin a, Marie Paulsson b and Petr Dejmek b ? CIRDC, Groupe DANONE, 15 avenue Galile& e, 92 350 Le Plessis Robinson, France @ Chemical Center, University of Lund, P.O. Box 124, 221 00 Lund, Sweden (Received 18 February 1998; accepted 2 November 1998) ABSTRACT In bilayer systems consisting of acidified sweetened agar gels and milk, mass transfer between the two layers led to the formation of a milk protein aggregate layer at the boundary, so called ‘hard layer’. The aim of this work was to understand main mechanisms involved in the formation of that ‘hard layer’. The kinetics of growth of the hard layer was followed using an experimetal set up described here, and in situ pH profile was determined by a microelectrode. The boundary of the hard layer with milk was found to coincide with pH 4.9. Increased acid concentration in the agar gel and preacidication of the milk increased the rate of formation of the ‘hard layer’. Increased sucrose concentration in the agar gel increased the rate of formation at longer contact times. This effect of sucrose was explained by a modification of water activity in the milk phase brought by mutual sucrose—water migration. It was concluded that the hard layer was induced by a pH drop of milk in contact of acidifed agar gels, below the gelation pH of milk in the boundary region. That pH drop was caused by acid and sugar migration from the agar gel into the milk phase. 1999 Elsevier Science Ltd. All rights reserved Keywords: bilayer products; aggregation; migration; pH microelectrode
INTRODUCTION
that this ‘skin’ became drier and harder as sugars migrated out from the fruit phase. They however did not study further the mechanisms involved in its formation. We observed similar phenomena in bilayer set yoghurt/ fruit preparation products, when milk fermentation occured in direct contact with the fruit layer, and no milk stabilizers were used. A ‘sandy’ dairy layer more compact than yoghurt appeared between the two layers, on the top of the fruit layer. This compact layer will be called ‘hard layer’ hereafter. When milk is slowly acidified, for example during fermentation of lactose to lactic acid by bacterial cultures, smooth casein gels without syneresis are formed. Faster acidification would however lead to coarser, less continuous networks, dense clusters of aggregated particles being formed (Lucey et al., 1997). A fast acidification of milk in contact with the fruit phase could therefore explain the formation of the sandy hard layer in set yoghurt/fruit preparation products. The aim of this work was to understand the main mechanisms involved in the formation of the hard layer. For this purpose, the influence of sugar and acid contents of the fruit phase on the formation of the hard layer was studied as well as the influence of the initial milk pH. Because food systems are very complex, a physical model that mimics the behavior of bilayer yoghurt/fruit preparation products was used. For simplification, and because the yoghurt phase can be considered as a milk phase in the early stages of contact with the fruit preparation, milk fermentation was omitted and the product was modelled by milk on top of agar gels. The gels contained different initial sucrose and citric acid concentrations, and two different initial milk pH (6.7 and 5.6) were chosen.
In some bilayer dairy products, a dairy layer is deposited on the top of and in direct contact with a fruit gel. Main components of fruit preparations are water, sugar (30—60% (w/w)), acids (pH 3—4), colorants and stabilizers, whereas milk contains proteins, fat, and much less sugar (about 5% (w/w)) and acids (pH 6.6). Because of differences in composition and concentration of components in fruit preparations and milk, chemical potential gradients exist between the two layers, which induce diffusional mass transfer. For example, in a bilayer yoghurt/fruit preparation product studied by La¨ngerer et al. (1980), the total sugars content (°Brix) of the fruit phase dropped from 70°B down to 50°B in 24 h. The osmotic pressure gradient caused by differences in solute concentrations between the fruit and the yoghurt phase induced exchanges of sugars and water. In bilayer sweet agar gel/milk model systems, the mutual disaccharide/ water migration was well described by Fickian diffusion with separated diffusivities in the two phases (Warin et al., 1997). Apparent disaccharide diffusivity into milk was only slightly affected by the initial sucrose content of the agar gel. In bilayer yoghurt/fruit preparation products, La¨ngerer et al. (1980) also mentioned the formation of a ‘skin’ on the fruit phase, during fermentation, caused by the local precipitation of casein in milk that was in contact with the fruit preparation. These authors also reported
*Corresponding author. Tel.:#33 1 41 07 85 08; Fax:#33 1 41 07 84 99; E-mail: [email protected] 801
802
F. Warin et al.
MATERIALS AND METHODS Materials In all experiments, 1.5% fat and 3.5% protein pasteurised commercial milk was used. 0.02% (w/w) sodium azide (Merck, Darmstadt, Germany) was added to avoid microbiological growth. Milk was heat treated at 85°C for 5 min in a water bath, and then stored at 4°C. It was warmed up to room temperature before use. Agar gels Agar gels were prepared by adding 1.5% (w/w) agar (Becton Dickinson, Cockeyville, USA) to sucrose solutions (SSA Sockerbolaget, Ka¨velinge, Sweden). These mixtures were then stirred to disperse agar particles, and heated for 15 min in a boiling water bath. Different amounts of a 30% (w/w) citric acid solution (monohydrate, Merck, Darmstadt, Germany) were added to reach final concentrations varying between 0.25 and 1.54% (w/w). Agar solutions were cooled down to 45°C before citric acid addition in order to obtain a firm gel at room temperature. Final sucrose concentrations varied between 5 and 60% (w/w). For brevity, agar gels were denoted G, followed by sucrose and citric acid contents. For example, an agar gel containing 40% sucrose and 0.5% citric acid was denoted G40-0.5. The pH values of the sweet acidified agar gels before addition of milk are given in Table 1.
Fig. 1. Experimental set up for measuring ‘hard layer’ growth on the top of agar gels.
slippery smooth surface of the agar gel, and it was weighed (see Fig. 1).
Kinetic study Hard layer analysis 6 mL agar solutions were poured at 40°C into 20 mL plastic syringes (Terumo, Leuven, Belgium) with the needle end cut off. Those syringes (21 mm in internal diameter and 60 mm long) provided columns of uniform cross section. Gels were allowed to set at room temperature. Around 10 mL milk was then poured in and left in contact with the gels between 0 and 30 h at room temperature (20—24°C). Milk was poured out after different contact times. The hard layer laid on the gel and stuck to the syringe wall. Using the plunger, remaining syringe contents were then pushed out. The hard layer was easily collected, as it separated easily from the
Protein content of the hard layer was measured according to the Kjeldahl method (FIL 20A, 1986) (Bu¨chi equipment, Flawil, Switzerland). Fat content was measured according to the butyrometric method GERBER ISO 2246, 1976. Total solid content, and thus water content, was measured by drying samples under low pressure. Hard layer samples were weighed to within plus or minus 0.01%, dried for 24 h at 70°C at absolute pressures less than 1.5 kPa, cooled down to room temperature under dry atmosphere, and reweighed. Preliminary tests showed that 24 h was adequate to reach
Table 1. Initial pH of the Agar Gels (Denoted G Sucrose—Citric Acid Content (%)). Disaccharide and Protein Contents in the Hard Layer Formed in Milk in Contact with those Gels at Room Temperature, and its Density Calculated from Eqn (1) Bilayer system
Initial pH of the agar gel
Disaccharide content of the hard layer (%) (w/w)
Protein content of the hard layer (%) (w/w)
Density
G5-0.5/milk G10-0.5/milk G20-0.5/milk G40-0.5/milk G60-0.5/milk G40-0.25/milk G40-0.3/milk G40-0.58/milk G40-0.72/milk G40-1/milk G40-1.54/milk G40-0.5/preacidified milk
2.3 2.3 2.3 2.3 2.2 2.5 2.4 2.2 2.2 2.1 2.0 2.3
6.1$0.6 8.8$0.7 12.8$0.7 21.9$0.4 27.6$1 — 22$1.5 21.6$0.4 21.1$0.6 21.1$0.8 16.3$1.2 17.4$0.3
5.2$0.3 5.6$0.4 5.7$0.3 5.6$0.1 4.9$0.4 — 5.6$0.5 4.9$0.2 4.5$0.3 4.5$0.2 4.2$0.2 4.6$0.1
1.04 1.05 1.07 1.12 1.15 — 1.12 1.11 1.11 1.11 1.08 1.1
Protein aggregation in bilayer systems
constant weight. All analyses were duplicated. The thickness of the hard layer was derived from its weight and density. To calculate the density of a hard layer, the average disaccharide (lactose plus sucrose) concentration of the layer was estimated from total solids by substracting protein and fat contents. The contribution of minerals to total solids was considered as negligible, milk containing less than 1% (w/w) salts. The density of a hard layer was calculated from eqn (1) (Walstra and Jenness, 1984), considering only the contribution of water, fat, proteins and disaccharides to the density.
m 1 " V (1) o o V where o was the density of the hard layer, m the mass V fraction of component x, and o its apparent density in V the hard layer. Taking arbitrarily for o the density of pure water (998.2 kg ) m\), o was about 918, o about 1400 and o about 1780 (Walstra and Jenness, 1984). o was taken equal to o . pH profiles pH profiles were determined in our bilayer systems by measuring pH at different distances from the interface, starting from the surface. A microelectrode was driven step by step from the surface into the systems. A micrometric screw was used to make steps of 500 km with a precision of 10 km. After each experiment, pH at the agar gel interface was measured by pouring out the dairy phase and plunging the microelectrode 20 km below the surface of the gel. The position of the interface was determined from that pH. This procedure allowed to calculate the position of the tip from the interface with a precision better than 200 km. Microelectrodes 1 km in diameter were made at department of Physiology and Neuroscience, University of Lund, Sweden (Ammann, 1986). These microelectrodes allowed pH measurement in volumes smaller than 10 km\. Each microelectrode was calibrated before and after use with pH 7, 6, 4 and 2 buffer solutions. Milk preacidification
803
RESULTS The formation of the hard layer was studied in three different types of model systems. First, milk was deposited on top of agar gels varying in sucrose content, with a constant citric acid content. Next, milk was deposited on top of agar gels varying in citric acid content, with a constant sucrose content. Finally, milk was preacidified with GDL to a stable pH value of 5.6, before contact with agar gels containing 40% sucrose and 0.5% citric acid. Composition of the hard layer In all bilayer systems studied, fat content of the hard layer was constant with time, did not depend on the system, and was equal to the initial fat content of milk (1.5%). In each bilayer system, disaccharide and protein contents of the hard layer were constant with time, eventhough the thickness of the layer increased. The disaccharide and protein contents of the hard layer however depended on the system considered, as shown in Table 1. The density of the hard layer was calculated from its composition (see Table 1). Influence of sucrose The thickness of the hard layer formed in milk in contact with agar gels initially containing 0.5% citric acid and different sucrose amounts was plotted against time in Fig. 2. During the first 2 h of contact, the initial sucrose concentration of the agar gel did not influence the kinetics of growth of the layer. Beyond 2 h, the hard layer did not grow further in G5-0.5/milk, whereas it did in the other systems. The layer grew faster and was thicker for higher initial sucrose concentrations in the agar gel. Influence of citric acid The thickness of the hard layer formed in milk in contact with agar gels initially containing 40% sucrose and different citric acid amounts was plotted against square root of time (Fig. 3). Below a critical initial citric acid content in the agar gel (0.272% citric acid in gels
When needed, milk was preacidified with glucono dlactone (GDL) (Roquette Fre`res, Lestrem, France) to slowly acidify milk without protein precipitation. The kinetic of pH decrease and the final pH reached depended on temperature and the amount of GDL added. To reach a final pH of 5.6, milk was acidified with 0.5% (w/w) GDL and kept at room temperature for 24 h (Arshad, 1991). The final pH values could be considered as stable and reproducible after 20 h at 20°C (Banon and Hardy, 1991). Milk acidification by citric acid To measure the impact of added sucrose on the citric acid titration curve of milk, different amounts of sucrose were added to 25 mL milk samples. The samples were then acidified by adding known amounts of citric acid under vigorous stirring and pH was recorded. Monohydrated citric acid additions between 0 and 0.16 g covered a pH range from 6.6 to 4.3 in milk. This protocol allowed milk acidification without dilution.
Fig. 2. Effect of initial sucrose content of agar gels on the kinetics of growth of the hard layer in G5-0.5/milk (䊏), G100.5/milk (;), G20-0.5/milk (䉱), G40-0.5/milk (#) and G600.5/milk (E) bilayer systems at room temperature. Notation: G sucrose—citric acid content.
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F. Warin et al.
Fig. 3. Effect of initial citric acid content of agar gels on the kinetics of growth of the hard layer in G40-0.25/milk (䉬), G400.3/milk (䊏), G40-0.5/milk (#), G40-0.58/milk (;), G400.74/milk (䉱), G40-1/milk (䊊) and G40-1.54/milk (E) bilayer systems at room temperature. Notation: G sucrose—citric acid content.
Fig. 5. pH profile in the bilayer system G40-0.5/milk after 2 min (E), 10 min (䊐), 90 min (;), 270 min (䉬) and 22 h (*) contact at room temperature. d: distance from the interface between agar gel and milk. 䉱: calculated thickness (see text). Notation: G sucrose—citric acid content.
Fig. 4. Effect of milk initial pH on the kinetics of growth of the hard layer in G40-0.5/milk bilayer systems at room temperature. pH 6.6 (#) and pH 5.6 (E). Notation: G sucrose—citric acid content.
containing 40% sucrose), the hard layer was not formed. Above this critical concentration, the hard layer grew faster for higher initial citric acid contents. Furthermore, at least during the first 24 h of contact, the thickness of the layer was proportional to the square root of time. So, the kinetic of growth of the hard layer followed a scaling law characteristic of diffusional phenomena (Crank, 1975).
Fig. 6. pH profile in the bilayer system G60-0.5/milk after 135 min (䊐) and 1350 min (*) contact at room temperature. d: distance from the interface between agar gel and milk. 䉱: calculated thickness (see text). Notation: G sucrose—citric acid content.
Influence of milk initial pH The thickness of the hard layer formed in the bilayer G40-0.5/preacidified milk was also proportional to the square root of time (Fig. 4). For comparison, the thickness of the hard layer formed in G40-0.5/milk was replotted on the same figure. The hard layer grew about 4 times faster when milk was preacidified. This result confirmed the importance of milk pH, and thus acid migration, in the formation of the hard layer. pH profiles pH profiles were measured in G40-0.5/milk, G600.5/milk and G40-0.5/preacidified milk bilayer systems after different contact times. Migration of protons from
Fig. 7. pH profile in the bilayer system G40-0.5/preacidified milk (pH 5.6) after 30 min (E), 2 h (䊐), 4 h (#), contact at room temperature. d: distance from the interface between agar gel and milk. 䉱: calculated thickness (see text). Notation: G sucrose— citric acid content.
Protein aggregation in bilayer systems
805
the agar gel into the milk phase led to a pH gradient in the systems, as shown in Figs 5—7. As expected, the pH gradient appeared initially in a narrow area, which widened with time. In each bilayer system, the pH at the interface remained constant during the experiments, equal to 4.3, 4 and 3.8 in G40-0.5/milk, G60-0.5/milk and G40-0.5/preacidified milk, respectively. The thickness of the hard layer previously measured in G40-0.5/milk, G60-0.5/milk and G40-0.5/preacidified milk after identical contact times was plotted (䉱 symbols) on the same graphs. In the three bilayer systems, the hard layer appeared in the region of milk where pH was below 4.9, on the top of the agar gel. Milk pH was thus proved to be the critical parameter for the formation of the hard layer. DISCUSSION The disaccharide content of the hard layer increased when the initial sucrose content of the agar gel increased, but slightly depended on the initial citric acid content. This was in agreement with previous results (Warin et al., 1997) that showed a Fickian migration of disaccharides in identical bilayer systems. The osmotic pressure gradient in G5-0.5/milk was negligible, so that water transfers did not occur. The osmotic pressure gradient did not significantly depend on the initial citric acid concentration of the agar gel. The difference of 6% in disaccharide content between the hard layer formed in G40-0.3/milk and in G40-1.54/milk was due to faster growth of the layer for higher initial citric acid content. As initial sucrose content of the gel was identical in the two systems, disaccharide concentration profiles were also identical. The average concentration was therefore lower in the thickest layer, formed in G40-1.54/milk. For the same reason, the hard layer disaccharide content was higher in G40-0.5/milk than in G40-0.5/preacidified milk, the hard layer being thicker in that last system. In our experimental conditions, the hard layer appeared systematically in the region of milk where pH was below 4.9. Protein particles in milk coalesce to form a gel network at room temperature at pH 4.8—4.7 (Gastaldi et al., 1996). In heated milk, gelation occurs at a slightly higher pH (Heertje et al., 1985). In the systems studied here, the hard layer was formed when pH was close to the gelation pH of milk. The uncontrolled and rapid pH drop in milk due to proton migration from the agar gel into the milk phase caused the acid precipitation of milk proteins, and thus the formation of a protein network in the boundary region. When milk had initially been preacidified, pH at the interface between agar gel and milk was lower (3.8 in G40-0.5/preacidified milk against 4.3 in G40-0.5/milk) and the critical pH for the hard layer growth (4.9) was reached faster. The hard layer grew therefore faster. As shown in Fig. 2, the initial sucrose concentration of the agar gel had an effect on the kinetics of formation of the hard layer after 2 h of contact. Skim milk osmolality is 326 mosmol/L (Neville and Jensen, 1995). For sucrose concentration of 10% or more in the gel, the osmotic pressure gradient between milk and gel phase drove water out of milk. Migration of sucrose in milk and water out of milk reduced water activity in milk, which increased ionic strength and further decreased pH (Grufferty and Fox, 1985). The presence of sucrose in milk modified its titration curve by citric acid, as shown on
Fig. 8. Effect of sucrose content of milk on its titration curve by citric acid. Sucrose content: 0% (#), 10% (䉬), and 20% (E).
Fig. 8. Therefore, migration of sucrose lowered milk buffering capacity, presumably by modifying dissociation equilibria and ion activities. Less citric acid was therefore needed to reduce milk pH when sucrose had migrated in milk. pH at the interface between agar gel and milk was lower (4.0 in G60-0.5/milk against 4.3 in G40-0.5/milk) and the critical pH for the hard layer growth (4.9) was reached faster. The hard layer grew therefore faster. In diluted system, proton diffusivity is about 20 times higher than sucrose diffusivity (90;10\ cm ) s\ for protons (Reid et al., 1977) against 5.2;10\ cm ) s\ for sucrose (Handbook of Chemistry and Physics, 1990)). Therefore, in the bilayer systems studied here, acid migration from gel phase into milk phase may have occured faster than sucrose migration. For short contact times, pH drop in milk was due to acid migration, and the initial sucrose content of the gel did not influence the kinetics of formation of the hard layer. For longer contact times, sucrose and water were exchanged between the two phases, which increased milk sucrose content in the boundary region, while decreasing water content. This mass transfer induced an increase in ion activities and a decrease in milk buffering capacity, contributing to further decrease pH in milk. This way, the mutual water/ sucrose migration increased the growth of the hard layer. ACKNOWLEDGEMENTS We thank Kristina Borglid and Pr Wolfgang Grampp, from the Department of Physiology and Neuroscience, University of Lund, for the preparation of microelectrodes and technical assistance in the measurement of pH profiles. REFERENCES Ammann, D. (1986) Ion-selective microelectrodes. Principles, design and applications. Springer, Berlin. Arshad, M. (1986) Breakdown of acid casein gel. Masters thesis, Department of Food Engineering, University of Lund, Sweden. Banon, S. and Hardy, J. (1991) Study of acid milk coagulation by an optical method using light reflection. Journal of Dairy Research 58, 75—84. Crank (1975) ¹he mathematics of diffusion, 2nd edn. Oxford University Press, London.
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Gastaldi, E., Lagaude, A. and Tarodo de la Fuente, B. (1996) Micellar transition state in casein between pH 5.5 and 5.0. Journal of Food Science 61, 59—64. Grufferty, M. B. and Fox, P. F. (1985) Effect of added NaCl on some physicochemical properties of milk. Irish Journal of Food Science ¹echnology 9, 1—9. Lide, D. R. (1990) Handbook of Chemistry and Physics, 71st edn. CRC Press, Boca Raton, FL. Heertje, I., Visser, J. and Smits, P. (1985) Structure formation in acid milk gels. Food Microstructure 4, 267—277. La¨ngerer, J., Werk, Z. Z. and Wild, R. (1980) Diffusionsvorga¨nge bei Zweischichtprodukten und ihre Bedeutung fu¨r die Produktqualita¨t. Die Molkerei-Zeitung ¼elt der Milch 34, 1585—1589.
Lucey, J. A., Van Vliet, T., Grolle, K., Geurts, T. and Walstra, P. (1997) Properties of acid casein gels made by acidification with glucono-d-lactone. 1. Rheological properties. International Dairy Journal 7, 381—388. Neville, M. C. and Jensen, R. G. (1995) Handbook of Milk Composition. Academic Press, San Diego, CA, p. 82. Reid, R. C., Prausnitz, J. M. and Sherwood, T. K. (1977) In ¹he Properties of Gases and ¸iquids, 3rd edn. Mc Graw Hill, New York, p. 594. Walstra, P. and Jenness, R. (1984) Dairy Chemistry and Physics. Wiley-Interscience, New York, p. 187. Warin, F., Gekas, V., Voirin, A. and Dejmek, P. (1997) Sugar diffusivity in agar gel/milk bilayer systems. Journal of Food Science 62(3), 454—456.
Int. Dairy Journal 8 (1998) 801—806 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/99/$ — see front matter
Formation of a Protein Aggregate Layer at a Milk/Acidified Gel Interface Franyoise Warina*, Alexandre Voirin a, Marie Paulsson b and Petr Dejmek b ? CIRDC, Groupe DANONE, 15 avenue Galile& e, 92 350 Le Plessis Robinson, France @ Chemical Center, University of Lund, P.O. Box 124, 221 00 Lund, Sweden (Received 18 February 1998; accepted 2 November 1998) ABSTRACT In bilayer systems consisting of acidified sweetened agar gels and milk, mass transfer between the two layers led to the formation of a milk protein aggregate layer at the boundary, so called ‘hard layer’. The aim of this work was to understand main mechanisms involved in the formation of that ‘hard layer’. The kinetics of growth of the hard layer was followed using an experimetal set up described here, and in situ pH profile was determined by a microelectrode. The boundary of the hard layer with milk was found to coincide with pH 4.9. Increased acid concentration in the agar gel and preacidication of the milk increased the rate of formation of the ‘hard layer’. Increased sucrose concentration in the agar gel increased the rate of formation at longer contact times. This effect of sucrose was explained by a modification of water activity in the milk phase brought by mutual sucrose—water migration. It was concluded that the hard layer was induced by a pH drop of milk in contact of acidifed agar gels, below the gelation pH of milk in the boundary region. That pH drop was caused by acid and sugar migration from the agar gel into the milk phase. 1999 Elsevier Science Ltd. All rights reserved Keywords: bilayer products; aggregation; migration; pH microelectrode
INTRODUCTION
that this ‘skin’ became drier and harder as sugars migrated out from the fruit phase. They however did not study further the mechanisms involved in its formation. We observed similar phenomena in bilayer set yoghurt/ fruit preparation products, when milk fermentation occured in direct contact with the fruit layer, and no milk stabilizers were used. A ‘sandy’ dairy layer more compact than yoghurt appeared between the two layers, on the top of the fruit layer. This compact layer will be called ‘hard layer’ hereafter. When milk is slowly acidified, for example during fermentation of lactose to lactic acid by bacterial cultures, smooth casein gels without syneresis are formed. Faster acidification would however lead to coarser, less continuous networks, dense clusters of aggregated particles being formed (Lucey et al., 1997). A fast acidification of milk in contact with the fruit phase could therefore explain the formation of the sandy hard layer in set yoghurt/fruit preparation products. The aim of this work was to understand the main mechanisms involved in the formation of the hard layer. For this purpose, the influence of sugar and acid contents of the fruit phase on the formation of the hard layer was studied as well as the influence of the initial milk pH. Because food systems are very complex, a physical model that mimics the behavior of bilayer yoghurt/fruit preparation products was used. For simplification, and because the yoghurt phase can be considered as a milk phase in the early stages of contact with the fruit preparation, milk fermentation was omitted and the product was modelled by milk on top of agar gels. The gels contained different initial sucrose and citric acid concentrations, and two different initial milk pH (6.7 and 5.6) were chosen.
In some bilayer dairy products, a dairy layer is deposited on the top of and in direct contact with a fruit gel. Main components of fruit preparations are water, sugar (30—60% (w/w)), acids (pH 3—4), colorants and stabilizers, whereas milk contains proteins, fat, and much less sugar (about 5% (w/w)) and acids (pH 6.6). Because of differences in composition and concentration of components in fruit preparations and milk, chemical potential gradients exist between the two layers, which induce diffusional mass transfer. For example, in a bilayer yoghurt/fruit preparation product studied by La¨ngerer et al. (1980), the total sugars content (°Brix) of the fruit phase dropped from 70°B down to 50°B in 24 h. The osmotic pressure gradient caused by differences in solute concentrations between the fruit and the yoghurt phase induced exchanges of sugars and water. In bilayer sweet agar gel/milk model systems, the mutual disaccharide/ water migration was well described by Fickian diffusion with separated diffusivities in the two phases (Warin et al., 1997). Apparent disaccharide diffusivity into milk was only slightly affected by the initial sucrose content of the agar gel. In bilayer yoghurt/fruit preparation products, La¨ngerer et al. (1980) also mentioned the formation of a ‘skin’ on the fruit phase, during fermentation, caused by the local precipitation of casein in milk that was in contact with the fruit preparation. These authors also reported
*Corresponding author. Tel.:#33 1 41 07 85 08; Fax:#33 1 41 07 84 99; E-mail: [email protected] 801
802
F. Warin et al.
MATERIALS AND METHODS Materials In all experiments, 1.5% fat and 3.5% protein pasteurised commercial milk was used. 0.02% (w/w) sodium azide (Merck, Darmstadt, Germany) was added to avoid microbiological growth. Milk was heat treated at 85°C for 5 min in a water bath, and then stored at 4°C. It was warmed up to room temperature before use. Agar gels Agar gels were prepared by adding 1.5% (w/w) agar (Becton Dickinson, Cockeyville, USA) to sucrose solutions (SSA Sockerbolaget, Ka¨velinge, Sweden). These mixtures were then stirred to disperse agar particles, and heated for 15 min in a boiling water bath. Different amounts of a 30% (w/w) citric acid solution (monohydrate, Merck, Darmstadt, Germany) were added to reach final concentrations varying between 0.25 and 1.54% (w/w). Agar solutions were cooled down to 45°C before citric acid addition in order to obtain a firm gel at room temperature. Final sucrose concentrations varied between 5 and 60% (w/w). For brevity, agar gels were denoted G, followed by sucrose and citric acid contents. For example, an agar gel containing 40% sucrose and 0.5% citric acid was denoted G40-0.5. The pH values of the sweet acidified agar gels before addition of milk are given in Table 1.
Fig. 1. Experimental set up for measuring ‘hard layer’ growth on the top of agar gels.
slippery smooth surface of the agar gel, and it was weighed (see Fig. 1).
Kinetic study Hard layer analysis 6 mL agar solutions were poured at 40°C into 20 mL plastic syringes (Terumo, Leuven, Belgium) with the needle end cut off. Those syringes (21 mm in internal diameter and 60 mm long) provided columns of uniform cross section. Gels were allowed to set at room temperature. Around 10 mL milk was then poured in and left in contact with the gels between 0 and 30 h at room temperature (20—24°C). Milk was poured out after different contact times. The hard layer laid on the gel and stuck to the syringe wall. Using the plunger, remaining syringe contents were then pushed out. The hard layer was easily collected, as it separated easily from the
Protein content of the hard layer was measured according to the Kjeldahl method (FIL 20A, 1986) (Bu¨chi equipment, Flawil, Switzerland). Fat content was measured according to the butyrometric method GERBER ISO 2246, 1976. Total solid content, and thus water content, was measured by drying samples under low pressure. Hard layer samples were weighed to within plus or minus 0.01%, dried for 24 h at 70°C at absolute pressures less than 1.5 kPa, cooled down to room temperature under dry atmosphere, and reweighed. Preliminary tests showed that 24 h was adequate to reach
Table 1. Initial pH of the Agar Gels (Denoted G Sucrose—Citric Acid Content (%)). Disaccharide and Protein Contents in the Hard Layer Formed in Milk in Contact with those Gels at Room Temperature, and its Density Calculated from Eqn (1) Bilayer system
Initial pH of the agar gel
Disaccharide content of the hard layer (%) (w/w)
Protein content of the hard layer (%) (w/w)
Density
G5-0.5/milk G10-0.5/milk G20-0.5/milk G40-0.5/milk G60-0.5/milk G40-0.25/milk G40-0.3/milk G40-0.58/milk G40-0.72/milk G40-1/milk G40-1.54/milk G40-0.5/preacidified milk
2.3 2.3 2.3 2.3 2.2 2.5 2.4 2.2 2.2 2.1 2.0 2.3
6.1$0.6 8.8$0.7 12.8$0.7 21.9$0.4 27.6$1 — 22$1.5 21.6$0.4 21.1$0.6 21.1$0.8 16.3$1.2 17.4$0.3
5.2$0.3 5.6$0.4 5.7$0.3 5.6$0.1 4.9$0.4 — 5.6$0.5 4.9$0.2 4.5$0.3 4.5$0.2 4.2$0.2 4.6$0.1
1.04 1.05 1.07 1.12 1.15 — 1.12 1.11 1.11 1.11 1.08 1.1
Protein aggregation in bilayer systems
constant weight. All analyses were duplicated. The thickness of the hard layer was derived from its weight and density. To calculate the density of a hard layer, the average disaccharide (lactose plus sucrose) concentration of the layer was estimated from total solids by substracting protein and fat contents. The contribution of minerals to total solids was considered as negligible, milk containing less than 1% (w/w) salts. The density of a hard layer was calculated from eqn (1) (Walstra and Jenness, 1984), considering only the contribution of water, fat, proteins and disaccharides to the density.
m 1 " V (1) o o V where o was the density of the hard layer, m the mass V fraction of component x, and o its apparent density in V the hard layer. Taking arbitrarily for o the density of pure water (998.2 kg ) m\), o was about 918, o about 1400 and o about 1780 (Walstra and Jenness, 1984). o was taken equal to o . pH profiles pH profiles were determined in our bilayer systems by measuring pH at different distances from the interface, starting from the surface. A microelectrode was driven step by step from the surface into the systems. A micrometric screw was used to make steps of 500 km with a precision of 10 km. After each experiment, pH at the agar gel interface was measured by pouring out the dairy phase and plunging the microelectrode 20 km below the surface of the gel. The position of the interface was determined from that pH. This procedure allowed to calculate the position of the tip from the interface with a precision better than 200 km. Microelectrodes 1 km in diameter were made at department of Physiology and Neuroscience, University of Lund, Sweden (Ammann, 1986). These microelectrodes allowed pH measurement in volumes smaller than 10 km\. Each microelectrode was calibrated before and after use with pH 7, 6, 4 and 2 buffer solutions. Milk preacidification
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RESULTS The formation of the hard layer was studied in three different types of model systems. First, milk was deposited on top of agar gels varying in sucrose content, with a constant citric acid content. Next, milk was deposited on top of agar gels varying in citric acid content, with a constant sucrose content. Finally, milk was preacidified with GDL to a stable pH value of 5.6, before contact with agar gels containing 40% sucrose and 0.5% citric acid. Composition of the hard layer In all bilayer systems studied, fat content of the hard layer was constant with time, did not depend on the system, and was equal to the initial fat content of milk (1.5%). In each bilayer system, disaccharide and protein contents of the hard layer were constant with time, eventhough the thickness of the layer increased. The disaccharide and protein contents of the hard layer however depended on the system considered, as shown in Table 1. The density of the hard layer was calculated from its composition (see Table 1). Influence of sucrose The thickness of the hard layer formed in milk in contact with agar gels initially containing 0.5% citric acid and different sucrose amounts was plotted against time in Fig. 2. During the first 2 h of contact, the initial sucrose concentration of the agar gel did not influence the kinetics of growth of the layer. Beyond 2 h, the hard layer did not grow further in G5-0.5/milk, whereas it did in the other systems. The layer grew faster and was thicker for higher initial sucrose concentrations in the agar gel. Influence of citric acid The thickness of the hard layer formed in milk in contact with agar gels initially containing 40% sucrose and different citric acid amounts was plotted against square root of time (Fig. 3). Below a critical initial citric acid content in the agar gel (0.272% citric acid in gels
When needed, milk was preacidified with glucono dlactone (GDL) (Roquette Fre`res, Lestrem, France) to slowly acidify milk without protein precipitation. The kinetic of pH decrease and the final pH reached depended on temperature and the amount of GDL added. To reach a final pH of 5.6, milk was acidified with 0.5% (w/w) GDL and kept at room temperature for 24 h (Arshad, 1991). The final pH values could be considered as stable and reproducible after 20 h at 20°C (Banon and Hardy, 1991). Milk acidification by citric acid To measure the impact of added sucrose on the citric acid titration curve of milk, different amounts of sucrose were added to 25 mL milk samples. The samples were then acidified by adding known amounts of citric acid under vigorous stirring and pH was recorded. Monohydrated citric acid additions between 0 and 0.16 g covered a pH range from 6.6 to 4.3 in milk. This protocol allowed milk acidification without dilution.
Fig. 2. Effect of initial sucrose content of agar gels on the kinetics of growth of the hard layer in G5-0.5/milk (䊏), G100.5/milk (;), G20-0.5/milk (䉱), G40-0.5/milk (#) and G600.5/milk (E) bilayer systems at room temperature. Notation: G sucrose—citric acid content.
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Fig. 3. Effect of initial citric acid content of agar gels on the kinetics of growth of the hard layer in G40-0.25/milk (䉬), G400.3/milk (䊏), G40-0.5/milk (#), G40-0.58/milk (;), G400.74/milk (䉱), G40-1/milk (䊊) and G40-1.54/milk (E) bilayer systems at room temperature. Notation: G sucrose—citric acid content.
Fig. 5. pH profile in the bilayer system G40-0.5/milk after 2 min (E), 10 min (䊐), 90 min (;), 270 min (䉬) and 22 h (*) contact at room temperature. d: distance from the interface between agar gel and milk. 䉱: calculated thickness (see text). Notation: G sucrose—citric acid content.
Fig. 4. Effect of milk initial pH on the kinetics of growth of the hard layer in G40-0.5/milk bilayer systems at room temperature. pH 6.6 (#) and pH 5.6 (E). Notation: G sucrose—citric acid content.
containing 40% sucrose), the hard layer was not formed. Above this critical concentration, the hard layer grew faster for higher initial citric acid contents. Furthermore, at least during the first 24 h of contact, the thickness of the layer was proportional to the square root of time. So, the kinetic of growth of the hard layer followed a scaling law characteristic of diffusional phenomena (Crank, 1975).
Fig. 6. pH profile in the bilayer system G60-0.5/milk after 135 min (䊐) and 1350 min (*) contact at room temperature. d: distance from the interface between agar gel and milk. 䉱: calculated thickness (see text). Notation: G sucrose—citric acid content.
Influence of milk initial pH The thickness of the hard layer formed in the bilayer G40-0.5/preacidified milk was also proportional to the square root of time (Fig. 4). For comparison, the thickness of the hard layer formed in G40-0.5/milk was replotted on the same figure. The hard layer grew about 4 times faster when milk was preacidified. This result confirmed the importance of milk pH, and thus acid migration, in the formation of the hard layer. pH profiles pH profiles were measured in G40-0.5/milk, G600.5/milk and G40-0.5/preacidified milk bilayer systems after different contact times. Migration of protons from
Fig. 7. pH profile in the bilayer system G40-0.5/preacidified milk (pH 5.6) after 30 min (E), 2 h (䊐), 4 h (#), contact at room temperature. d: distance from the interface between agar gel and milk. 䉱: calculated thickness (see text). Notation: G sucrose— citric acid content.
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the agar gel into the milk phase led to a pH gradient in the systems, as shown in Figs 5—7. As expected, the pH gradient appeared initially in a narrow area, which widened with time. In each bilayer system, the pH at the interface remained constant during the experiments, equal to 4.3, 4 and 3.8 in G40-0.5/milk, G60-0.5/milk and G40-0.5/preacidified milk, respectively. The thickness of the hard layer previously measured in G40-0.5/milk, G60-0.5/milk and G40-0.5/preacidified milk after identical contact times was plotted (䉱 symbols) on the same graphs. In the three bilayer systems, the hard layer appeared in the region of milk where pH was below 4.9, on the top of the agar gel. Milk pH was thus proved to be the critical parameter for the formation of the hard layer. DISCUSSION The disaccharide content of the hard layer increased when the initial sucrose content of the agar gel increased, but slightly depended on the initial citric acid content. This was in agreement with previous results (Warin et al., 1997) that showed a Fickian migration of disaccharides in identical bilayer systems. The osmotic pressure gradient in G5-0.5/milk was negligible, so that water transfers did not occur. The osmotic pressure gradient did not significantly depend on the initial citric acid concentration of the agar gel. The difference of 6% in disaccharide content between the hard layer formed in G40-0.3/milk and in G40-1.54/milk was due to faster growth of the layer for higher initial citric acid content. As initial sucrose content of the gel was identical in the two systems, disaccharide concentration profiles were also identical. The average concentration was therefore lower in the thickest layer, formed in G40-1.54/milk. For the same reason, the hard layer disaccharide content was higher in G40-0.5/milk than in G40-0.5/preacidified milk, the hard layer being thicker in that last system. In our experimental conditions, the hard layer appeared systematically in the region of milk where pH was below 4.9. Protein particles in milk coalesce to form a gel network at room temperature at pH 4.8—4.7 (Gastaldi et al., 1996). In heated milk, gelation occurs at a slightly higher pH (Heertje et al., 1985). In the systems studied here, the hard layer was formed when pH was close to the gelation pH of milk. The uncontrolled and rapid pH drop in milk due to proton migration from the agar gel into the milk phase caused the acid precipitation of milk proteins, and thus the formation of a protein network in the boundary region. When milk had initially been preacidified, pH at the interface between agar gel and milk was lower (3.8 in G40-0.5/preacidified milk against 4.3 in G40-0.5/milk) and the critical pH for the hard layer growth (4.9) was reached faster. The hard layer grew therefore faster. As shown in Fig. 2, the initial sucrose concentration of the agar gel had an effect on the kinetics of formation of the hard layer after 2 h of contact. Skim milk osmolality is 326 mosmol/L (Neville and Jensen, 1995). For sucrose concentration of 10% or more in the gel, the osmotic pressure gradient between milk and gel phase drove water out of milk. Migration of sucrose in milk and water out of milk reduced water activity in milk, which increased ionic strength and further decreased pH (Grufferty and Fox, 1985). The presence of sucrose in milk modified its titration curve by citric acid, as shown on
Fig. 8. Effect of sucrose content of milk on its titration curve by citric acid. Sucrose content: 0% (#), 10% (䉬), and 20% (E).
Fig. 8. Therefore, migration of sucrose lowered milk buffering capacity, presumably by modifying dissociation equilibria and ion activities. Less citric acid was therefore needed to reduce milk pH when sucrose had migrated in milk. pH at the interface between agar gel and milk was lower (4.0 in G60-0.5/milk against 4.3 in G40-0.5/milk) and the critical pH for the hard layer growth (4.9) was reached faster. The hard layer grew therefore faster. In diluted system, proton diffusivity is about 20 times higher than sucrose diffusivity (90;10\ cm ) s\ for protons (Reid et al., 1977) against 5.2;10\ cm ) s\ for sucrose (Handbook of Chemistry and Physics, 1990)). Therefore, in the bilayer systems studied here, acid migration from gel phase into milk phase may have occured faster than sucrose migration. For short contact times, pH drop in milk was due to acid migration, and the initial sucrose content of the gel did not influence the kinetics of formation of the hard layer. For longer contact times, sucrose and water were exchanged between the two phases, which increased milk sucrose content in the boundary region, while decreasing water content. This mass transfer induced an increase in ion activities and a decrease in milk buffering capacity, contributing to further decrease pH in milk. This way, the mutual water/ sucrose migration increased the growth of the hard layer. ACKNOWLEDGEMENTS We thank Kristina Borglid and Pr Wolfgang Grampp, from the Department of Physiology and Neuroscience, University of Lund, for the preparation of microelectrodes and technical assistance in the measurement of pH profiles. REFERENCES Ammann, D. (1986) Ion-selective microelectrodes. Principles, design and applications. Springer, Berlin. Arshad, M. (1986) Breakdown of acid casein gel. Masters thesis, Department of Food Engineering, University of Lund, Sweden. Banon, S. and Hardy, J. (1991) Study of acid milk coagulation by an optical method using light reflection. Journal of Dairy Research 58, 75—84. Crank (1975) ¹he mathematics of diffusion, 2nd edn. Oxford University Press, London.
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Gastaldi, E., Lagaude, A. and Tarodo de la Fuente, B. (1996) Micellar transition state in casein between pH 5.5 and 5.0. Journal of Food Science 61, 59—64. Grufferty, M. B. and Fox, P. F. (1985) Effect of added NaCl on some physicochemical properties of milk. Irish Journal of Food Science ¹echnology 9, 1—9. Lide, D. R. (1990) Handbook of Chemistry and Physics, 71st edn. CRC Press, Boca Raton, FL. Heertje, I., Visser, J. and Smits, P. (1985) Structure formation in acid milk gels. Food Microstructure 4, 267—277. La¨ngerer, J., Werk, Z. Z. and Wild, R. (1980) Diffusionsvorga¨nge bei Zweischichtprodukten und ihre Bedeutung fu¨r die Produktqualita¨t. Die Molkerei-Zeitung ¼elt der Milch 34, 1585—1589.
Lucey, J. A., Van Vliet, T., Grolle, K., Geurts, T. and Walstra, P. (1997) Properties of acid casein gels made by acidification with glucono-d-lactone. 1. Rheological properties. International Dairy Journal 7, 381—388. Neville, M. C. and Jensen, R. G. (1995) Handbook of Milk Composition. Academic Press, San Diego, CA, p. 82. Reid, R. C., Prausnitz, J. M. and Sherwood, T. K. (1977) In ¹he Properties of Gases and ¸iquids, 3rd edn. Mc Graw Hill, New York, p. 594. Walstra, P. and Jenness, R. (1984) Dairy Chemistry and Physics. Wiley-Interscience, New York, p. 187. Warin, F., Gekas, V., Voirin, A. and Dejmek, P. (1997) Sugar diffusivity in agar gel/milk bilayer systems. Journal of Food Science 62(3), 454—456.