Use of a nucleotide for improving the heat stability of an aqueous micellar casein composition

Abstract

The invention relates to the use of one or more nucleotides for improving the heat stability of an aqueous micellar casein composition comprising 6 to 20 g per 100 ml of micellar casein, and having a pH of about 6 to 8. The invention also relates to heat-treated liquid nutritional compositions comprising 6 to 20 g, preferably 9-20 g, of protein per 100 ml of the composition and having a pH of about 6 to 8, in which all or a major part of said protein comprises micellar casein, further comprising one or more nucleotides.

Claims

1. A method for improving the heat stability of an aqueous micellar casein composition having a pH of about 6 to 8 and comprising (a) 11 to 20 g of protein per 100 ml of the composition, in which at least 80 wt % of the protein comprises micellar casein, and wherein 0-15 weight % of the protein present in the nutritional composition comprises whey, the method comprising adding (b) 45 to 120 mEq.L.sup.?1 of one or more nucleotides to the composition, wherein the composition has a heat coagulation time (HCT) value which is at least 10% higher than the HCT value for a reference composition not including the one or more nucleotides.

2. The method according to claim 1, wherein the nucleotides are selected from the group consisting of uridine monophosphate (UMP), cytidine monophosphate (CMP), thymidine monophosphate (TMP), guanosine monophosphate (GMP), adenosine monophosphate (AMP), and inosine monophosphate (IMP).

3. The method according to claim 2, wherein the monophosphate is a sodium phosphate, a potassium phosphate, or a mixture thereof.

4. The method according to claim 3, wherein the nucleotide monophosphate is disodium uridine monophosphate or disodium cytidine monophosphate.

5. The method according to claim 1, wherein 45 to 100 mEq.L.sup.?1 of the one or more nucleotides is added to the composition.

6. The method according to claim 5, wherein 45 to 60 mEq.L.sup.?1 of the one or more nucleotides is added to the composition.

7. The method according to claim 1, wherein at least 80 wt % of the proteins of the composition is micellar casein proteins of the composition are micellar casein.

8. A liquid nutritional composition having a pH of about 6 to 8 and comprising: (a) 11 to 20 g of protein per 100 ml of the composition, in which at least 80 wt % of the protein comprises micellar casein, and wherein 0-15 weight % of the protein present in the nutritional composition comprises whey, and (b) 45 to 120 mEq.L.sup.?1 of one or more nucleotides, wherein the composition has a heat coagulation time (HCT) value which is at least 10% higher than the HCT value for a reference composition not including the one or more nucleotides.

9. The composition according to claim 8, wherein the nucleotides are selected from the group consisting of uridine monophosphate (UMP), cytidine monophosphate (CMP), thymidine monophosphate (TMP), guanosine monophosphate (GMP), adenosine monophosphate (AMP), and inosine monophosphate (IMP).

10. The composition according to claim 9, wherein the monophosphate is a sodium phosphate, a potassium phosphate, or a mixture thereof.

11. The composition according to claim 10, wherein the nucleotide monophosphate is disodium uridine monophosphate or disodium cytidine monophosphate.

12. The composition according to claim 8, wherein the composition comprises 45 to 100 mEq.L.sup.?1 of the one or more nucleotides.

13. The composition according to claim 12, wherein the composition comprises 45 to 60 mEq.L.sup.?1 of the one or more nucleotides.

14. The composition according to claim 8, wherein at least 90 wt % of the protein comprises micellar casein.

15. The composition according to claim 8, further comprising one or more of fat, digestible and non-digestible carbohydrates.

16. The composition according to claim 8, wherein the composition is pasteurized or sterilized.

17. The composition according to claim 8, wherein the composition is subjected to a temperature of at least 60? C. for at least a time t (in seconds)=(500/(T?59))?4, in which temperature T is expressed in ? C. and t is at least 0.1 sec.

18. The composition according to claim 8, wherein the composition has a sterilizing value or Fzero value of at least 2.8 minutes.

19. A process for the heat treatment of an aqueous micellar casein composition having a pH of about 6 to 8 and comprising (a) 11 to 20 g of protein per 100 ml of the composition, in which at least 80 wt % of the protein comprises micellar casein, and wherein 0-15 weight % of the total protein present in the composition comprises whey, the process comprising adding (b) 45 to 120 mEq.L.sup.?1 of one or more nucleotides to the composition prior to the heat treatment, wherein the composition has a heat coagulation time (HCT) value which is at least 10% higher than the HCT value for a reference composition not including the one or more nucleotides.

20. The process according to claim 19, wherein 45 to 100 mEq.L.sup.?1 of the nucleotide(s) is added to the composition.

21. A method of providing nutrition to a person in need thereof, comprising administering to said person the nutritional composition according to claim 8, wherein the person is an elderly person, a person who is in a disease state, a person who is recovering from a disease state, a person who is malnourished, or a healthy person.

22. A liquid nutritional composition having a pH of about 6 to 8 and comprising: (a) 11 to 20 g of protein per 100 ml of the composition, in which at least 80 wt % of the protein comprises micellar casein, and wherein 0-15 weight % of the protein present in the nutritional composition comprises whey, and (b) 45 to 120 mEq.L.sup.?1 of one or more nucleotides, wherein the composition does not show coagulation after heating for 90 minutes in a Klarograph using a 126? C. oil bath.

23. The liquid nutritional composition according to claim 8, wherein the composition has an energy density of at least 1.0 kcal/ml.

Description

LIST OF FIGURES

(1) FIG. 1: HCT, calcium-ion activity, viscosity, turbidity, and zeta potential of the MCI solution at pH 6.7 as function of calcium chelator concentration. Results are the means for at least duplicates with standard deviations as error bars. (?) and (---) indicate that no coagulation appeared after heating for 90 min.

(2) FIG. 2: HCT, calcium-ion activity, viscosity, turbidity, and zeta potential of the MCI solution at pH 7.0 as function of calcium chelator concentration. Results are the means for at least duplicates with standard deviations as error bars. (?) and (---) indicate that no coagulation appeared after heating for 90 min.

(3) FIG. 3: HCT, calcium-ion activity, viscosity, turbidity, and zeta potential of the MCI solution at pH 7.3 as function of calcium chelator concentration. Results are the means for at least duplicates with standard deviations as error bars. (?) and (---) indicate that no coagulation appeared after heating for 90 min.

(4) FIG. 4: Turbidity, viscosity, and zeta potential of the MCI solution at pH 7.3 of the reference samples and those with 60 mEq L.sup.?1 Na.sub.2HPO.sub.4, TSC, and SP as function of the heating time in the oil bath. Results are the means for duplicates with standard deviations as error bars.

1. MATERIALS AND METHODS

(5) 1.1 Sample Preparation

(6) MCI powder (Nutripro?) was supplied by DairyGold Food Ingredients (Cork, Ireland). This powder contains 85 w/w % protein of which ?5 w/w % is whey. A MCI solution with 9% w/v protein was prepared by dissolving the MCI powder in 80% of the total demineralised water at ambient temperature, while stirring at 600 rpm with a laboratory stirrer (RW 20.n, IKA Labortechnik, Staufen, Germany). A 9% w/v MCI solution contains approximately 8.5 mmol.Math.L?1 sodium, 4.2 mmol.Math.L?1 potassium, 2.5 mmol.Math.L?1 chloride, 59.8 mmol.Math.L?1 calcium, 43.5 mmol.Math.L?1 phosphorus, and 3.1 mmol.Math.L?1 magnesium. The protein solution was homogenised with a high pressure laboratory homogeniser (NS2006L, GEA Niro Soari S.P.A., Parma, Italy) at 350+50 bar to obtain single casein micelles with a diameter D[4,3] of 0.15 ?m as determined with a Mastersizer 2000 containing a hydro 2000G water bath (Malvern Instruments, Worcestershire, England). The temperature of the protein solution was 40? C. after homogenization.

(7) Stock solutions were prepared of disodium uridine monophosphate (Na.sub.2UMP) (Yamasa Corporation, Chiba, Japan), disodium hydrogen phosphate (Na.sub.2HPO.sub.4) (Merck & Co. Inc, Darmstadt, Germany), sodium hexametaphosphate (SHMP) (VWR International Ltd, Poole, England), phytic acid dodecasodium salt hydrate (SP) (Sigma-Aldrich GmbH, Steinheim, Germany), and trisodium citrate (TSC) (Gadot Biochemical Industries Ltd., Haifa Bay, Israel). Different amounts of these stock solutions were added to the MCI solutions in order to obtain final chelator concentrations of 0, 15, 30, 45, or 60 mEq L.sup.?1 in the samples. These chelators contain a different amount of negative charges, which gives them different calcium-binding capacities (De Kort, E. J. P., Minor, M., Snoeren, T. H. M., van Hooijdonk, A. C. M., & van der Linden, E. (2009). Journal of Dairy Science and Technology, 89, 283-299). Therefore, the concentration ranges of the calcium chelators were based on milliequivalents so as to add a similar amount of charges to the samples. Only sodium sources were used, because the type of counter-ion may also influence protein-mineral interactions.

(8) The pH of the samples was adjusted, after stirring for 30 minutes, to 6.7?0.05, 7.0?0.05, and 7.3?0.05 with 1 mol L.sup.?1 sodium hydroxide (Sigma-Aldrich GmbH, Steinheim, Germany) or 1 mol L.sup.?1 hydrochloric acid (Merck & Co. Inc, Darmstadt, Germany). Finally, samples were brought to their final protein concentration of 9% w/v with demineralized water. Samples were stored overnight at 20? C. for approximately 17 hours to let them equilibrate. The pH of the samples was readjusted the next morning to 6.7?0.05, 7.0?0.05, or 7.3?0.05 in case deviations had occurred during storage. Deviations in pH were always small and samples did not show any visible spoilage. Samples with 0, 15, 30, 45, and 60 mEq L.sup.?1 phosphate or citrate were analyzed at least in duplicate for their HCT in the Klarograph and samples with 0, 15, and 60 mEq L.sup.?1 phosphate or citrate were heated for 0, 15, 35, and 55 minutes in an oil bath. The samples were analyzed in duplicate before and after heating in the oil bath for their pH, calcium-ion activity, turbidity, viscosity, and zeta potential.

(9) 1.2 HCT Measurements; Klarograph

(10) The Klarograph was used to determine the HCT of the samples. The Klarograph is based on the principle of the falling-ball viscometer (Cruijsen, J. M. M. 1996. Wageningen Agricultural University, Wageningen; De Wit, J. N., Klarenbeek, G., & De Graaf, C. (1986). Voedingsmiddelentechnologie, 19 (3), 25-27; Van Mil, P. J. J. M., & De Koning, J. (1992). Netherlands Milk and Dairy Journal, 40, 351-368). Samples are inserted in the inner part of a double walled glass tube. The inner diameter of the tubes is 9.3 mm and the volume is 20 ml from the bottom to the expansion chamber. Two glass balls with a diameter 9.0 mm are put in the tubes. The tubes are placed in the system and silicone oil is circulated around the tubes. The silicone oil is connected to a thermostatic oil bath, which is set at 126? C. The apparatus allows the use of eight tubes at the same time (2 times 4 tubes). The tubes are placed 10? from upright, so that the balls roll along the wall of the tubes. The tubes are rotated 180? clockwise and anti-clockwise during the measurement. The tubes are rotated as soon as the balls reach the bottom of the tubes, which is approximately 20 s. When the samples become unstable, the balls are stopped by coagulated particles. The time needed to reach coagulation is recorded as the HCT. The reported heating times do not include the heating-up period, which is approximately 4 min. Hence, the HCT is only determined once the temperature reaches its constant value.

(11) 1.3. Oil Bath

(12) We used an oil bath to determine heat-induced changes, because larger sample volumes could be heated than in the Klarograph. The samples were inserted in heat-resistant glass tubes of 15 ml (at least three tubes per sample) and heated for 15, 35, and 55 min in the oil bath. Similar samples were pooled after heating to obtain sufficient volume for analyses. The oil bath was set at 126? C. The time of heating did not include the heating-up of the samples, which was approximately 6 min. The samples were cooled in cold water until they reached ambient temperature, which was within 30 min, before analyses were done.

(13) 1.4. pH

(14) The pH was measured at ambient temperature with an Inlab? Expert Pro pH meter (Mettler Toledo, Greifensee, Switzerland), which is part of the calcium-ion measuring device. The pH meter was calibrated with stock solutions of pH 4.0 and pH 7.0. The pH value was read after gently stirring for 5 min.

(15) 1.5. Calcium-Ion Activity

(16) The calcium-ion activity was measured with a Mettler Toledo Seven Multi? (with an Inlab? Expert Pro pH-meter) calcium measuring device (Mettler Toledo, Greifensee, Switzerland) using an Orion 9300BH electrode and an Orion 900100 reference electrode. Calibration of the electrodes, sample measurements, and calculations of the calcium-ion activities were performed as described in De Kort, E. J. P., Minor, M., Snoeren, T. H. M., van Hooijdonk, A. C. M., & van der Linden, E. (2009). Journal of Dairy Science and Technology, 89, 283-299.

(17) 16. Turbidity

(18) The turbidity was measured with a spectrophotometer (4053 Kinetics, LKB 0, Midland, Canada). Plastic cuvettes with a path length of 1 cm were used. Measurements were carried out at ambient temperature using a wavelength of 700 nm. Samples were measured for their turbidity by diluting the samples to 10% of their initial dry matter in demineralized water so as to be within the detection limits of the spectrophotometer.

(19) 1.7. Viscosity

(20) Samples were analyzed with a MCR 300 rheometer (Anton Paar Physica, Graz, Austria) using a cup (CC27 cylinder) and bob geometry. The viscosity was measured at shear rates of 1 s.sup.?1 to 1000 s.sup.?1. In this paper viscosity results at a shear rate of 50 s.sup.?1 are given. Most of the samples behaved very similarly to Newtonian liquids.

(21) 1.8. Zeta Potential

(22) The zeta potential was measured with the Zetasizer Nano Z (Malvern Instruments, Worcestershire, England) by using disposable folded capillary Zetasizer Nano cells of 1.5 ml (DTS1060, Malvern Instruments). Measurement of negative charges is based on the electrophoretic mobility in the samples. The zeta potential is calculated with the Smoluchowski approximation. Prior to analysis, samples were diluted to 1% of their initial dry matter in demineralized water and subsequently filtered through disposable Nalgene? Syringe celluloseacetate filters with a pore size of 0.8 ?m (Nalgene Nunc International Corporation, Rochester, USA). Analyses were performed in duplicate at a cell temperature of 25? C. and voltage of 100 V.

2. RESULTS

(23) In the first part the HCT results obtained with the Klarograph and the differences in calcium-ion activity, viscosity, turbidity, and zeta potential of the samples before heating are described. In the second part the heat-induced changes that were measured after heating the samples for various time periods in the oil bath are described.

(24) 2.1. Heat Coagulation Time

(25) The HCT of the MCI solution with and without phosphates or citrate was measured at pH 6.7, 7.0, and 7.3 with the Klarograph for maximally 90 min. Calcium-ion activity, viscosity, turbidity, and zeta potential analyses were performed before heating to obtain information about changes in the concentration of free calcium ions, integrity of the micellar structure, and charge distribution on the micellar surface after addition of calcium chelators. Overviews of the results are shown for pH 6.7 in FIG. 1, pH 7.0 in FIG. 2, and pH 7.3 in FIG. 3. The HCT markedly increased upon addition of the calcium chelators, an effect which was most pronounced at pH 6.7. The differences in HCT were investigated in relation to the initial calcium-ion activity, viscosity, turbidity, and zeta potential of the samples. We can divide the results into four groups: 1) reference samples; 2) Na.sub.2UMP; 3) Na.sub.2HPO.sub.4, TSC, and SP; 4) SHMP.

(26) 2.1.1. Reference Samples

(27) The HCT of the reference samples (without chelators) increased with increasing pH: 2 min at pH 6.7, 40 min at pH 7.0, and 55 min at pH 7.3. This monotonic increase in HCT is in agreement with the HCT as function of pH for whey-protein-free casein micelle dispersions. The increase in HCT of the reference samples is due to the higher initial pH and concomitant lower calcium-ion activity. Besides these effects, the net negative charge of the casein micelles increases at higher pH. This induces more electrostatic repulsion between the negatively charged caseins, which gave an increase in heat stability. However, the changes in net negative charge were too small to detect with the zetasizer.

(28) 2.1.2. Addition of Na.sub.2UMP

(29) Na.sub.2UMP is very effective in increasing the heat stability of the MCI solution at all three pH values (FIGS. 1 to 3). At higher pH a lower concentration of Na.sub.2UMP was needed to give the MCI solution a HCT of more than 90 min. Na.sub.2UMP reduced the concentration of free calcium ions by approximately 40% at pH 6.7 and 7.0, which greatly reduced protein aggregation. It is probable that below a calcium-ion activity of ?2 mmol.Math.L.sup.?1, enough free calcium ions were bound to give a strong increase in HCT. The viscosity, turbidity, and zeta potential of the solutions remained constant at all pH values. Therefore, it is most likely that the decrease in calcium-ion activity was the main driver for the increase in HCT in Na.sub.2UMP samples.

(30) 2.1.3. Addition of Na.sub.2HPO.sub.4, TSC, and SP

(31) Addition of Na.sub.2HPO.sub.4, TSC, and SP induced large increases in HCT at pH 6.7 and 7.0 (FIGS. 1 and 2). The increase in HCT was less pronounced at pH 7.3 (FIG. 3), because the HCT was already high for the reference sample. Addition of Na.sub.2HPO.sub.4 resulted in HCTs of more than 90 min at pH 7.0 and 7.3, whereas slightly lower HCTs were obtained for TSC and SP at these pH values. The calcium-ion activities, viscosities, and zeta potentials measured for Na.sub.2HPO.sub.4, TSC, and SP samples were of the same order at all three pH values. At pH 6.7 the large increase in HCT is most likely due to the strong decrease in calcium-ion activity. At pH 7.0 and 7.3 the calcium-ion activities stayed below 1.5 mmol L.sup.?1 and 1.0 mmol L.sup.?1, respectively, and this was sufficiently low to give the samples a high HCT.

(32) The slight differences in HCT that were measured for Na.sub.2HPO.sub.4, TSC, and SP might be related to their differences in turbidity before heating. The decrease in turbidity is due to dissociation of the casein micelles into smaller structures upon addition of calcium chelators. It was concluded that micellar dissociation most probably occurred in the order SP>TSC>Na.sub.2HPO.sub.4. Hence, the MCI solutions with Na.sub.2HPO.sub.4, TSC, and SP contain different concentrations of dissociated and intact casein micelles. A decreasing trend in heat stability was measured in the order SP>TSC>Na.sub.2HPO.sub.4 at pH 7.0 and 7.3, which suggests that the small micellar particles formed have a negative impact on the heat stability of the MCI solution. However, these small micellar particles are also present in sodium caseinate at high ionic strength, whereas sodium caseinate is known for its high heat stability. Nevertheless, it is known that the heat stability of sodium caseinate can be markedly reduced in the presence of ionic calcium, It is also known that the heat stability of sodium caseinate and CCP-free milk shows a greater reduction in the presence of heat-precipitated calcium phosphate than milk containing unaltered casein micelles. As our samples contained a high concentration of calcium (phosphate) it is likely that the smaller micellar particles formed upon chelator addition were more susceptible to calcium-induced protein-aggregation than intact micelles. As a result, lower heat stabilities were measured for SP and TSC than for Na.sub.2HPO.sub.4. It is also known that the decrease in heat stability of recombined concentrated milk containing Na.sub.2HPO.sub.4, TSC, or EDTA was more pronounced when more casein micelles were dissociated.

(33) The HCT of both Na.sub.2HPO.sub.4 and Na.sub.2UMP samples increased to approximately 90 min or more at pH 7.0 and 7.3, whereas the HCT of Na.sub.2HPO.sub.4 samples was considerably lower than for Na.sub.2UMP samples at pH 6.7. Addition of 15 mEq L.sup.?1 Na.sub.2HPO.sub.4 or Na.sub.2UMP at pH 6.7 reduced the concentration of free calcium ions by approximately 55% and 25%, respectively, because Na.sub.2HPO.sub.4 has a stronger calcium-binding capacity than Na.sub.2UMP. The decrease in free calcium ions was sufficient to obtain a HCT of more than 70 min for Na.sub.2HPO.sub.4, but for Na.sub.2UMP a HCT of just 40 min was measured. The HCT increased more for Na.sub.2UMP than for Na.sub.2HPO.sub.4 at higher chelator concentrations at pH 6.7. The calcium-ion activity in both samples was sufficiently low to increase the HCT. However, in Na.sub.2HPO.sub.4 samples the amount of CCP in the micelles increased, most likely because of precipitation of calcium phosphate complexes in the casein micelle, whereas in Na.sub.2UMP samples the amount of CCP was negligibly affected. This increase in amount of CCP decreased the HCT of Na.sub.2HPO.sub.4 samples.

(34) 2.1.4. Addition of SHMP

(35) The lowest HCTs were measured for addition of SHMP at pH 6.7 and 7.0 in comparison to the other calcium chelators. The SHMP samples became very viscous with increasing SHMP concentration, which made it difficult to determine coagulation, because the glass balls could not freely move in the Klarograph tubes. The high viscosities are due to the cross-links formed between the caseins by SHMP. Samples were gelled upon addition of more than 45 mEq L.sup.?1 SHMP at all three pH values. Addition of ?45 mEq L.sup.?1 SHMP at pH 7.3 caused a sharp decrease in the HCT, which is probably due to the high initial viscosity. A strong decrease in zeta potential was also observed for these samples. The net negative charge of the casein micelles and depletion of CCP from the casein micelles could have reached a critical value, at which ?-casein could not be retained on the micellar surface and the micellar structures could not be kept intact during heating. Moreover, the turbidity results indicate that most of the casein micelles were already dissociated at >45 mEq L.sup.?1 SHMP before heating. This may have caused a strong increase in coagulation for the SHMP samples, because the small micellar particles formed upon calcium chelator addition are more susceptible to protein-aggregation.

(36) It is remarkable that approximately ?6 to ?10 mV more negative zeta potentials were measured for SHMP than for SP samples at all three pH values (see zeta potentials in FIGS. 1 to 3). An equal amount of charges was added to the solutions and both polyphosphates can bind to the casein micelles, increasing the net negative charge of the casein micelles. It was hypothesized that SHMP interacts with the caseins and calcium ions (i.e. forms cross-links), whereas SP initially interacts more strongly with the calcium ions than with the casein micelles. This is related to the pK.sub.a, form, and charge distribution of the SHMP and SP molecules. SHMP has more homogeneously distributed charges around its molecule, whereas SP has twelve negative charges, clustered in pairs, around its molecule. This might have resulted in more negatively charged casein micelles in SHMP samples.

(37) 2.2 Heat-Induced Changes

(38) Samples with 0, 15, and 60 mEq L.sup.?1 phosphate or citrate were selected and heated for 15, 35, and 55 min in the oil bath to determine heat-induced changes. A concentration of 15 mEq L.sup.?1 was selected, because the largest increase in HCT was measured between 0 and 15 mEq L.sup.?1. The samples were analyzed for their pH, calcium-ion activity, turbidity, viscosity, and zeta potential after heating. The results can be divided in three groups: 1) reference samples; 2) Na.sub.2UMP, Na.sub.2HPO.sub.4, TSC, and SP; 3) SHMP. This classification is based on the fact that comparable heat-induced changes were measured for Na.sub.2UMP and Na.sub.2HPO.sub.4, TSC, and SP, although they showed different HCTs (FIGS. 1 to 3).

(39) 2.2.1. Reference Samples

(40) The results of the reference samples, without chelator addition, are summarized in Table 1.

(41) TABLE-US-00001 TABLE 1 Reference samples at pH 6.7, 7.0, and 7.3 heated in the oil bath for 0-55 min Calcium-ion Zeta Time Measured activity Turbidity Viscosity potential pH (min) pH () (mmol L.sup.?1) () (mPa s) (mV) 6.7 0 6.70 2.57 2.65 3.31 ?22.83 15 6.48 1.39 3.00 coagulated ?27.85 35 6.46 1.44 3.00 coagulated ?28.00 55 6.41 1.34 3.00 coagulated ?26.60 7.0 0 7.00 1.47 2.51 4.18 ?23.25 15 6.71 0.91 2.93 3.04 ?22.30 35 6.71 0.99 2.95 3.14 ?22.95 55 6.58 0.97 2.98 3.72 ?21.73 7.3 0 7.30 0.71 2.30 4.45 ?21.55 15 6.94 0.80 2.53 3.12 ?19.56 35 6.85 0.80 2.50 3.01 ?22.63 55 6.67 0.84 2.60 3.10 ?24.04

(42) The pH decreased by 0.3 to 0.6 units during heating and it decreased more in the samples with higher initial pH. This decrease in pH is also observed for skim milk. The pH decrease is attributed to calcium phosphate precipitation rather than formation of formic acid, because MCI contains a negligible amount of lactose. The initial calcium-ion activity was higher than ?2 mmol.Math.L.sup.?1 at pH 6.7, which likely caused coagulation within 15 min of heating and a pH decrease to 6.5. The strong decrease in calcium-ion activity at pH 6.7 during heating also indicates calcium phosphate precipitation and protein aggregation, which resulted in a more negative zeta potential, increase in turbidity, and coagulation of the sample. These heat-induced changes were also observed at pH 7.0 and 7.3.

(43) 2.2.2. Addition of Na.sub.2UMP, Na.sub.2HPO.sub.4, TSC, or SP

(44) The pH decrease after heating for 55 min in the oil bath for 15 and 60 mEq L.sup.?1 Na.sub.2UMP, Na.sub.2HPO.sub.4, TSC, and SP samples at pH 6.7, 7.0, and 7.3 was comparable to the pH decrease that was measured for the reference samples (see Table 1). None of these samples showed visible coagulation after heating for 55 min in the oil bath. The calcium-ion activities of these samples remained constant or slightly decreased. The calcium-ion activities before heating were already sufficiently low because of the calcium-binding capacity of the chelators and the stronger calcium phosphate binding in the micelles with increasing pH (FIGS. 1 to 3). Small changes could be detected for the zeta potential of these samples. The turbidity increased and viscosity decreased in the samples during heating, because calcium phosphate precipitation and decomposition of the caseins occurred.

(45) In FIG. 4 it is shown that the turbidity of the MCI solution with 60 mEq L.sup.?1 SP at pH 7.3 only slightly increased during heating. This sample behaved remarkably differently than the samples with 0 or 60 mEq L.sup.?1 Na.sub.2HPO.sub.4 or TSC. SP probably binds the calcium ions so strongly (also at lower pH) that only a low concentration of calcium ions is available for heat-induced calcium phosphate precipitation or calcium-induced protein-aggregation. Moreover, the electrostatic repulsion between the micelles is stronger at lower calcium-ion activity, which reduces protein-aggregation. The strongly charged anionic SP molecules might also bind to the positively charged amino acid residues, increasing the electrostatic repulsion between the casein micelles as well. This resulted in a HCT of more than 90 min for addition of 60 mEq L.sup.?1 SP at pH 7.3 (FIG. 3). Only a slight decrease in viscosity and increase in zeta potential was measured for this SP sample during heating (FIG. 4), because the strong repulsion between the caseins and strong calcium binding capacity of SP was probably maintained during heating.

(46) FIG. 4 shows that the viscosity of the 60 mEq L.sup.?1 Na.sub.2HPO.sub.4 or TSC samples at pH 7.3 strongly decreased during heating, to values that were slightly higher than the reference samples. The decrease in viscosity is related to the changes that occur in the micelles during heating. It is known for milk that during heating the viscosity decreases because of dissociation of the micelles (i.e. solubilization of casein and CCP and release of ?-casein). These heat-induced changes make the dissociated casein micelles more susceptible to coagulation. Hence, with the onset of coagulation, the viscosity strongly increases. In the Na.sub.2UMP, Na.sub.2HPO.sub.4, TSC, and SP samples this strong increase in viscosity was not measured, because the samples did not coagulate in the oil bath.

(47) 2.2.3. Addition of SHMP

(48) SHMP gave a more pronounced decrease in pH during heating than the reference samples and the other calcium chelators at all three pH values: a pH decrease of 0.7-0.9 (Table 2) versus 0.3-0.6 (Table 1). This caused an increase in the concentration of free calcium ions, which made the samples more susceptible to calcium-induced protein aggregation. As a result, coagulation was measured after heating 55 min in the oil bath upon addition of 15 or 60 mEq L.sup.?1 SHMP at pH 6.7. These low heat stabilities are in agreement with the low HCTs that were measured for these samples (FIG. 1). In a previous study (De Kort, E. J. P., Minor, M., Snoeren, T. H. M., van Hooijdonk, A. C. M., & van der Linden, E. (2009). Journal of Dairy Science and Technology, 89, 283-299), a strong decrease in pH for SHMP in a calcium chloride solution upon heating was observed. SHMP hydrolyzes into sodium trimetaphosphate and sodium orthophosphate in acidic conditions and this hydrolysis probably occurred in the MCI solutions during heating as well. This induced, besides the strong pH decrease, the release of calcium ions, which can cause calcium-induced protein aggregation. The calcium-ion activity was lower at higher pH and upon addition of 60 mEq L.sup.?1 SHMP, as more calcium ions were part of the CCP complexes or bound to SHMP, respectively. Moreover, less SHMP will be hydrolyzed at higher pH.

(49) The strong decrease in viscosity and increase in zeta potential (e.g. from ?33.10 to ?21.15 mV at pH 6.7 for 60 mEq L.sup.?1) in the SHMP samples at all three pH values indicate that the cross-links formed before heating between the caseins and SHMP were broken during heating. As an increase in calcium-ion activity was measured during heating, it is likely that calcium ions were involved in the cross-links as well. The increase in the concentration of free calcium ions during heating most probably initiated calcium-induced protein-aggregation. The strong increase in zeta potential may be caused by the release of SHMP from the micelles or by release of ?-casein from the casein micelles. ?-Casein depletion is more pronounced at higher pH, which reduced the net negative charge of the casein micelles and increased the sensitivity to protein-aggregation. As a result, a strong decrease in HCT was measured upon addition of ?45 mEq L.sup.?1 SHMP at pH 7.3 (FIG. 3). The turbidity also strongly increased during heating in all SHMP samples, which is attributed to calcium-induced protein-aggregation. Overall, the MCI solutions with SHMP are more susceptible to heat coagulation than those with the other calcium chelators because of the strong decrease in pH and increase in calcium-ion activity during heating.

(50) TABLE-US-00002 TABLE 2 SHMP samples at pH 6.7, 7.0, and 7.3 heated in the oil bath for 0-55 min. 15 mEq L.sup.?1 SHMP 60 mEq L.sup.?1 SHMP Calcium-ion Zeta Calcium-ion Zeta Time Measured activity Turbidity Viscosity potential Measured activity Turbidity Viscosity potential pH (min) pH () (mmol L.sup.?1) () (mPa s) (mV) pH () (mmol L.sup.?1) () (mPa s) (mV) 6.7 0 6.70 1.13 2.08 6.04 ?25.85 6.70 0.30 0.11 144 ?33.10 15 6.21 1.14 2.91 5.66 ?24.78 6.06 0.40 2.79 5.53 ?22.93 35 6.24 1.37 2.56 coagulated ?24.13 5.94 0.49 2.86 6.53 ?24.40 55 6.04 1.92 2.63 coagulated ?22.10 5.85 0.54 2.52 coagulated ?21.15 7.0 0 7.00 0.72 1.79 14.0 ?27.40 7.00 0.21 0.13 331 ?37.93 15 6.50 0.61 1.76 5.04 ?27.57 6.29 0.30 2.07 5.54 ?27.87 35 6.36 0.74 2.13 3.26 ?24.52 6.17 0.26 2.58 4.30 ?28.57 55 6.27 0.42 2.66 3.77 ?25.13 6.10 0.31 2.73 coagulated ?23.25 7.3 0 7.30 0.41 0.75 115 ?31.33 7.30 0.14 0.09 593 ?43.00 15 6.76 0.56 2.62 4.08 ?26.58 6.78 0.54 1.86 11.1 ?27.60 35 6.74 0.30 1.99 3.89 ?27.40 6.65 0.28 2.44 10.8 ?26.53 55 6.60 0.23 2.36 4.27 ?24.95 6.57 0.15 2.49 9.39 ?24.53

3. DISCUSSION

(51) Without being bound by theory, this research has shown that the influence of the various calcium chelators on the heat stability of the MCI solution is determined by the initial calcium-ion activity, the amount of CCP in the casein micelle, and the extent of dissociation of the casein micelle.

(52) A low calcium-ion activity was the most important parameter to effectively increase the HCT of the MCI solution. The weak calcium chelator Na.sub.2UMP is a very effective heat stabilizer, because it decreased the calcium-ion activity sufficiently without affecting the micellar structure. As a result, the highest HCTs were measured for Na.sub.2UMP at all three pH values. An effect was also obtained with increasing pH: the calcium-ion activity decreased, the protein charge increased and, consequently, the HCT increased. Na.sub.2HPO.sub.4, TSC, and SP also increased the HCT of the MCI solution by decreasing the calcium-ion activity to comparable levels, but their effect on the HCT was smaller than for Na.sub.2UMP. This is probably because these chelators affected the amount of CCP in the casein micelle and integrity of the micellar structure as well. Reduction of the level of CCP is known to increase the heat stability of milk below pH 7.0. However, when a critical level of CCP is removed from the micelles, it is known they start to dissociate, which decreases heat stability. Na.sub.2HPO.sub.4 precipitates with calcium on the micelle and thereby the amount of CCP in the micelle increases. This implies that a lower HCT should be measured for Na.sub.2HPO.sub.4 than for TSC or SP. However, a decrease in heat stability occurred in the order SP>TSC>Na.sub.2HPO.sub.4. It was concluded that these chelators most probably dissociate the micelles in the order SP>TSC>Na.sub.2HPO.sub.4. Therefore, it is likely that slight differences in heat stability for these samples are mainly attributable to the extent of micellar dissociation and not to the amount of CCP present in the casein micelles. The small micellar particles formed upon micelle dissociation in the MCI solutions seem more susceptible to calcium-induced protein-aggregation than intact casein micelles.

(53) Heat-induced changes that occurred in the reference, Na.sub.2UMP, Na.sub.2HPO.sub.4, TSC, and SP samples during heating were of the same order. This implies that the differences in HCT of these samples were mainly determined by the state of the MCI solutions before heating.

(54) Contrary to the other calcium chelators, the heat-induced changes that occurred in the SHMP samples did play an important role for their heat stability. Of course the calcium-ion activity and state of the micellar structure before heating were also important for the heat stability of these SHMP samples. However, the results indicate that the strong decrease in pH, increase in calcium-ion activity, and breakdown of SHMP cross-links between the caseins during heating were mainly responsible for the strong decrease in the HCT of SHMP samples.

4. CONCLUSIONS

(55) The heat stability of a MCI solution can be improved by addition of calcium chelators. Na.sub.2UMP is the most effective heat stabilizer, as it binds sufficient free calcium ions to reduce protein aggregation without affecting the integrity of the micellar structure. The HCT of the MCI solutions with Na.sub.2HPO.sub.4, TSC, and SP increased to comparable levels compared to one another, but the increase in HCT was much smaller than for Na.sub.2UMP. The slight differences in HCT that were measured for these samples other than Na.sub.2UMP were due to the extent to which the casein micelles were dissociated. This made the MCI solutions more susceptible to coagulation. SHMP was the least effective heat stabilizer. SHMP cross-linked the caseins, but these cross-links were broken during heating. This decreased the pH and increased the calcium-ion activity during heating, which reduced the heat stability of the SHMP samples.

(56) In conclusion, calcium chelators increase the heat stability of the MCI solution to different extents and these differences are attributable to the calcium-ion activity and state of the micellar structure before heating. Optimization of heat stability of dairy systems is complex and therefore selection of the type and concentration of calcium chelator requires careful investigation. Surprisingly, pronounced effects on heat stability are observed with nucleotides.