Article pubs.acs.org/JAFC
Calcium Phosphates in Ca2+-Fortified Milk: Phase Identification and Quantification by Raman Spectroscopy Martha Arifin,† Peter J. Swedlund,*,† Yacine Hemar,† and Ian R. McKinnon‡ †
School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand School of Chemistry Monash University, P. O. Box 23, Clayton, Victoria 3800, Australia
‡
S Supporting Information *
ABSTRACT: Calcium phosphate nanoclusters (CPNs) are important for the structure, function, and nutrient density of many dairy products. Phosphorylated amino acids in caseins stabilize calcium phosphate as nanoclusters which are amorphous to X-ray diffraction and exist within casein micelles, and these CPNs play a key role in micelle stability. Addition of calcium to milk results in further calcium phosphate removal from the serum, and there is uncertainty about the nature of the material formed and its stability. In this work we investigate both the solution and colloidal phases in CaCl2 enriched bovine milk to identify, quantify, and determine the solubility of the calcium phosphate material formed in response to calcium addition to milk. The P−O stretching bands are quite distinct in the Raman spectra of the main synthetic calcium phosphate mineral phases, including the amorphous calcium phosphate phase. In response to adding between 5 and 40 mM CaCl2 to milk, the serum phosphate concentration decreased asymptotically from 7.5 ± 0.2 to 0.54 ± 0.05 mM. Using Raman spectroscopy with a combination of internal and external standards, it was possible to show that the calcium phosphate material formed after Ca2+ addition to milk was the same as amorphous calcium phosphate nanoclusters present in the absence of added calcium. The use of an internal standard allowed a quantitative analysis of the spectra which demonstrated that the amorphous calcium phosphate formed accounted for all of the calcium and phosphate that was removed from solution in response to calcium addition. KEYWORDS: quantitative Raman, milk, calcium fortification, calcium phosphate, phosphoserine
■
INTRODUCTION The bones and teeth of mammalian infants impose a significant nutritional requirement for calcium and phosphate. Because calcium phosphate minerals have an inherently low solubility, the way in which milks meet this need relies upon a complex interaction between calcium ions and phosphorylated amino acids present in the casein group of milk proteins.1 Proteins that contain centers of phosphorylated amino acid residues, typically phosphoserines, can bind strongly to calcium ions on the surface of calcium phosphate particles.2,3 The presence of phosphopeptides stabilizes calcium phosphate as particles of amorphous calcium phosphate (ACP) with diameters of just a few nanometers. The phosphoserine−calcium interaction is an important factor, along with the presence of magnesium, in preventing precipitation of crystalline calcium phosphate phases, such as hydroxyapatite (HAP), which are thermodynamically more stable but are not able to be transported in milk.1,4,5 Even though the calcium phosphate particles are amorphous in terms of X-ray diffraction, it has been proposed that the calcium phosphate particles are structurally analogous to the nascent nuclei of dicalcium phosphate3 (DCP) with stoichiometry CaHPO4·2H2O. Note that dicalcium refers to the number of Ca2+ per two phosphates as in tricalcium phosphate, Ca3(PO4)2. In fact while the ratio of calcium to inorganic phosphate (Pi) in the particles is approximately 2, the particles contain almost all of the phoshpopeptide residues yielding a total Ca/P ratio of approximately 1.25.3 These phosphopeptide-stabilized nanometer sized particles are referred to as calcium phosphate nanoclusters (CPNs). The CPNs exist © 2014 American Chemical Society
within casein micelles which are on the order of 100 nm in diameter,6,7 and several structural models have been proposed for the arrangement of the casein−calcium−phosphate networks in these micelles.8−10 While CPNs were once thought to be metastable and in a state of arrested precipitation, it is now considered that they represent equilibrium complexes with casein phosphopeptides in which the serum is undersaturated with respect to the sequestered ACP phase.11,12 A key aspect of stabilizing CPNs is preventing the nucleation of HAP. Suspensions of ACP in the absence of phosphopeptides are metastable and will convert to HAP via a process of ACP dissolution followed by reprecipitataion at HAP nuclei. The inhibition of HAP nucleation is a requirement for the stability of the milk casein−calcium phosphate system. While the form of the calcium phosphate in native milk has been studied extensively, less attention has been paid to milks with added calcium. Therefore, uncertainty remains in terms of the identification, quantification, and stability of the calcium phosphate that forms in response to calcium addition in the production of calcium-fortified milks. Raman spectroscopy is a powerful probe for the study of poorly ordered phases13 because the vibrational bands depend upon the local environment of the molecular moiety and are not necessarily dependent on long-range order. Raman is also applicable to aqueous systems such as milk,14 unlike infrared Received: Revised: Accepted: Published: 12223
July 29, 2014 November 12, 2014 November 21, 2014 November 21, 2014 dx.doi.org/10.1021/jf503602n | J. Agric. Food Chem. 2014, 62, 12223−12228
Journal of Agricultural and Food Chemistry
Article
to 11 with ≈4 mL of 28% NH4OH and followed by dilution to 180 mL) over 30−40 min.19 The resultant suspension was stirred for 24 h and centrifuged at 2000 rpm for 10 min and then the solid washed with water and recentrifuged. The resultant white pellet was vacuum filtered for ≈3 h, and then the filter cake was dried at 90 °C. Solid Phase Characterization. Raman spectra were measured using a Renishaw Raman Imaging Microscope System 1000 equipped with a confocal Leica microscope and CCD detector. Spectra were collected using either the 785 or the 488 nm laser as discussed in Results and Discussion. The instrument was calibrated daily using the 1086 cm−1 calcite peak. To allow for quantitative information from the spectra, a range of compounds were assessed for suitability as internal standards. The ideal internal standard should have a strong Raman signal but have no peaks in the 910−1110 cm−1 region of the spectrum where the characteristic calcium phosphate phases are observed. The internal standard should not interfere with the chemistry of the system, for example by acting as a ligand toward Ca2+. Powder X-ray diffraction (XRD) patterns were measured using a Model D5000 Siemens instrument with a Cu Kα source operating at 40 kV and 30 mA. Solid samples were crushed to a fine powder and mounted in an aluminum well. XRD data were collected between 2 and 70° 2θ with a step size of 0.02° 2θ. The morphology and topography were examined using the environmental scanning electron microscopy (ESEM; FEI Quanta 200F, Hillsboro, OR, USA) with large-field detector (LFD) at 0.58 Torr pressure. Samples were mounted on black carbon tape and sputter coated with platinum (Pt) for 15 min using a Quorum Q150RS at 20 mA current and 1 × 10−1 mBar chamber pressure. Solution Phase Characterization. Prior to cation analysis, the milk and milk sera samples were treated using the procedure from Fox et al.20 Initially 1 mL of 24% TCA was added to 2 mL of a 10-fold diluted sample and the mixture shaken for 30 min. Following this, 1 mL of 5% LaCl3·7H2O was added as an ionization suppressor for the atomic absorption measurement and the samples were diluted to 50.0 mL with water. All samples were centrifuged at 3500 rpm for 30 min, and the resultant supernatants were collected for measurement. Calcium and magnesium were quantified using atomic absorption spectroscopy (AAS) while atomic emission spectroscopy (AES) was used for sodium and potassium. All measurements were made using an air−acetylene flame and parameters described by de la Fuente and Juarez21 on a Varian AA-1275 series atomic absorption spectrophotometer (Varian Techron Pty. Ltd., Springvale, Australia). The standards for AAS and AES were prepared from TraceCERT grade 1000 ppm reference standards that were diluted and pretreated using the same method as the samples. Phosphorus in the sera was quantified using a colorimetric method based on that by Allen.22 Initially protein was precipitated by adding 2.5 mL of 24% TCA to 5 mL of a 10-fold diluted sample and the mixture shaken for 30 min, then diluted to 25 mL, and centrifuged at 3500 rpm for 30 min. Supernatant (18 mL) was removed and to this was added 1.17 mL of 70% HNO3, 2 mL of amidol reagent (2 g of 2,4-diaminophenol dihydrochloride and 40 g of sodium bisulphite in 200 mL water) and 1 mL of an 8.3% (w/v) ammonium molybdate reagent. Samples were mixed for 5 min and left to stand for 15 min, and then absorbance was measured at 695 nm and compared to matrix matched standards. All milk samples were prepared in duplicate, and the error bars on the figures represent one standard deviation. The milk pellet samples with the internal standard for Raman analysis were prepared in duplicate, and the relationship between Raman band intensity and the amount of phosphate removed from the serum was determined by regression analysis using the Data Analysis Tools facility in EXCEL and a 95% confidence interval. Modeling the Serum Chemistry. The distribution of calcium and Pi in the milk without added CaCl2 was modeled using the framework from Holt.3 The model input concentrations for fat were 0 g L−1, while lactose, total protein, and casein were the measured values of 50.1, 35.5, and 27.3 g L−1, respectively, and the total concentration of phosphate centers (PCs) was 1.51 mM based on the distribution of phosphate centers per mole of each casein from Holt.3 The ratios of Ca2+, Pi, Mg2+, and citrate per PC in the nanoclusters were 13.2, 6.5, 1.0, and 0.637, respectively. The model input total cation
(IR) spectroscopy which is limited by the strong IR absorbance of water. The objective of this study is to use Raman spectroscopy with a range of external and internal standards to identify and quantify the calcium phosphate that forms in response to CaCl2 addition to bovine milk. The study combines the Raman analysis with standard wet chemical analyses to determine the re-distribution of calcium, phosphate, and the major inorganic milk constituents in response to CaCl2 addition to milk and compares this to the solubility product of the calcium phosphate minerals.
■
MATERIALS AND METHODS
Chemicals. All water used in the experiments was Milli-Q water with a resistivity of 18.2 MΩ·cm. The CaCl2, NaOH, trichloroacetic acid (TCA), and LaCl3·7H2O were purchased from Sigma-Aldrich. The NH4OH, (NH4)2HPO4, Na2HPO4, CaCl2·2H2O, H3PO4, and boric acid were purchased from AJAX Finechem while the NaNO3 was from Fluka. Milk Samples. The milk was prepared from a low-heat skim milk powder sample supplied by Westland Co-operative Dairy Co. Ltd., Hokitika, New Zealand, and had a composition of 35.5% protein and 1.25% fat. A stock skim milk was prepared with 20% (w/w) skim milk powder in Milli-Q water and was stirred for 2 h on a magnetic stirrer to ensure complete dispersal of the powder. Calcium fortification between 5 and 50 mM was achieved by adding the appropriate amounts of 0.2 M CaCl2 solution to the milk. After 3 h at room temperature, the pH was adjusted back to 6.7, water was added to take the milk to 11.1% (w/w) solids, and this was left overnight at room temperature. The pH was again adjusted to 6.7 and water added to produce the final milk with 10% (w/w) solids. Changes in pH over time were manually corrected, and the sample was held until there was no further drift in pH. To achieve phase separation between the micellar and serum phases, the milk samples were ultracentrifuged at 20 °C and 38000g for 1 h using an Avanti-J-30I instrument with JA25.50 rotor (Beckman Coulter, Brea, CA, USA). The supernatants were immediately collected by decantation and centrifuged again at 3000 rpm for 30 min in Vivaspin Turbo 15 containing poly(ether sulfone) membranes with molecular weight cutoff inferior to 10 kDa (Sartorius Stedim Biotech GmbH, Goettingen, Germany). These samples are termed the milk sera, and the pellets remaining after ultracentrifugation are the milk pellet samples. Preparation of Reference Calcium Phosphate Phases. Standard methods were used to prepare the reference calcium phosphate phases. Amorphous calcium phosphate was prepared by rapidly adding a calcium solution (46.3 g of Ca(NO3)2·4H2O in 550 mL of water containing 40 mL of 28% NH4OH) to a phosphate solution (27.2 g of (NH4)2HPO4 in 1,300 mL of water containing 40 mL of 28% NH4OH).15 The resulting precipitate was immediately filtered, washed, and freeze-dried. Dicalcium phosphate dihydrate (DCPD, CaHPO4·2H2O) was prepared by rapidly adding a calcium solution (4.014 g of CaCl2·2H2O in 200 mL of water at pH 5.5) to a phosphate solution (0.825 g of KH2PO4 in 700 mL of water and then addition of 3.01 g of Na2HPO4, pH 7.4), aging for 80 min, and then filtration and drying at 65 °C.16 The β-tricalcium phosphate (β-TCP, β-Ca3(PO4)2) was prepared by dropwise addition of a calcium solution (71.9 g of Ca(NO3)2·4H2O in 250 mL of water at pH 6.30) to a phosphate solution (27.0 g of (NH4)2HPO4 in 250 mL of water at pH 8.28) over 150 min while vigorously stirring and maintaining the pH at 8 by manually adding NaOH.17 The suspension was stirred for 12 h, and solids were filtered, washed, dried at 80 °C, and then calcined at 700 °C for 2 h. Octacalcium phosphate (OCP, Ca8H2(PO4)6·5H2O) was prepared by adding 11.69 g of concentrated H3PO4 and 13.3 g of CaCO3 to 1 L of water, stirring for 6 h at 60 °C, and followed by filtration and drying at 60 °C.18 Hydroxylapatite (HAP, Ca5OH(PO4)3) was prepared by dropwise addition of a phosphate solution (25.87 g of (NH4)2HPO4 in 150 mL of water, pH to 11 with ≈80 mL of NH4OH and then dilution to 320 mL) to a vigorously stirred calcium solution (43.15 g of Ca(NO3)2·4H2O in 90 mL of water, pH 12224
dx.doi.org/10.1021/jf503602n | J. Agric. Food Chem. 2014, 62, 12223−12228
Journal of Agricultural and Food Chemistry
Article
concentrations for Mg2+, Na+, and K+ were the measured values of 4.29, 13.9, and 31.3 mM, respectively, while the total anion concentrations for phosphate, sulfate, citrate, and a generic carboxylate (ROO−) were 17.2, 0.12, 4.66, and 3.1 mM, respectively. The calcium and chloride values varied with the added CaCl2, the pH was 6.7, and the partial pressure of CO2 was set at 0. Activity coefficients were calculated using the Davies equation except in the cases of Ca2+, Mg2+, H+, and the −3 charged citrate anion where the ion specific Truesdell−Jones equation was used. The solution phase complexation and protonation constants are from Holt.3
milk serum contrasts with inorganic ACP in an aqueous suspension for which the transformation to HAP can occur on the time scale of minutes to hours. Table SI1 (SI) also gives the KSP and IAP for two ACP phases from Christoffersen et al.,24 where mixing CaCl2 and K2HPO4 solutions initially formed an amorphous phase denoted ACP1 followed after a few minutes by formation of a less soluble amorphous phase termed ACP2. The milk is undersaturated with respect to ACP1 but is oversaturated with respect to ACP2. Serum Chemistry in Response to Added Calcium. The measured concentrations of calcium and Pi in the sera of milk with added CaCl2 are shown in Figure 1a. In response to CaCl2
■
RESULTS AND DISCUSSION Serum Chemistry. Serum Chemistry without Added Ca2+. The measured total concentrations of calcium and Pi in the milk were 28.0 ± 0.8 mM and 17.2 ± 0.1 mM, respectively. These values are at the low end or slightly below the typical ranges of 26−32 mM and 19−23 mM for total calcium and Pi, respectively.23 In the serum phase the corresponding measured concentrations were 7.0 ± 0.5 mM for calcium and 7.5 ± 0.2 mM for the Pi which represent concentrations inferior to 10 kDa fraction. While some of the nondiffusible calcium will be associated with nonmiscellar proteins, the “great majority of the nondiffusible salts are in the form of colloidal calcium phosphate”3 indicating that approximately 21 mM calcium and 10 mM Pi were contained within the calcium phosphate nanoclusters. The 2:1 ratio of calcium to Pi in the nanoclusters is typical3 of milk CPNs but does not reflect the calcium phosphate phase because of the inclusion of the casein based phosphorylated serine residues with the structure. The distribution of calcium and Pi between the serum and the CPN was in good agreement with the ion activity product (IAP) of Holt3 which is (Ca2+)(HPO42−)0.7(PO43−)0.2 where (X) is the activity of species X and the IAP has a value of 10−6.80, whereas the value calculated from the measured parameters in this work was 10−6.81. Based on the PC concentration in the milk (1.51 mM) and the Ca:Pi:PC ratio in the CPN (13.2:6.5:1), almost 100% of the PCs are incorporated into the CPNs in the milk without added CaCl2. It is instructive to consider this system with regard to the individual terms in the equation prior to discussing the systems with added calcium. The 7.5 mM Pi in the serum is predominantly present as either H2PO4− (4.3 mM) or as complexes with the group I or II cations present in the milk (1.7 mM). The calculated concentration of HPO42− is 1.3 mM and the activity coefficient (γ2) = 0.45 so ((HPO42−)0.7) = 10−2.26. Because the pH of the milk (6.7) is much lower than the pKa of HPO42− (12.4), the calculated concentration of PO43− is very low at 7.4 nM. The activity coefficient (γ3) is 0.15 so ((PO43−)0.2) = 10−1.8. In the case of the 7.0 mM serum calcium approximately 3 mM is complexed by citrate while another 1 mM is complexed by inorganic ligands including HPO4− and Cl−. This leaves 3.5 mM free Ca2+ in solution and with a γ2 of 0.45 the (Ca2+) = 10−2.80. Therefore, the presence of citrate in the serum approximately doubles the solubility of calcium. Table SI1 (Supporting Information (SI)) gives the algebraic expressions and values for KSP and the calculated serum IAP for each of the calcium phosphate phases. The milk serum is oversaturated with respect to all of the main crystalline calcium phosphate phases and is most oversaturated with respect to HAP with a ratio of the IAP to the KSP of 1010.7. This means that, all else being equal, the (Ca2+) would have to be 140 times lower, i.e., (1010.7)0.2, for the solution to be saturated with respect to HAP. The failure of HAP to precipitate from the
Figure 1. Response of milk sera to added CaCl2: (a) measured calcium and Pi in the
