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All issues Volume 89 / No 5 (September-October 2009) Dairy Sci. Technol., 89 5 (2009) 501-510 Full HTML
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Dairy Sci. Technol.
Volume 89, Number 5, September-October 2009
Page(s) 501 - 510
DOI https://doi.org/10.1051/dst/2009026
Published online 25 July 2009

© INRA, EDP Sciences, 2009

1. INTRODUCTION

Bovine milk is a complex biological fluid containing protein, emulsified fat, minerals, carbohydrates and water. The casein proteins, much of the calcium and phosphate, and low levels of other minerals, are found as large macromolecular assemblies called casein micelles [17]. The mineral components that are associated with the casein micelles (predominantly calcium and phosphate) are commonly referred to as colloidal calcium phosphate (CCP) and this CCP is in a dynamic equilibrium with the mineral components in the soluble phase. The soluble calcium (Casol) and the soluble inorganic phosphate (Psol) are present in the soluble phase as free ions or are associated with other ions, as has been described in detail [6].

The equilibrium state of the calcium and phosphate in milk is markedly dependent on the temperature, concentration and pH of the milk [6]. Increasing the temperature of milk results in the transfer of the Casol and Psol to the colloidal phase, with a concomitant decrease in milk pH [14, 16]. Most of this decrease in Casol and Psol occurs within the first minutes of heating, with smaller changes on prolonged holding [14, 16]. Under mild heating conditions (below 95 °C/several minutes), the changes in Casol, Psol and milk pH are largely reversible when the temperature of the milk is restored to its initial value [7, 15].

Concentration of milk by evaporation retains all the milk salts, and, as milk is saturated with respect to calcium phosphate, some of the Casol and Psol is transferred to the colloidal phase. However, as the milk pH decreases on increasing milk concentration, and, as increasing ionic strengths reduce the activity coefficients of the Casol and Psol, the concentrations of calcium and phosphate in the soluble phase do increase but this increase is not proportional to the concentration factor of the milk [6, 12].

Milk concentration can be markedly increased when milk is processed to dairy products, and these concentration changes are usually accompanied by increases or decreases in the temperature of the milk [17]. For example, during the manufacture of skim milk powder, the milk can be concentrated from the natural level (about 10% total solids (TS)) to about 50% TS before spray drying, and the temperature during this processing can range from about 4 °C to temperatures above 100 °C depending on the specification of the powder product [17]. Milk can be concentrated up to 2.5 times to produce evaporated liquid milk products, during which the milk temperature can be varied from about 4 °C to over 140 °C depending on the product being produced. There can be problems while processing milk concentrates (e.g. fouling of surfaces, thickening or coagulation) or storing concentrated milks (e.g. age gelation or sedimentation), and the milk minerals, particularly calcium and inorganic phosphate, are often implicated in these issues [17]. Despite this, there have been few studies on the effect of temperature on the Casol, Psol and pH for concentrated skim milk. This study, therefore, examined the effect of milk concentration and temperature on the pH of the milk and the partition of calcium and phosphate between colloidal and soluble phases.

2. MATERIALS AND METHODS

2.1. Milk supply

Skim milk samples of 9.6%, 19.2%, 28.8% and 38.4% TS (w/w) were prepared by dissolving the appropriate quantity of low-heat skim milk powder (Fonterra Co-operative Group Ltd., New Zealand: contains about 34% protein, 4% moisture, 54% carbohydrate, 8% minerals and 0.8% fat) in high purity water. Sodium azide was added to each milk sample at a rate of 0.1 mg·L−1. The milks were allowed to equilibrate at 20 °C for at least 24 h.

2.2. Heat treatment and ultrafiltration permeate collection

A sample of each milk sample was heated from ambient temperature (~ 20 °C) to temperatures ranging from 30 to 80 °C. Heating commenced when the milk sample was pumped from a vessel at room temperature, through the ultrafiltration (UF) unit (10 000 Da (nominal) hollow fibre membrane cartridge and pumping equipment (Amicon, Inc., Beverly, MA, USA)) and a tubular heat-exchange device (at the desired temperature) to a second vessel held in a temperature-controlled water bath set to the desired temperature. Once the required volume of milk had passed through to the second vessel, it was recirculated between the UF unit, the heat-exchange device and the second vessel for the duration of the experiment, thus maintaining the milk at the desired temperature. The heat-up time to the required temperature was < 2 min for all samples. For each sample, a series of UF permeate samples (about 2.5 mL) was collected during a 60-min heating-recirculation period. Not more than about 10% of the total milk volume was extracted as permeate.

2.3. Measurement of pH

The pH of the milk samples was measured across a range of temperatures and holding times using a combination glass electrode coupled with a temperature probe. At each temperature, standard pH buffers were allowed to equilibrate for about 30 min before being used for calibration of the pH meter. The pH shift of buffers due to temperature was taken into account during calibration. Milk samples were pumped through a heat exchanger to a vessel held at the desired temperature in a thermostatically controlled water bath as described in Section 2.2. The pH of these samples was monitored against time and temperature.

2.4. Calcium, phosphate and TS analysis

Calcium in the milk and permeate samples was determined by an inductively coupled plasma emission spectroscopy method [1]. Phosphate was determined as inorganic phosphate using the molybdo-vanadate method [2]. The TS content of the milk samples was determined by drying samples at 102 °C for 5 h [13].

3. RESULTS

3.1. Time-dependent changes in Casol and Psol on heating milk

The results for the time-dependent changes in Casol and Psol levels when milk samples were heated from 20 °C to temperatures in the range from 30 to 80 °C are shown in Figures 1A and 1B for the 9.6% and 28.8% TS milk samples, respectively. Similar general results were observed for the milks at the other concentrations. On heating, there was an initial steep decrease in Casol and Psol levels over the first 5 min of heating, followed by a period where only small further changes in Casol and Psol were observed. The extent of the decrease in the Casol and Psol was greater at higher temperatures. As only small changes in Casol and Psol are observed on heating beyond about 10 min, the Casol and Psol levels obtained after 60 min heating are referred to as the final Casol and final Psol at each particular temperature. Figure 1C shows the effect of temperature on the final Casol and final Psol levels. For each milk concentration, there was an inverse linear relationship between the final Casol or the final Psol level and temperature. The relative effect of temperature on the change in final Casol or final Psol was more pronounced as the milk concentration increased from 9.6% to 28.8% TS, but was similar at milk concentrations of 28.8% and 38.4% TS.

thumbnail Figure 1.

Changes in soluble calcium (i) and soluble phosphate (ii) on heating milk. (A) Changes in soluble calcium or soluble phosphate with heating time for the 9.6% TS milk heated at 20 °C (●), 30 °C (○), 40 °C (▼), 50 °C (▽), 60 °C (■), 70 °C (□) and 80 °C (♦). (B) Changes in soluble calcium or soluble phosphate with heating time for the 28.8% TS milk heated at 20 °C (●), 30 °C (○), 40 °C (▼), 50 °C (▽), 60 °C (■), 70 °C (□) and 80 °C (♦). (C) Changes in final soluble calcium and final soluble phosphate after 60-min heating at a range of temperatures for the 9.6% TS (●), 19.2% TS (○), 28.8% TS (▼) and 38.4% TS (▽) milk samples.

3.2. Relationship between Casol and Psol

The relationship between the final Casol and the final Psol levels is shown in Figure 2A. At each milk solids concentration, there was a linear relationship between the final Casol and the final Psol levels, with the lines displaced to higher Casol and Psol as the milk concentration increased. The changes in final Casol and final Psol with increasing temperature (relative to the sample at 20 °C) can be plotted against each other to give an indication of the composition of the calcium phosphate material that is transferred from the soluble to the colloidal state (Fig. 2B). With the exception of a couple of points (the 80 °C points for the 28.2% and 38.4% TS milks), all the data fell close to a single line with a slope very close to unity (1.02).

thumbnail Figure 2.

Relationships between soluble calcium and soluble phosphate for milks of different concentrations. (A) Relationship between soluble calcium and soluble phosphate for the 9.6% TS (●), 19.2% TS (○), 28.8% TS (▼) and 38.4% TS (▽) milk samples that were heated at temperatures between 20 and 80 °C for 60 min. (B) Relationship between the change in soluble calcium and the change in soluble phosphate (relative to the samples held at 20 °C) for the 9.6% TS (●), 19.2% TS (○), 28.8% TS (▼) and 38.4% TS (▽) milk samples that were heated at temperatures between 20 and 80 °C for 60 min.

3.3. Time-dependent changes in pH on heating milk

The results for the time-dependent changes in pH when milk samples were heated from 20 °C to temperatures in the range from 30 to 80 °C are shown in Figures 3A and 3B for the 9.6% and 28.8% TS milk samples, respectively. Similar results were observed for the milks at the other concentrations. For each milk concentration, the pH decreased rapidly on initial heating followed by a period where only small further changes in pH were observed. For each milk concentration, a greater decrease in pH was observed at higher temperatures. Only small changes in pH were observed on heating beyond 10 min, therefore, the pH at 60-min heating was referred to as the final pH. At any particular milk concentration, the final pH decreased with increasing temperature, and at any particular temperature, the final pH of the milk decreased with increasing milk solids concentration (Fig. 3).

thumbnail Figure 3.

Changes in pH on heating milk. (A) Changes in pH with heating time for the 9.6% TS milk heated at 20 °C (●), 30 °C (○), 40 °C (▼), 50 °C (▽), 60 °C (■), 70 °C (□) and 80 °C (♦). (B) Changes in pH with heating time for the 28.8% TS milk heated at 20 °C (●), 30 °C (○), 40 °C (▼), 50 °C (▽), 60 °C (■), 70 °C (□) and 80 °C (♦). (C) Changes in final pH after 60-min heating at a range of temperatures for the 9.6% TS (●), 19.2% TS (○), 28.8% TS (▼) and 38.4% TS (▽) milk samples.

Figure 3C shows the final pH plotted against temperature. For each milk concentration, the pH decreased essentially linearly with increasing temperature. The observed effect of temperature on the final pH for the 9.6% milk was almost identical to that reported by Chaplin and Lyster [4]. Increasing the milk concentration resulted in a shift in the position of the curve to lower pH; however, the slopes of the curves at all milk concentrations, and therefore the relative effect of temperature on the pH, were very similar.

3.4. Relationship between pH and Casol, and pH and Psol

The relationships between the final pH and the final Casol, and between the final pH and the final Psol are shown in Figure 4. For each milk concentration, there was a strong linear correlation between the final pH and the final Casol or the final Psol, with the curves displaced to higher Casol or Psol as the milk concentration is increased. The relative increase in final Casol or final Psol with increasing pH was more pronounced (i.e. steeper slope) as the milk concentration increased from 9.6% to 28.8% TS, but was similar at milk concentrations of 28.8% and 38.4% TS.

thumbnail Figure 4.

Relationships between pH and soluble calcium or soluble phosphate for milks of different concentrations. (A) Relationship between pH and soluble calcium for the 9.6% TS (●), 19.2% TS (○), 28.8% TS (▼) and 38.4% TS (▽) milk samples that were heated at temperatures between 20 and 80 °C for 60 min. (B) Relationship between pH and soluble phosphate for the 9.6% TS (●), 19.2% TS (○), 28.8% TS (▼) and 38.4% TS (▽) milk samples that were heated at temperatures between 20 and 80 °C for 60 min.

4. DISCUSSION

The results in Figure 1 indicate that, on heating, the new equilibrium position between soluble and colloidal calcium and phosphate is rapidly approached at all milk concentrations. For the 9.6% TS milks, the changes in Casol and Psol with heating time at various temperatures were comparable with those reported by Pouliot et al. [14], and their final Casol and Psol levels were very similar to those obtained in this study (Fig. 1). At any particular temperature, the final Casol or final Psol increased, but this increase was markedly less than the concentration factor of the milk (Figs. 1 and 2). Although milk is saturated with respect to Casol and Psol, the increased ionic strength on concentration of the milk will reduce the ion activity coefficients, and this, coupled with the reduced pH on increasing the milk concentration, will result in higher Casol and Psol concentrations, while maintaining the solubility product relationship between soluble and colloidal calcium and phosphate [6, 12].

Figure 3 shows that the final pH of the milks varied from about pH 6.7 to pH 5.9 depending on the milk concentration and temperature. It may have been expected that this variation in pH would change the nature of the calcium phosphate that is precipitated from solution. However, the results in Figure 2 indicate that the calcium phosphate transferred from the soluble phase during heating milk is similar in composition, regardless of the milk concentration and the temperature to which the milk is heated. This may be explained by the studies of Holt [5, 6] who found that the solubility product of calcium phosphate from milk was independent of pH over the range from pH 6.4 to pH 7.1 and suggested that the solubility product for calcium and phosphate in milk is dictated by the stoichiometry of the equilibrium calcium phosphate phase.

The observation that the heat-precipitated calcium phosphate has a calcium to phosphate ratio close to 1 suggests that this material may have a similar composition to dicalcium phosphate (CaHPO4). These results are consistent with literature reports as Pouliot et al. [14] and Rose and Tessier [16] found a calcium to phosphate ratio close to 1 for the calcium phosphate transferred to the colloidal phase when milk at its natural concentration was heated. Walstra et al. [17] suggested that transformation of Casol and Psol to colloidal calcium and phosphate involved the reaction: Ca2+ + H2PO4− → CaHPO4 + H+, which also suggests that the heat-precipitated calcium phosphate has a calcium to phosphate ratio close to unity.

It should be noted that milk serum is a complex mixture of mineral components, and some of these other mineral components may also associate with the Casol and Psol (such as citrate and magnesium) on heating to form insoluble salts that are transferred to the colloidal phase [6, 7]. However, Rose and Tessier [16] indicated that there was little change in soluble citrate or soluble magnesium on heating milk at a temperature of ~ 95 °C for up to 80 min, which suggests that little calcium will be precipitated as the citrate salt and little phosphate will be precipitated as the magnesium salt.

The nature of the CCP in milk, the mode of association of CCP with the casein micelles and the relationship between the CCP and the heat-precipitated calcium phosphate have not been unequivocally established [5, 8]. Although CCP has a calcium to inorganic phosphate ratio of about 1.5, when the Ca to (inorganic + organic phosphate) is considered, the ratio is close to unity [8]. This has led to the suggestion that the native CCP in milk may resemble dicalcium phosphate [8, 9]. Holt et al. [10, 11] have prepared calcium phosphate nano-clusters that are similar in size, composition, short-range order and dynamics to the native CCP. Through chemical analyses and infrared spectroscopy, it was suggested that these nano-clusters are composed of amorphous dicalcium phosphate complexed to the organic phosphate groups of the casein proteins. As the heat-precipitated calcium phosphate has a similar calcium to phosphate ratio as the native CCP, it has been suggested that the heating of milk resulted in an increase in the size of the CCP particles within the casein micelles in order to accommodate the precipitated calcium phosphate [7]. It is possible that the native CCP acts as nucleation sites for the heat-precipitated calcium phosphate. This is supported by the observation that the level of casein cross-linked by CCP is not increased by moderate heating [3].

The decrease in pH on increasing milk concentration (Fig. 3) is due to the increase in ionic components in the milk and possibly due to the precipitation of calcium phosphate. The decrease in milk pH under mild heating conditions (Fig. 3) is reportedly due to the changes in the dissociation of ions within the milk [6, 7, 17] and the conversion of Casol and Psol to the colloidal phase (through the reaction Ca2+ + H2PO4− → CaHPO4 + H+). On extensive heating, other reactions, such as the degradation of lactose or Maillard reactions, may cause more substantial and irreversible decreases in pH [4, 6]; however, these types of reactions are unlikely to occur to any great extent under the mild heating temperatures used in this study.

This study has demonstrated that the distribution of calcium and phosphate between soluble and colloidal phases is markedly dependent on the temperature and concentration of the milk. Increasing the temperatures resulted in a rapid conversion of Casol and Psol to the colloidal phase, and this was accompanied by a concomitant decrease in pH. The Casol, Psol and milk pH decreased within the first few minutes of heating and then remained relatively constant at longer heating times. Although the concentrations of Casol, Psol and pH were affected by the milk concentration, a similar general effect was observed at all milk concentrations, and as a consequence, inter-relationships were observed between Casol, Psol and pH. It appears that, at all milk concentrations, the predominant calcium phosphate phase precipitated from milk on increasing the temperature has a calcium to phosphate ratio close to unity. These results indicate that during the processing of milk, rapid decreases in the pH and the Casol and Psol occur when the milk is heated. If calcium or phosphate components are implicated in instability problems during the processing or storage of concentrated milks, it will be necessary to consider the concentration of these components as well as the effects of temperature, heating time and milk concentration on the equilibria of calcium and phosphate between the soluble and the colloidal phases.

Acknowledgments

This study was supported by funding from the New Zealand Foundation for Research, Science and Technology (Contract No. DRIX0701).

References

  1. Alkanani T., Friel J.K., Jackson S.E., Longerich H.P., Comparison between digestion procedures for the multielemental analysis of milk by inductively-coupled plasma-mass spectrometry, J. Agric. Food Chem. 42 (1994) 1965–1970 [CrossRef].
  2. Allen R.J.L., The estimation of phosphorus, Biochem. J. 34 (1940) 858–865 [PubMed].
  3. Aoki T., Umeda T., Kako Y., Cleavage of the linkage between colloidal calcium phosphate and casein on heating milk at high temperature, J. Dairy Res. 57 (1990) 349–354 [CrossRef].
  4. Chaplin L.C., Lyster R.L.J., Effect of temperature on the pH of skim milk, J. Dairy Res. 55 (1988) 277–280 [CrossRef].
  5. Holt C., Inorganic constituents of milk. III. The colloidal calcium phosphate of cow's milk, J. Dairy Res. 49 (1982) 29–38 [CrossRef] [PubMed].
  6. Holt C., The milk salts: their secretion, concentrations and physical chemistry, in: Fox P.F. (Ed.), Developments in Dairy Chemistry – 3. Lactose and Minor Constituents, Elsevier Applied Science Publishers, London, England, 1985, pp. 143–181.
  7. Holt C., Effect of heating and cooling on the milk salts and their interaction with casein, in: Heat-Induced Changes in Milk, Int. D. Fed. Special Issue 9501, International Dairy Federation, Brussels, Belgium, 1995, pp. 105–133.
  8. Holt C., An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein micelles and its application to the calculation of the partition of salts in milk, Eur. Biophys. J. 33 (2004) 421–434 [PubMed].
  9. Holt C., Hasnain S.S., Hukins D.W.L., Structure of bovine milk calcium phosphate determined by X-ray absorption spectroscopy, Biochim. Biophys. Acta – Gen. Subj. 719 (1982) 299–303 [CrossRef].
  10. Holt C., Timmins P.A., Errington N., Leaver J., A core-shell model of calcium phosphate nanoclusters stabilized by $\beta$-casein phosphopeptides derived from sedimentation equilibrium and small-angle X-ray and neutron-scattering measurements, Eur. J. Biochem. 252 (1998) 73–78 [CrossRef] [PubMed].
  11. Holt C., Wahlgren N.M., Drakenberg T., Ability of a $\beta$-casein phosphopeptide to modulate the precipitation of calcium phosphate by forming amorphous dicalcium phosphate nanoclusters, Biochem. J. 314 (1996) 1035–1039 [PubMed].
  12. Le Graet Y., Brule G., Effect of concentration and drying on mineral equilibria of skim-milk and retentates, Lait 62 (1982) 113–125 [CrossRef].
  13. McDowell A.K., Comparison of various methods for estimation of solids-not-fat in milk and whey, J. Dairy Res. 39 (1972) 251–259 [CrossRef].
  14. Pouliot Y., Boulet M., Paquin P., Observations on the heat-induced salt balance changes in milk. 1. Effect of heating time between 4 °C and 90 °C, J. Dairy Res. 56 (1989) 185–192 [CrossRef].
  15. Pouliot Y., Boulet M., Paquin P., Observations on the heat-induced salt balance changes in milk. 2. Reversibility on cooling, J. Dairy Res. 56 (1989) 193–199 [CrossRef].
  16. Rose D., Tessier H., Composition of ultra-filtrates from milk heated at 80 to 230 °F in relation to heat stability, J. Dairy Sci. 42 (1959) 969–980.
  17. Walstra P., Geurts T.J., Noomen A., Jellema A., van Boekel M.A.J.S., Dairy Technology: Principles of Milk Properties and Processes, Marcel Dekker, Inc., New York, USA, 1999.

All Figures

thumbnail Figure 1.

Changes in soluble calcium (i) and soluble phosphate (ii) on heating milk. (A) Changes in soluble calcium or soluble phosphate with heating time for the 9.6% TS milk heated at 20 °C (●), 30 °C (○), 40 °C (▼), 50 °C (▽), 60 °C (■), 70 °C (□) and 80 °C (♦). (B) Changes in soluble calcium or soluble phosphate with heating time for the 28.8% TS milk heated at 20 °C (●), 30 °C (○), 40 °C (▼), 50 °C (▽), 60 °C (■), 70 °C (□) and 80 °C (♦). (C) Changes in final soluble calcium and final soluble phosphate after 60-min heating at a range of temperatures for the 9.6% TS (●), 19.2% TS (○), 28.8% TS (▼) and 38.4% TS (▽) milk samples.
In the text
thumbnail Figure 2.

Relationships between soluble calcium and soluble phosphate for milks of different concentrations. (A) Relationship between soluble calcium and soluble phosphate for the 9.6% TS (●), 19.2% TS (○), 28.8% TS (▼) and 38.4% TS (▽) milk samples that were heated at temperatures between 20 and 80 °C for 60 min. (B) Relationship between the change in soluble calcium and the change in soluble phosphate (relative to the samples held at 20 °C) for the 9.6% TS (●), 19.2% TS (○), 28.8% TS (▼) and 38.4% TS (▽) milk samples that were heated at temperatures between 20 and 80 °C for 60 min.
In the text
thumbnail Figure 3.

Changes in pH on heating milk. (A) Changes in pH with heating time for the 9.6% TS milk heated at 20 °C (●), 30 °C (○), 40 °C (▼), 50 °C (▽), 60 °C (■), 70 °C (□) and 80 °C (♦). (B) Changes in pH with heating time for the 28.8% TS milk heated at 20 °C (●), 30 °C (○), 40 °C (▼), 50 °C (▽), 60 °C (■), 70 °C (□) and 80 °C (♦). (C) Changes in final pH after 60-min heating at a range of temperatures for the 9.6% TS (●), 19.2% TS (○), 28.8% TS (▼) and 38.4% TS (▽) milk samples.
In the text
thumbnail Figure 4.

Relationships between pH and soluble calcium or soluble phosphate for milks of different concentrations. (A) Relationship between pH and soluble calcium for the 9.6% TS (●), 19.2% TS (○), 28.8% TS (▼) and 38.4% TS (▽) milk samples that were heated at temperatures between 20 and 80 °C for 60 min. (B) Relationship between pH and soluble phosphate for the 9.6% TS (●), 19.2% TS (○), 28.8% TS (▼) and 38.4% TS (▽) milk samples that were heated at temperatures between 20 and 80 °C for 60 min.
In the text