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Calcium carbonate–hydrolyzed soy protein complexation in the presence of citric acid
Journal of Colloid and Interface Science 345 (2010) 88–95
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Calcium carbonate–hydrolyzed soy protein complexation in the presence of citric acid Latha-Selvi Canabady-Rochelle a,*, Christian Sanchez a,1, Michel Mellema b, Sylvie Banon a,2 a
Nancy-Université, Institut National Polytechnique de Lorraine, Ecole Nationale Supérieure d’Agronomie et des Industries Alimentaires, Laboratoire d’Ingénierie des Biomolécules, 2, Avenue de la forêt de Hayes, 54 505 Vandoeuvre-Lès-Nancy, France b Unilever R&D Vlaardingen, PO Box 114, Olivier van Noortlaan 120, 3130 AC Vlaardingen, The Netherlands
a r t i c l e
i n f o
Article history: Received 13 November 2009 Accepted 13 January 2010 Available online 18 January 2010 Keywords: Calcium carbonate Soy protein Complexation Amorphous phase ITC SEM XPS
a b s t r a c t The influence of hydrolyzed soy proteins on calcium carbonate stabilization was studied in citric acid solution. Calcium–soy proteins interactions were characterized using a calcium ion selective electrode, turbidity, and Isothermal Titration Calorimetry. Once the meta-stable phase was reached or just after soy protein addition, spray-drying was performed and SEM, XRD, and XPS analysis were carried out on spray-dried powders. In citric acid solution calcite crystals were eroded giving rise to smaller amorphous particles. In the presence of soy proteins, complexation exothermic in nature occurred with the mineral phase, which prevented CaCO3 from recrystallisation and kept the system in an amorphous state. SEM performed on spray-dried powder showed that soy proteins were swollen in presence of mineral phase and resulted in a decrease of calcium concentration at the extreme surface of the studied powders as demonstrated by XPS. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Recent studies on consumer habits reported that some populations at risk do not satisfy their Recommended Daily Allowances (RDA) in calcium (Ca). More specifically, teenage girls from 10 to 19 years old, women older than 55 years, men older than 65 years and elderly people are concerned by this Ca deficiency [1]. According to an INCA survey [2], even in western countries, these specific populations at risk do not satisfy the RDA for Ca. Yet Ca is a mineral essential for humans, especially during growth [3] and during old-age to act against osteoporosis [4–8]. Hence, this nutritional deficiency in Ca presents a world-wide health stake that can be overcome by Ca supplementation of food. Dairy products, naturally rich in Ca and with a high Ca bioavailability, are appropriate foods for Ca supplementation [9]. Nevertheless for various nutritional, ethical, religious or economic reasons, cow’s milk cannot be consumed by specific populations. Hence, soy-based drinks
* Corresponding author. Present address: Biomolecular and Materials Interface Research Group, School of Science and Technology, Nottingham Trent University, Clifton lane, Nottingham NG11 8NS, United Kingdom. E-mail address: [email protected] (L.-S. Canabady-Rochelle). 1 Present address: Laboratoire Ingénierie des Agropolymères et Technologies Emergentes, Université Montpellier II, cc 023 Pl. E. Bataillon, 34 095 Montpellier cedex 05, France. 2 Present address: Nancy-Université, Université des Sciences et Techniques Henri Poincaré, LIMOS UMR CNRS 7137, Campus Victor Grignard BP 239, 54 506 Vandoeuvre-Lès-Nancy, France. 0021-9797/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2010.01.037
supplemented with Ca constitute an alternative to dairy drinks, especially for populations suffering from Cow’s Milk protein Allergy [10] and/or lactose intolerance [11]. Various Ca salts, among them Ca carbonate, are used for soy-based drink supplementation, which present various characteristics in regards to the physical–chemical stability of the system (solubility/sedimentation of salt, aggregation and precipitation of proteins). Crystalline Ca carbonate has three different polymorphs, vaterite, aragonite and calcite, whose solubility is also in accordance with this sequence. The least stable form is vaterite followed by aragonite and calcite, which is thermodynamically the most stable phase [12]. In addition, there also exist three hydrated forms, i.e. Amorphous Calcium Carbonate (ACC), highly unstable at room temperature and pressure as reported by Herman et al. [13], monohydrate calcium carbonate and calcium carbonate hexahydrate (CaCO36H2O). ACC can function as a transient precursor of the more stable crystalline aragonite and calcite and draws much attention due to its role in biomineralisation as reviewed by Addadi et al. [14]. Mineral crystallization, and especially CaCO3 crystallization, is often shaped by macromolecules [15]. Dutour-Sikiric and Füredi-Milhofer [16] reported the influence of surface active molecules on the crystallization of biominerals in solution. Modification of Ca carbonate powder characteristics can be obtained by the use of various additives such as carboxylic acids [17] and various polymers [18], while carrying out the reaction without any additive leads to difficulties in controlling the
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shape and the precipitated crystallographic form [19]. The dissolution of these minerals can lead to the meta-stable zone defined as a supersaturation subregion located between the precipitation straight line obtained experimentally and the equilibrium curve of the least soluble form of Ca carbonate, which is calcite in the CaCO3–CO2–H2O system [20]. In this phase characterized by its transparency, nanosized Ca carbonate could remain in suspension and serve as a ‘‘seed” for subsequent interactions with biopolymers [19]. Amorphous calcium carbonate could also be present and could interact with biopolymers as in biomineralization mechanisms [21,22]. Other interaction mechanisms could occur via citrate, a chelator of Ca, which can reduce the crystallization growth rate [23]. In a previous study [24], Ca chloride was supplemented in a soy-based drink and Ca–soy protein interactions as the origin of Ca-induced soy protein aggregates were studied. The effect of pH cycle was also investigated on the restructuring and stability of these former aggregates, which could then be used as possible vectors for further Ca delivery. This former work was followed by the study of another type of Ca source, Ca carbonate (CaCO3) commonly used in the food industry with advantageous economies (cheapest source of Ca). Moreover, Ca carbonate, due to its neutral taste, is often preferred to Ca chloride, which can involve bitterness perception by consumers in supplemented products. The major drawback of Ca carbonate is its low solubility (0.014 g L 1) in comparison to the high solubility of Ca chloride (745 g L 1) [25]. In an approach to decrease the sedimentation phenomenon observed for Ca carbonate and improve its solubility, we have acidified the reaction medium and added soy proteins as chelating agents. The scientific aim of this work was to understand the mechanism occurring upon Ca carbonate solubilization in citric acid in the presence of soy proteins. Interactions involved in such systems (CaCO3/citric acid/soy proteins) were studied in solution using the microcalorimetric method, i.e. Isothermal Titration Calorimetry. Once the meta-stable phase was reached or just after the soy protein addition, spray-drying was carried out and the system was analyzed. 2. Materials and methods 2.1. Calcium–soy protein interactions 2.1.1. Dispersion preparation Citric acid (monohydrate, analytical reagent, VWR Prolabo, Leuven, Belgium) was solubilized in distilled water at 0.57% w/w at room temperature, followed by the addition of 0.23% w/w calcium carbonate (CaCO3, Rectapur, Prolabo, Merk, Briare le Canal, France). Chemical reactions between the different molecular species led to a meta-stable system characterized by its transparency and named the ‘‘acid mineral solution”. Hydrolyzed soy proteins (H-SP, non-GM soy protein isolate 219, The Solae company, Barcelona, Spain) were supplemented to stabilize the ‘‘acid mineral solution” against sedimentation. The final concentration of the acid mineral–soy protein dispersion was composed of 70% w/w of the acid mineral solution and 1% w/w of soy proteins. This acid mineral–soy protein dispersion was completed with distilled water (up to 100% w/w). 2.1.2. Reactor experimental set up The preparation of the acid mineral solution and acid mineral– soy protein dispersion was followed using the experimental set up described previously [26]. A pH-meter (Radiometer analytical, Remiremont, France), and Calcium Ion Selective Electrode (CaISE, Sentek, Estate, Braintree, United Kingdom) were immersed into the bulk. A turbidity sensor (Analite NEP 160, McVan Instruments,
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Mulgrave, Australia) was placed through the vessel wall to avoid disturbances during stirring. Continuous monitoring (Almemo 8990-8 V5, Ahlborn, Holzkirchen, Germany) of pH, ionized Ca (Ca2+) and turbidity was performed using a data logger coupled with a PC equipped with software (AMR WinControl for Almemo, Akrobit, Gera, Germany). Ca ISE was used to evaluate Ca ionization [27–30]. The pH-meter and Ca ISE were daily calibrated at the experimental temperature (Texptl = 20 °C). pH-meter calibration was performed at pH 7.00 and pH 4.00 and the accuracy of the measurement was ±0.02 pH unit. For the acid mineral solution study, Ca carbonate was added after 5 min of probe stabilization in the citric acid solution. For the study of calcium–soy protein interactions in citric acid solution, a soy protein dispersion was prepared in distilled water and added to the acid mineral solution, in which probes were stabilized, up to reach the final concentration described previously. 2.1.3. Thermodynamic characterization of calcium–soy protein interactions For the study of Ca–soy protein interactions in citric acid solution, the acid mineral solution and soy protein dispersion were prepared as described above. Each solution was diluted three times in milliQ water to obtain an ITC signal, within the range of detection of the apparatus. Protein concentration in the soy protein dispersion was determined according to the Bradford method. A calibration curve was performed with Bovine Serum Albumin solution at 1.35 mg mL 1. A VP-ITC microcalorimeter (MicroCal, North-Hampton, MA, USA) is composed of two identical spherical cells, a reference cell and a sample cell, both with a volume of 1.449 mL, which are enclosed in an adiabatic jacket. Before each experiment, both solutions (the acid mineral solution and the soy protein dispersion) were degassed for 7 min to eliminate air bubbles, which could perturb the baseline. Then, the sample and the reference cells were, respectively, filled with the acid mineral solution and the solvent used to prepare the sample solution (i.e., milliQ water). The titrant, the soy protein dispersion, was prepared in milliQ water, and injected in a single step (250 lL; duration: 75.3 s) into the sample cell. The measurement was performed under stirring (300 rpm) at constant temperature (Texptl = 20 °C). The spacing time between each consecutive measurement was of 300 s. Due to the lack of information about the expected heats of the studied system, the reference power of the ITC instrument was set at 15 lcal s 1. The experiment lasted 3600 s. Each experiment was performed in triplicate. In the analysis, the injection of soy protein dispersion into milliQ water corresponded to the reference, and was used to determine the heat of dilution of soy proteins. Data for this reference experiment, carried out in the same way as the sample experiment (soy protein dispersion titrated in acid mineral solution), was subtracted from the sample data. Binding curves were plotted in kcal mol 1 of injectant as a function of time. 2.2. Spray-dried powder characterization 2.2.1. Spray-drying Once the meta-stable phase was reached in solution or just after the soy protein addition, spray-drying was performed (Büchi mini spray-dryer B191, Switzerland. The inlet and the outlet temperatures were set at 180 °C and 80 °C, respectively. The pump and the aspirator activity were set at 55% and at 70%, respectively. Acid mineral powders were prepared in the absence or in the presence of soy proteins. Calcium carbonate and soy proteins (both solubilized in distilled water and then spray-dried) were studied as references powders.
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2.2.2. Electron microscopy (EM) Scanning Electron Microscopy (SEM). The samples were attached to double-sided adhesive tape attached to SEM stubs, covered with a carbon layer of 10 nm and then a mix of gold/ palladium by sputtering (Biorad type SC 502). The samples were then examined with a SEM S2500 instrument (Hitachi, Science Systems, Ibaraki, Japan) operated at 10 kV. Measurements were carried out under secondary vacuum (about 10 5 torr) by retro diffusion of secondary electrons. Energy dispersive X-ray analysis carried out during SEM experiments gave some information on the local composition of the studied sample over a depth of a few lm. The detection limits for the elements varied between 0.1% and 0.5% of the total elements detected and was determined by the continuous background noise of the spectrum. Environmental scanning electron microscopy (ESEM). ESEM was carried out using a SEM Quanta 600 FEG apparatus (FEI Company, Eindhoven, Netherlands). This experiment was performed to determine whether or not vacuum induced artifacts would be seen in the standard SEM imagery. 2.2.3. X-ray diffraction (XRD) XRD experiments were carried out using an X’pert PRO diffractometer apparatus (PANalytical, Almelo, Netherlands). The instrument used a Bragg–Brentano geometry. The X-ray beam used a copper anticathode with a wavelength k of 0.70926 Å. The beam was converged with a Germanium monochromator selecting a raw Ka1, with the Soller slit having a divergence of 0.02 Rad. The goniometer was used in h 2h geometry and the measurement was done in reflexion mode, with the sample located onto a spinner. The detector used was an Xcelerator, the measurement step was of 0.017° with a time by step of 712 s. 2.2.4. X-photoelectron spectroscopy (XPS) XPS analyzes were carried out with a Kratos Axis Ultra spectrometer (Kratos analytical, Manchester, UK) using a set monochromatic radiation A1Ka source with incident energy of 1486.8 eV. The power applied at the X-ray anode was reduced to 90 W in order to avoid X-ray induced degradation of the sample. The instrument work function was calibrated to give a binding energy (EB) of 83.96 eV for the Au 4f7/2 line for metallic gold and the spectrometer dispersion was adjusted to give a binding energy of 932.65 eV for Cu 2p3/2 for metallic copper. All spectra were recorded at a take-off angle of 90°, which enabled analysis to about a 5 nm-depth over an area of about 700 lm 300 lm. Survey spectra were recorded with 1.0 eV steps and 160 eV analyzer pass energy. The high resolution regions (peaks of C, O, and N) were recorded with 0.1 eV steps for O 1s and N 1s, and with 0.05 eV step for C 1s. In both cases, the hybrid lens mode was employed. During data acquisition the Kratos charge neutralizer system was used to avoid the overcompensation phenomenon (peak shift towards higher levels of energy due to isolating samples) and the following settings were used: filament current 1.6 A, charge balance 2.4 V, filament bias 1.0 V and magnetic lens trim coil 0.375 A. As overcompensation was always observed, the C 1s line for adventious carbon and C–C carbon was set to 284.60 eV and therefore used as an internal energy reference to enable comparison of results for various samples. With these parameters we could obtain a C 1s signal with sharp, symmetric components with FWHM of 1.2 eV. Spectra were analyzed using the Vision software from Kratos (Vision 2.2.6). A Shirley baseline allowed background noise subtraction whereas Gaussian (70%) and Lorentzian (30%) models were used for spectral decomposition. Indeed, this former treatment enables a chemical analysis through the determination of
the nature of the surface binding. Quantification was performed using the photoemission cross-sections and the transmission coefficients provided in the Vision package.
3. Results and discussion 3.1. Calcium–soy protein interactions 3.1.1. Ionized calcium (Ca2+) variations In the absence of soy proteins, Ca carbonate (CaCO3) dissolved in citric acid solution. The ionized Ca concentration (M) increased from 0 to about 8 mM and was stabilized at this concentration for the whole reaction period (24 h, Fig. 1A). This equilibrium resulted from an ionic balance between dissolution and precipitation phenomena. Simultaneously the pH value measured changed from pH 2.5 (pH of the citric acid solution) to pH 4.2 (pH of the acid mineral solution). This former pH value slightly decreased over the time, which may be due to the CO2 solubilization from ambient air upon stirring and formation of HCO3 , with H+ ions into the bulk. Similarly, Watanabe and Akashi [31] observed CaCO3 dissolution in an acidic environment, especially in citric acid solution and their dissolution rate measured represented up to 80% of the initial Ca ion content. Upon soy protein supplementation into the acid mineral solution, the ionized Ca concentration decreased first from about 8 mM to 5 mM, with final equilibrate ion at around 6 mM (Fig. 1B). Simultaneously the pH value changed from pH 4.2 in the acid mineral solution to pH 4.5 in the presence of soy proteins. The ionized Ca concentration decrease observed upon soy protein addition is due to the interaction of ionized Ca (Ca2+) with soy proteins. Soy proteins are rich in residues of acidic amino-acids and contain 11.6% and 19.1% (w/w) of aspartic acid and glutamic acid, respectively. In the range pH 4.2–pH 4.5 and taking into account the pKR value of the acidic amino-acids (3.86 and 4.25 for aspartate and glutamate, respectively), the acidic group of the sidechain will be negatively charged and can bind Ca2+, even if the global charge of the soy protein is close to a zero value. Indeed, pH 4.5 agrees with the isoelectrical pH (pHi) of soy protein commonly reported in the literature [32,33]. During the first hour, ionized Ca concentration slightly increased from 5 mM to 6 mM, which may be due to equilibrium of Ca2+ between the soluble and the protein colloidal phase as reported by Canabady-Rochelle et al. [24].
3.1.2. Turbidity (s) variations Turbidity variations were followed during the whole process to characterize the colloidal phase. The turbidity signal depends on three main factors: size, concentration and optical properties of the particles in solution. In the absence of soy proteins, Ca carbonate (CaCO3) dissolved in citric acid solution. The initial turbidity of the citric acid solution of 10 NTU (NTU, for Nephelometric Turbidity Unit) increased to about 400 NTU upon CaCO3 addition. After complete CaCO3 dissolution, turbidity decreased to its initial value (10 NTU, Fig. 1C). Hence, the turbidity peak corresponds to the kinetic of dissolution of CaCO3 and is time dependent. On the contrary, upon soy protein addition in the acid mineral solution (Citric acid/CaCO3), the turbidity increased from 10 NTU (transparency of the meta-stable phase) to 5600 NTU (Fig. 1D) and remained stable over the remaining time of the experiment (no peak observed). This turbidity increase could be due to the calcium-induced aggregation, as previously reported. Moreover at such pH, equivalent to the isoelectrical pH of soy proteins (pHi = 4.5), soy proteins can also aggregate.
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(A)
Ionized calcium concentration (M)
8.0x10-3
(C)
1000 900
-3
7.0x10
800
Turbidity (NTU)
6.0x10-3 5.0x10-3 4.0x10-3 3.0x10-3 2.0x10-3 1.0x10
-3
700 600 500 400 300 200 100
0.0
0
-3
-1.0x10
0.1
1
0.1
10
1
10
Time (hours) 6000
(B)
1.0x10-2 -3
9.0x10
(D)
5000
8.0x10-3
Turbidity (NTU)
Ionized calcium concentration (M)
Time (hours)
7.0x10-3 -3
6.0x10
5.0x10-3 4.0x10-3 3.0x10-3
4000 3000 2000 1000
2.0x10-3 1.0x10-3
0
0.0 0.1
1
10
0.1
Time (hours)
1
10
Time (hours)
Fig. 1. Ionized calcium concentration (A and B) and turbidity variations (C and D) versus time (hours) in the absence or in the presence of soy proteins, respectively. At 0.1 h, CaCO3 was added in citric acid solution (panels A and C). At 0.1 h, soy proteins were added in the acid mineral solution (citric acid/CaCO3; panels B and D).
Heat of binding (Cp, µcal.sec-1)
3.1.3. Thermodynamic characterization of Ca–soy protein interactions The soy protein dispersion was injected in a single step into the acid mineral solution. A scaled down reaction was followed with ITC and the thermodynamic signal obtained is presented in Fig. 2. A highly exothermic signal (DH < 0) was observed, which could be decomposed into two parts, corresponding to two exothermic peaks. This result was unexpected since an endothermic phenomenon was observed in Canabady-Rochelle et al. [24] on Ca–soy proteins interactions. Nevertheless, in the former study, the Ca source was the highly soluble Ca chloride without any citric acid in solution.
0 -10 -20 -30 -40
Wan et al. [34] studied by microcalorimetry the precipitation of calcium carbonate from solution as a function of L-aspartic acid (LAsp) concentration. They observed that the precipitation of CaCO3 was an endothermic process with the thermal spectrum divided into two peaks: first a major endothermic event and then, a second minor endothermic peak whose intensity was inversely related to the amount of L-Asp in solution. By means of FT-IR data, former authors have shown that as the amount of L-Asp increased, the amount of vaterite in the precipitate increased accordingly. Indeed, L-Asp is known to induce the formation and stabilize the existence of vaterite crystals [35]. Wan et al. proposed that the first major peak can be related to the precipitation process and that the second peak (endothermic process) fits with the transition from a meta-stable phase to calcite, which formed by recrystallization of the meta-stable phase (combination of dissolution and recrystallization). With higher L-Asp concentration, less of the meta-stable phase (vaterite) would transform into calcite and the minor endothermic peak resulting from the recrystallization event decreased. In accordance with these results, we propose that in our case the two peaks observed relate to exothermic processes with the transition from crystalline forms to meta-stable phases in the presence of soy proteins, which are rich in acidic amino-acids.
-50
3.2. Spray-dried powder characterization -60 0
500
1000
1500
2000
2500
3000
3500
4000
Time (sec) Fig. 2. Thermodynamic signal obtained upon soy protein titration into acid mineral solution (citric acid/CaCO3). The reference signal (soy protein titrated into milliQ water) was substracted.
3.2.1. Electron microscopy (EM) Scanning Electron Microscopy (SEM) was performed on various spray-dried powders. Spray-dried powder, obtained from CaCO3 solubilized in distilled water, was studied as the CaCO3 control (Fig. 3A). Cubic-shaped crystals of Ca 10 lm side were observed.
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Fig. 3. SEM (A–D) and ESEM (E and F) micrographs. (A) Spray-dried powder of CaCO3 control; (B) acid mineral spray-dried powder in the absence of soy protein; and (C) Spray-dried powder of soy protein control; (D–F) Acid mineral spray-dried powder in the presence of soy proteins.
This crystal form was characteristic of calcite. On some crystals, further nucleation and growth had occurred (center of the Fig. 3A). The particles obtained from spray-dried acid mineral solution (CaCO3 dissolved in citric acid) in Fig. 3B were not characteristic of crystals of CaCO3 dissolved from water alone (Fig. 3A). These amorphous particles (confirmed by XRD) had a mean diameter between 1 and 4 lm. Citric acid allows the erosion and dispersion of such crystallites since that acidic pH, characteristic of citric acid solution, the solubility of calcium carbonate is increased. This interpretation is in accordance with the study of Donnet et al. [19] on the use of PolyAcrylic Acid (PAA) to control CaCO3 precipitation. We can assume that CaCO3 crystals are colloidally stabilized in presence of citric acid. In the center of Fig. 3B, a larger particle is observed, which seems to be an aggregation of smaller particles. This particle size polydispersity could be due to the per-
iod of time, during which the spray-drying process lasted (about 2 h). During this period a variation in the meta-stable solution may occur, involving variation of particle size, and also significant changes in the characteristic both of the protein and of the inorganic phase but the stability is preserved. The control spray-dried soy protein dispersion (soy protein dissolved in distilled water) was also studied by SEM (Fig. 3C) and Environmental SEM (ESEM) experiments (results not shown). In the two cases, similar pictures were obtained showing collapsed spray-dried particles of soy proteins. In the spray-dried powder obtained from acid mineral solution in the presence of soy proteins (Fig. 3D), it would appear that soy proteins bind to the mineral phase, giving a roughness to their surface. Considering the SEM pictures of the soy protein control (collapsed particles, Fig. 3C), we could expect that soy proteins were
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swollen in the presence of minerals until completely filled (Fig. 3E and F). To conclude, calcite crystals (Fig. 3A) of about 10 lm become eroded in citric acid solution (Fig. 3B), giving rise to a majority of smaller particles of around 1 lm and a minority of 3 lm-diameter particles, with a few aggregates, probably arising from the disappearance of the meta-stable phase over time during the spray-drying step. In presence of soy proteins, a mechanism of complexation may occurs with mineral phase particle of about 1 lm (Fig. 3D–F), preventing from CaCO3 reprecipitation. Energy Dispersive X-ray Spectrometry (EDX) spectra was also performed on spray-dried acid mineral powders in the absence and in the presence of soy proteins (Fig. 4A and B, respectively). The main difference observed in the EDX spectra, is related to the two calcium peaks in Ka and Kb. Indeed, upon the soy protein addition, these two calcium peaks significantly decreased in their intensity. First, in mineral–soy protein dispersion, there was a dilution effect with the soy protein dispersion added before the spray-drying step. Secondly, the Ca peak decrease may also be related to the complexation phenomenon of soy proteins with calcium and in this case, less calcium may be accessible at the powder surface. Similarly to our study, Rautarey et al. [36] observed with EDX spectrum that synthesized vaterite crystals were stabilized by proteins secreted during the root growth from the chickpea seeds.
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3.2.2. X-ray diffraction (XRD) X-ray diffraction analysis was performed on spray-dried acid mineral powder, in the absence or in the presence of soy proteins. Both XRD diffractograms were characteristic of an amorphous phase (Fig. 5A). Hence the meta-stable solution of CaCO3 dissolved into citric acid, which was spray-dried, was specifically that of an amorphous phase (in accordance with SEM results, Fig. 3B). The addition of soy proteins at that step of the kinetic allowed the former solution to remain in an amorphous state. We can expect that whether the meta-stable phase of the kinetic would have been left, the XRD diffractogram would have been characteristic of a crystalline phase (presence of well-defined peaks). XRD diffractogram of CaCO3 precipitated from water shows a crystalline phase (Fig. 5B) contrary to the XRD diffractogram of CaCO3 dissolved into citric acid solution. These XRD results confirm the SEM interpretation on calcite crystal erosion in the presence of citric acid, leading to an amorphous phase. Yet, the transition of such a meta-stable amorphous phase into a crystalline form over the time would be naturally favoured while leading to a lower energetic level. Nevertheless, the presence of soy proteins prevents this transient amorphous phase from being recrystallised. As reported in literature [14,34,37], in biominerals, the crystalline calcium carbonate phase with uniform orientation, size and complex structures is transformed from a transient amorphous phase and this transformation process can be modified and tailored by various organic templates.
Fig. 4. EDX spectrum of acid mineral spray-dried powder in absence (A, up) or in presence (B, down) of soy proteins.
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With soy protein
2500
Without soy protein 2000
Counts
Peak 1500
O 1s N 1s Ca 2p C 1s S 2p
1000
500
0 0
20
40
60
80
2 theta
Atomic concentration (%) Acid mineral spray-dried powder no soy proteins
Acid mineral spray-dried powder soy proteins
36.40 0.00 3.74 59.85 0.00
20.46 9.26 0.59 69.48 0.20
In our study, soy proteins, rich in aspartic acid and glutamic acid residues, may prevent mineral phase to be recrystallised probably through potential electrostatic interactions.
60000 50000
Control CaCO3
Counts
40000 30000 20000 10000 0 0
20
40
60
80
2 theta Fig. 5. XRD diffractograms. (Up) Acid mineral spray-dried powder in the absence or in the presence of soy proteins. (Down) CaCO3 control spray-dried powder.
3.2.3. X-Photoelectron Spectroscopy (XPS) Global XPS spectra (Fig. 6) are presented for spray-dried acid mineral powders, in the absence (bottom spectrum) or in the presence of soy protein (top spectrum). The following peaks O1s, Ca2p and C1s were identified in the absence of proteins while in presence of protein, the spectrum allowed the identification of the following peaks: O1s, N1s, Ca2p, C1s and S2p. The peaks of O1s, N1s, Ca2p, C1s and S2p are, respectively, localized at 531 eV, 400 eV, 347 eV, 284.60 eV and 162.85 eV. In the presence of soy protein, the first peak of Ca set at 447 eV has almost disappeared and the second peak of Ca (347 eV) significantly decreased in intensity as well. The atomic concentration (%) corresponding to each peak was quantified (Table 1). In the presence of soy protein isolate, the intensity of the peak corresponding to Ca1s is almost disappeared (447 eV) or decreased (347 eV), which can be related to Ca–soy protein complexation. Calcium has disappeared from the extreme surface layer and may be probably stabilized inside soy proteins considering SEM results. XPS analysis also enables the determination of the nature of bonds involved at the surface of the powder through the deconvolution of each peak. In our study, each peak of a specific element (O1s, N1s, Ca2p, C1s, and S2p) was deconvoluted using the model published by Gerin et al. [38]. Whatever the spray-dried powders, each peak were decomposed as follows (results not shown). The peak O1s was decomposed into three peaks attributed to O@C, O–C, and O–C'O bounds. The peak N1s (specific to the presence of soy proteins) was deconvoluted into NH, NH2, N–(C@O) and C–NH3 bounds. Finally, the peak C1s was decomposed into four peaks attributed to C–(C, H), C–(O,N), C@O, and O–C@O. The absence of bonds involving Ca at the extreme surface of the spray-dried powder, would indicate that Ca might be rather absorbed inside soy proteins or would interact through electrostatic interactions rather than covalent interactions. Further investigations should be performed to support these hypotheses. 4. Conclusion In citric acid solution calcite crystals were submitted to erosion giving rise to smaller amorphous particles. In presence of soy proteins a complexation mechanism, exothermic in nature, occurred with mineral phase, which would prevent from CaC03 recrystallisation and kept the system in an amorphous state. SEM pictures performed on spray-dried powder showed soy proteins would bind to the mineral phase and XPS investigations on the same samples demonstrated that the calcium concentration decreased at the extreme surface of studied powder. Acknowledgment
Fig. 6. XPS spectra of acid mineral spray-dried powder in the absence (down) or in the presence (up) of soy proteins.
We would like to thank the laboratory of Mineral Chemistry at the University of Sciences and Techniques (LSCM laboratory, UHP
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Nancy 1, Vandoeuvre-Lès-Nancy), the laboratory of Mines French Engineering School in Nancy and the laboratory of Physical Chemistry and Microbiology for Environment (LCPME, UMR 7564 CNRS, Henry Poincaré University Nancy 1, Vandoeuvre-Lès-Nancy). We are indebted to Unilever Research and Development center (Vlaardingen, the Netherlands) for their financial support. The authors would like to thank Prof. Carole C. Perry for the final reading of the manuscript. References [1] L. Guéguen, Nutr. Clin. Metab. 14 (2000) 206. [2] J.L. Volatier, J. Maffre, A. Couvreur, Enquête Individuelle et Nationale Sur Les Consommations Alimentaires (INCA), TEC & DOC Lavoisier, Paris, 2000. [3] J.P. Bonjour, T. Chevalley, S. Ferrari, R. Rizzoli, Cah. Nutr. Diet. 40 (2005). [4] E. Renner, J. Dairy Sci. 77 (1994) 3498. [5] B.E.C. Nordin, Nutrition 13 (1997) 664. [6] R.P. Heaney, J. Am. Coll. Nutr. 19 (2000) 83S. [7] R.P. Heaney, J. Am. Coll. Nutr. 20 (2001) 192S. [8] M.B. O’Connell, P.L. Stamm, Clin. Rev. Bone Miner. Metab. 2 (2004) 357. [9] L.S. Canabady-Rochelle, C. Sanchez, M. Mellema, S. Banon, Dairy Sci. Technol. 89 (2009) 257. [10] B.M. Exl, R. Fritsché, Nutrition 17 (2001) 642. [11] D.L. Swagerty, A.D. Walling, Am. Fam. Phys. 65 (2002) 1845. [12] H.A. Lowenstam, S. Weiner, On Biomineralization, Oxford University Press, New York, 1989. [13] H. Herman, L. Addadi, S. Weiner, Nature 331 (1988) 546. [14] L. Addadi, S. Raz, S. Weiner, Adv. Mater. 15 (2003) 959. [15] T. Kato, T. Suzuki, T. Amamiya, T. Irie, M. Komiyama, Supramol. Sci. 5 (1998) 411.
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[16] M. Dutour-Sikiric, H. Füredi-Milhofer, Adv. Colloid Interface Sci. 128 (2006) 135. [17] N. Wada, K. Yamashita, T. Umegaki, J. Colloid Interface Sci. 212 (1999) 357. [18] D. Verdoes, D. Kashchiev, G.M. Van Rosmalen, J. Cryst. Growth 118 (1992) 401. [19] M. Donnet, P. Bowen, N. Jongen, J. Lemaitre, H. Hofmann, Langmuir 21 (2005) 100. [20] H. Elfil, H. Roques, AIChE J. 50 (2004) 1908. [21] S. Weiner, Bone 39 (2006) 431. [22] S. Weiner, J. Mahamid, Y. Politi, Y. Ma, L. Addadi, Front. Mater. Sci. China 3 (2009) 104. [23] K.J. Westin, Å.C. Rasmuson, J. Colloid Interface Sci. 282 (2005) 370. [24] L.S. Canabady-Rochelle, C. Sanchez, M. Mellema, S. Banon, J. Agric. Food Chem. 57 (2009) 5339. [25] R.C. Weast, M.J. Astle, W.H. Bayer, Handbook of Chemistry and Physics, CRC Press, Boca Raton, Florida, 1987. [26] L.S. Canabady-Rochelle, C. Sanchez, M. Mellema, A. Bot, S. Desobry, S. Banon, J. Dairy Sci. 90 (2007) 2155. [27] D.G. Dalgleish, T.G. Parker, J. Dairy Res. 47 (1980) 113. [28] C. Holt, D.G. Dalgleish, R. Jenness, Anal. Biochem. 113 (1981) 154. [29] M.B. Assoumani, Agro-Food Indus. Hi-Technol. September/October (1998) 33. [30] H.K. Vyas, P.S. Tong, J. Dairy Sci. 87 (2004) 1177. [31] J. Watanabe, M. Akashi, Cryst. Growth Des. 8 (2008) 478. [32] J.L. Shen, J. Agric. Food Chem. 24 (1976) 784. [33] Y.J. Yuan, O.D. Velev, K. Chen, B.E. Campbell, E.W. Kaler, A.M. Lenhoff, J. Agric. Food Chem. 50 (2002) 4953. [34] P. Wan, H. Tong, Z. Zhu, X. Shen, J. Yan, J. Hu, Mater. Sci. Eng. A 458 (2007) 244. [35] H. Tong, W. Ma, L.L. Wang, P. Wan, J.M. Hu, L.X. Cao, Biomaterials 25 (2004) 3923. [36] D. Rautaray, A. Sanyal, A. Bharde, A. Ahmad, M. Sastry, Cryst. Growth Des. 5 (2005) 399. [37] J. Aizenberg, Adv. Mater. 16 (2004) 1295. [38] P.A. Gerin, P.B. Dengis, P.G. Rouxhet, J. Chem. Phys. 92 (1995) 1043.
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Calcium carbonate–hydrolyzed soy protein complexation in the presence of citric acid Latha-Selvi Canabady-Rochelle a,*, Christian Sanchez a,1, Michel Mellema b, Sylvie Banon a,2 a
Nancy-Université, Institut National Polytechnique de Lorraine, Ecole Nationale Supérieure d’Agronomie et des Industries Alimentaires, Laboratoire d’Ingénierie des Biomolécules, 2, Avenue de la forêt de Hayes, 54 505 Vandoeuvre-Lès-Nancy, France b Unilever R&D Vlaardingen, PO Box 114, Olivier van Noortlaan 120, 3130 AC Vlaardingen, The Netherlands
a r t i c l e
i n f o
Article history: Received 13 November 2009 Accepted 13 January 2010 Available online 18 January 2010 Keywords: Calcium carbonate Soy protein Complexation Amorphous phase ITC SEM XPS
a b s t r a c t The influence of hydrolyzed soy proteins on calcium carbonate stabilization was studied in citric acid solution. Calcium–soy proteins interactions were characterized using a calcium ion selective electrode, turbidity, and Isothermal Titration Calorimetry. Once the meta-stable phase was reached or just after soy protein addition, spray-drying was performed and SEM, XRD, and XPS analysis were carried out on spray-dried powders. In citric acid solution calcite crystals were eroded giving rise to smaller amorphous particles. In the presence of soy proteins, complexation exothermic in nature occurred with the mineral phase, which prevented CaCO3 from recrystallisation and kept the system in an amorphous state. SEM performed on spray-dried powder showed that soy proteins were swollen in presence of mineral phase and resulted in a decrease of calcium concentration at the extreme surface of the studied powders as demonstrated by XPS. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Recent studies on consumer habits reported that some populations at risk do not satisfy their Recommended Daily Allowances (RDA) in calcium (Ca). More specifically, teenage girls from 10 to 19 years old, women older than 55 years, men older than 65 years and elderly people are concerned by this Ca deficiency [1]. According to an INCA survey [2], even in western countries, these specific populations at risk do not satisfy the RDA for Ca. Yet Ca is a mineral essential for humans, especially during growth [3] and during old-age to act against osteoporosis [4–8]. Hence, this nutritional deficiency in Ca presents a world-wide health stake that can be overcome by Ca supplementation of food. Dairy products, naturally rich in Ca and with a high Ca bioavailability, are appropriate foods for Ca supplementation [9]. Nevertheless for various nutritional, ethical, religious or economic reasons, cow’s milk cannot be consumed by specific populations. Hence, soy-based drinks
* Corresponding author. Present address: Biomolecular and Materials Interface Research Group, School of Science and Technology, Nottingham Trent University, Clifton lane, Nottingham NG11 8NS, United Kingdom. E-mail address: [email protected] (L.-S. Canabady-Rochelle). 1 Present address: Laboratoire Ingénierie des Agropolymères et Technologies Emergentes, Université Montpellier II, cc 023 Pl. E. Bataillon, 34 095 Montpellier cedex 05, France. 2 Present address: Nancy-Université, Université des Sciences et Techniques Henri Poincaré, LIMOS UMR CNRS 7137, Campus Victor Grignard BP 239, 54 506 Vandoeuvre-Lès-Nancy, France. 0021-9797/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2010.01.037
supplemented with Ca constitute an alternative to dairy drinks, especially for populations suffering from Cow’s Milk protein Allergy [10] and/or lactose intolerance [11]. Various Ca salts, among them Ca carbonate, are used for soy-based drink supplementation, which present various characteristics in regards to the physical–chemical stability of the system (solubility/sedimentation of salt, aggregation and precipitation of proteins). Crystalline Ca carbonate has three different polymorphs, vaterite, aragonite and calcite, whose solubility is also in accordance with this sequence. The least stable form is vaterite followed by aragonite and calcite, which is thermodynamically the most stable phase [12]. In addition, there also exist three hydrated forms, i.e. Amorphous Calcium Carbonate (ACC), highly unstable at room temperature and pressure as reported by Herman et al. [13], monohydrate calcium carbonate and calcium carbonate hexahydrate (CaCO36H2O). ACC can function as a transient precursor of the more stable crystalline aragonite and calcite and draws much attention due to its role in biomineralisation as reviewed by Addadi et al. [14]. Mineral crystallization, and especially CaCO3 crystallization, is often shaped by macromolecules [15]. Dutour-Sikiric and Füredi-Milhofer [16] reported the influence of surface active molecules on the crystallization of biominerals in solution. Modification of Ca carbonate powder characteristics can be obtained by the use of various additives such as carboxylic acids [17] and various polymers [18], while carrying out the reaction without any additive leads to difficulties in controlling the
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shape and the precipitated crystallographic form [19]. The dissolution of these minerals can lead to the meta-stable zone defined as a supersaturation subregion located between the precipitation straight line obtained experimentally and the equilibrium curve of the least soluble form of Ca carbonate, which is calcite in the CaCO3–CO2–H2O system [20]. In this phase characterized by its transparency, nanosized Ca carbonate could remain in suspension and serve as a ‘‘seed” for subsequent interactions with biopolymers [19]. Amorphous calcium carbonate could also be present and could interact with biopolymers as in biomineralization mechanisms [21,22]. Other interaction mechanisms could occur via citrate, a chelator of Ca, which can reduce the crystallization growth rate [23]. In a previous study [24], Ca chloride was supplemented in a soy-based drink and Ca–soy protein interactions as the origin of Ca-induced soy protein aggregates were studied. The effect of pH cycle was also investigated on the restructuring and stability of these former aggregates, which could then be used as possible vectors for further Ca delivery. This former work was followed by the study of another type of Ca source, Ca carbonate (CaCO3) commonly used in the food industry with advantageous economies (cheapest source of Ca). Moreover, Ca carbonate, due to its neutral taste, is often preferred to Ca chloride, which can involve bitterness perception by consumers in supplemented products. The major drawback of Ca carbonate is its low solubility (0.014 g L 1) in comparison to the high solubility of Ca chloride (745 g L 1) [25]. In an approach to decrease the sedimentation phenomenon observed for Ca carbonate and improve its solubility, we have acidified the reaction medium and added soy proteins as chelating agents. The scientific aim of this work was to understand the mechanism occurring upon Ca carbonate solubilization in citric acid in the presence of soy proteins. Interactions involved in such systems (CaCO3/citric acid/soy proteins) were studied in solution using the microcalorimetric method, i.e. Isothermal Titration Calorimetry. Once the meta-stable phase was reached or just after the soy protein addition, spray-drying was carried out and the system was analyzed. 2. Materials and methods 2.1. Calcium–soy protein interactions 2.1.1. Dispersion preparation Citric acid (monohydrate, analytical reagent, VWR Prolabo, Leuven, Belgium) was solubilized in distilled water at 0.57% w/w at room temperature, followed by the addition of 0.23% w/w calcium carbonate (CaCO3, Rectapur, Prolabo, Merk, Briare le Canal, France). Chemical reactions between the different molecular species led to a meta-stable system characterized by its transparency and named the ‘‘acid mineral solution”. Hydrolyzed soy proteins (H-SP, non-GM soy protein isolate 219, The Solae company, Barcelona, Spain) were supplemented to stabilize the ‘‘acid mineral solution” against sedimentation. The final concentration of the acid mineral–soy protein dispersion was composed of 70% w/w of the acid mineral solution and 1% w/w of soy proteins. This acid mineral–soy protein dispersion was completed with distilled water (up to 100% w/w). 2.1.2. Reactor experimental set up The preparation of the acid mineral solution and acid mineral– soy protein dispersion was followed using the experimental set up described previously [26]. A pH-meter (Radiometer analytical, Remiremont, France), and Calcium Ion Selective Electrode (CaISE, Sentek, Estate, Braintree, United Kingdom) were immersed into the bulk. A turbidity sensor (Analite NEP 160, McVan Instruments,
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Mulgrave, Australia) was placed through the vessel wall to avoid disturbances during stirring. Continuous monitoring (Almemo 8990-8 V5, Ahlborn, Holzkirchen, Germany) of pH, ionized Ca (Ca2+) and turbidity was performed using a data logger coupled with a PC equipped with software (AMR WinControl for Almemo, Akrobit, Gera, Germany). Ca ISE was used to evaluate Ca ionization [27–30]. The pH-meter and Ca ISE were daily calibrated at the experimental temperature (Texptl = 20 °C). pH-meter calibration was performed at pH 7.00 and pH 4.00 and the accuracy of the measurement was ±0.02 pH unit. For the acid mineral solution study, Ca carbonate was added after 5 min of probe stabilization in the citric acid solution. For the study of calcium–soy protein interactions in citric acid solution, a soy protein dispersion was prepared in distilled water and added to the acid mineral solution, in which probes were stabilized, up to reach the final concentration described previously. 2.1.3. Thermodynamic characterization of calcium–soy protein interactions For the study of Ca–soy protein interactions in citric acid solution, the acid mineral solution and soy protein dispersion were prepared as described above. Each solution was diluted three times in milliQ water to obtain an ITC signal, within the range of detection of the apparatus. Protein concentration in the soy protein dispersion was determined according to the Bradford method. A calibration curve was performed with Bovine Serum Albumin solution at 1.35 mg mL 1. A VP-ITC microcalorimeter (MicroCal, North-Hampton, MA, USA) is composed of two identical spherical cells, a reference cell and a sample cell, both with a volume of 1.449 mL, which are enclosed in an adiabatic jacket. Before each experiment, both solutions (the acid mineral solution and the soy protein dispersion) were degassed for 7 min to eliminate air bubbles, which could perturb the baseline. Then, the sample and the reference cells were, respectively, filled with the acid mineral solution and the solvent used to prepare the sample solution (i.e., milliQ water). The titrant, the soy protein dispersion, was prepared in milliQ water, and injected in a single step (250 lL; duration: 75.3 s) into the sample cell. The measurement was performed under stirring (300 rpm) at constant temperature (Texptl = 20 °C). The spacing time between each consecutive measurement was of 300 s. Due to the lack of information about the expected heats of the studied system, the reference power of the ITC instrument was set at 15 lcal s 1. The experiment lasted 3600 s. Each experiment was performed in triplicate. In the analysis, the injection of soy protein dispersion into milliQ water corresponded to the reference, and was used to determine the heat of dilution of soy proteins. Data for this reference experiment, carried out in the same way as the sample experiment (soy protein dispersion titrated in acid mineral solution), was subtracted from the sample data. Binding curves were plotted in kcal mol 1 of injectant as a function of time. 2.2. Spray-dried powder characterization 2.2.1. Spray-drying Once the meta-stable phase was reached in solution or just after the soy protein addition, spray-drying was performed (Büchi mini spray-dryer B191, Switzerland. The inlet and the outlet temperatures were set at 180 °C and 80 °C, respectively. The pump and the aspirator activity were set at 55% and at 70%, respectively. Acid mineral powders were prepared in the absence or in the presence of soy proteins. Calcium carbonate and soy proteins (both solubilized in distilled water and then spray-dried) were studied as references powders.
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2.2.2. Electron microscopy (EM) Scanning Electron Microscopy (SEM). The samples were attached to double-sided adhesive tape attached to SEM stubs, covered with a carbon layer of 10 nm and then a mix of gold/ palladium by sputtering (Biorad type SC 502). The samples were then examined with a SEM S2500 instrument (Hitachi, Science Systems, Ibaraki, Japan) operated at 10 kV. Measurements were carried out under secondary vacuum (about 10 5 torr) by retro diffusion of secondary electrons. Energy dispersive X-ray analysis carried out during SEM experiments gave some information on the local composition of the studied sample over a depth of a few lm. The detection limits for the elements varied between 0.1% and 0.5% of the total elements detected and was determined by the continuous background noise of the spectrum. Environmental scanning electron microscopy (ESEM). ESEM was carried out using a SEM Quanta 600 FEG apparatus (FEI Company, Eindhoven, Netherlands). This experiment was performed to determine whether or not vacuum induced artifacts would be seen in the standard SEM imagery. 2.2.3. X-ray diffraction (XRD) XRD experiments were carried out using an X’pert PRO diffractometer apparatus (PANalytical, Almelo, Netherlands). The instrument used a Bragg–Brentano geometry. The X-ray beam used a copper anticathode with a wavelength k of 0.70926 Å. The beam was converged with a Germanium monochromator selecting a raw Ka1, with the Soller slit having a divergence of 0.02 Rad. The goniometer was used in h 2h geometry and the measurement was done in reflexion mode, with the sample located onto a spinner. The detector used was an Xcelerator, the measurement step was of 0.017° with a time by step of 712 s. 2.2.4. X-photoelectron spectroscopy (XPS) XPS analyzes were carried out with a Kratos Axis Ultra spectrometer (Kratos analytical, Manchester, UK) using a set monochromatic radiation A1Ka source with incident energy of 1486.8 eV. The power applied at the X-ray anode was reduced to 90 W in order to avoid X-ray induced degradation of the sample. The instrument work function was calibrated to give a binding energy (EB) of 83.96 eV for the Au 4f7/2 line for metallic gold and the spectrometer dispersion was adjusted to give a binding energy of 932.65 eV for Cu 2p3/2 for metallic copper. All spectra were recorded at a take-off angle of 90°, which enabled analysis to about a 5 nm-depth over an area of about 700 lm 300 lm. Survey spectra were recorded with 1.0 eV steps and 160 eV analyzer pass energy. The high resolution regions (peaks of C, O, and N) were recorded with 0.1 eV steps for O 1s and N 1s, and with 0.05 eV step for C 1s. In both cases, the hybrid lens mode was employed. During data acquisition the Kratos charge neutralizer system was used to avoid the overcompensation phenomenon (peak shift towards higher levels of energy due to isolating samples) and the following settings were used: filament current 1.6 A, charge balance 2.4 V, filament bias 1.0 V and magnetic lens trim coil 0.375 A. As overcompensation was always observed, the C 1s line for adventious carbon and C–C carbon was set to 284.60 eV and therefore used as an internal energy reference to enable comparison of results for various samples. With these parameters we could obtain a C 1s signal with sharp, symmetric components with FWHM of 1.2 eV. Spectra were analyzed using the Vision software from Kratos (Vision 2.2.6). A Shirley baseline allowed background noise subtraction whereas Gaussian (70%) and Lorentzian (30%) models were used for spectral decomposition. Indeed, this former treatment enables a chemical analysis through the determination of
the nature of the surface binding. Quantification was performed using the photoemission cross-sections and the transmission coefficients provided in the Vision package.
3. Results and discussion 3.1. Calcium–soy protein interactions 3.1.1. Ionized calcium (Ca2+) variations In the absence of soy proteins, Ca carbonate (CaCO3) dissolved in citric acid solution. The ionized Ca concentration (M) increased from 0 to about 8 mM and was stabilized at this concentration for the whole reaction period (24 h, Fig. 1A). This equilibrium resulted from an ionic balance between dissolution and precipitation phenomena. Simultaneously the pH value measured changed from pH 2.5 (pH of the citric acid solution) to pH 4.2 (pH of the acid mineral solution). This former pH value slightly decreased over the time, which may be due to the CO2 solubilization from ambient air upon stirring and formation of HCO3 , with H+ ions into the bulk. Similarly, Watanabe and Akashi [31] observed CaCO3 dissolution in an acidic environment, especially in citric acid solution and their dissolution rate measured represented up to 80% of the initial Ca ion content. Upon soy protein supplementation into the acid mineral solution, the ionized Ca concentration decreased first from about 8 mM to 5 mM, with final equilibrate ion at around 6 mM (Fig. 1B). Simultaneously the pH value changed from pH 4.2 in the acid mineral solution to pH 4.5 in the presence of soy proteins. The ionized Ca concentration decrease observed upon soy protein addition is due to the interaction of ionized Ca (Ca2+) with soy proteins. Soy proteins are rich in residues of acidic amino-acids and contain 11.6% and 19.1% (w/w) of aspartic acid and glutamic acid, respectively. In the range pH 4.2–pH 4.5 and taking into account the pKR value of the acidic amino-acids (3.86 and 4.25 for aspartate and glutamate, respectively), the acidic group of the sidechain will be negatively charged and can bind Ca2+, even if the global charge of the soy protein is close to a zero value. Indeed, pH 4.5 agrees with the isoelectrical pH (pHi) of soy protein commonly reported in the literature [32,33]. During the first hour, ionized Ca concentration slightly increased from 5 mM to 6 mM, which may be due to equilibrium of Ca2+ between the soluble and the protein colloidal phase as reported by Canabady-Rochelle et al. [24].
3.1.2. Turbidity (s) variations Turbidity variations were followed during the whole process to characterize the colloidal phase. The turbidity signal depends on three main factors: size, concentration and optical properties of the particles in solution. In the absence of soy proteins, Ca carbonate (CaCO3) dissolved in citric acid solution. The initial turbidity of the citric acid solution of 10 NTU (NTU, for Nephelometric Turbidity Unit) increased to about 400 NTU upon CaCO3 addition. After complete CaCO3 dissolution, turbidity decreased to its initial value (10 NTU, Fig. 1C). Hence, the turbidity peak corresponds to the kinetic of dissolution of CaCO3 and is time dependent. On the contrary, upon soy protein addition in the acid mineral solution (Citric acid/CaCO3), the turbidity increased from 10 NTU (transparency of the meta-stable phase) to 5600 NTU (Fig. 1D) and remained stable over the remaining time of the experiment (no peak observed). This turbidity increase could be due to the calcium-induced aggregation, as previously reported. Moreover at such pH, equivalent to the isoelectrical pH of soy proteins (pHi = 4.5), soy proteins can also aggregate.
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Fig. 1. Ionized calcium concentration (A and B) and turbidity variations (C and D) versus time (hours) in the absence or in the presence of soy proteins, respectively. At 0.1 h, CaCO3 was added in citric acid solution (panels A and C). At 0.1 h, soy proteins were added in the acid mineral solution (citric acid/CaCO3; panels B and D).
Heat of binding (Cp, µcal.sec-1)
3.1.3. Thermodynamic characterization of Ca–soy protein interactions The soy protein dispersion was injected in a single step into the acid mineral solution. A scaled down reaction was followed with ITC and the thermodynamic signal obtained is presented in Fig. 2. A highly exothermic signal (DH < 0) was observed, which could be decomposed into two parts, corresponding to two exothermic peaks. This result was unexpected since an endothermic phenomenon was observed in Canabady-Rochelle et al. [24] on Ca–soy proteins interactions. Nevertheless, in the former study, the Ca source was the highly soluble Ca chloride without any citric acid in solution.
0 -10 -20 -30 -40
Wan et al. [34] studied by microcalorimetry the precipitation of calcium carbonate from solution as a function of L-aspartic acid (LAsp) concentration. They observed that the precipitation of CaCO3 was an endothermic process with the thermal spectrum divided into two peaks: first a major endothermic event and then, a second minor endothermic peak whose intensity was inversely related to the amount of L-Asp in solution. By means of FT-IR data, former authors have shown that as the amount of L-Asp increased, the amount of vaterite in the precipitate increased accordingly. Indeed, L-Asp is known to induce the formation and stabilize the existence of vaterite crystals [35]. Wan et al. proposed that the first major peak can be related to the precipitation process and that the second peak (endothermic process) fits with the transition from a meta-stable phase to calcite, which formed by recrystallization of the meta-stable phase (combination of dissolution and recrystallization). With higher L-Asp concentration, less of the meta-stable phase (vaterite) would transform into calcite and the minor endothermic peak resulting from the recrystallization event decreased. In accordance with these results, we propose that in our case the two peaks observed relate to exothermic processes with the transition from crystalline forms to meta-stable phases in the presence of soy proteins, which are rich in acidic amino-acids.
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Time (sec) Fig. 2. Thermodynamic signal obtained upon soy protein titration into acid mineral solution (citric acid/CaCO3). The reference signal (soy protein titrated into milliQ water) was substracted.
3.2.1. Electron microscopy (EM) Scanning Electron Microscopy (SEM) was performed on various spray-dried powders. Spray-dried powder, obtained from CaCO3 solubilized in distilled water, was studied as the CaCO3 control (Fig. 3A). Cubic-shaped crystals of Ca 10 lm side were observed.
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Fig. 3. SEM (A–D) and ESEM (E and F) micrographs. (A) Spray-dried powder of CaCO3 control; (B) acid mineral spray-dried powder in the absence of soy protein; and (C) Spray-dried powder of soy protein control; (D–F) Acid mineral spray-dried powder in the presence of soy proteins.
This crystal form was characteristic of calcite. On some crystals, further nucleation and growth had occurred (center of the Fig. 3A). The particles obtained from spray-dried acid mineral solution (CaCO3 dissolved in citric acid) in Fig. 3B were not characteristic of crystals of CaCO3 dissolved from water alone (Fig. 3A). These amorphous particles (confirmed by XRD) had a mean diameter between 1 and 4 lm. Citric acid allows the erosion and dispersion of such crystallites since that acidic pH, characteristic of citric acid solution, the solubility of calcium carbonate is increased. This interpretation is in accordance with the study of Donnet et al. [19] on the use of PolyAcrylic Acid (PAA) to control CaCO3 precipitation. We can assume that CaCO3 crystals are colloidally stabilized in presence of citric acid. In the center of Fig. 3B, a larger particle is observed, which seems to be an aggregation of smaller particles. This particle size polydispersity could be due to the per-
iod of time, during which the spray-drying process lasted (about 2 h). During this period a variation in the meta-stable solution may occur, involving variation of particle size, and also significant changes in the characteristic both of the protein and of the inorganic phase but the stability is preserved. The control spray-dried soy protein dispersion (soy protein dissolved in distilled water) was also studied by SEM (Fig. 3C) and Environmental SEM (ESEM) experiments (results not shown). In the two cases, similar pictures were obtained showing collapsed spray-dried particles of soy proteins. In the spray-dried powder obtained from acid mineral solution in the presence of soy proteins (Fig. 3D), it would appear that soy proteins bind to the mineral phase, giving a roughness to their surface. Considering the SEM pictures of the soy protein control (collapsed particles, Fig. 3C), we could expect that soy proteins were
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swollen in the presence of minerals until completely filled (Fig. 3E and F). To conclude, calcite crystals (Fig. 3A) of about 10 lm become eroded in citric acid solution (Fig. 3B), giving rise to a majority of smaller particles of around 1 lm and a minority of 3 lm-diameter particles, with a few aggregates, probably arising from the disappearance of the meta-stable phase over time during the spray-drying step. In presence of soy proteins, a mechanism of complexation may occurs with mineral phase particle of about 1 lm (Fig. 3D–F), preventing from CaCO3 reprecipitation. Energy Dispersive X-ray Spectrometry (EDX) spectra was also performed on spray-dried acid mineral powders in the absence and in the presence of soy proteins (Fig. 4A and B, respectively). The main difference observed in the EDX spectra, is related to the two calcium peaks in Ka and Kb. Indeed, upon the soy protein addition, these two calcium peaks significantly decreased in their intensity. First, in mineral–soy protein dispersion, there was a dilution effect with the soy protein dispersion added before the spray-drying step. Secondly, the Ca peak decrease may also be related to the complexation phenomenon of soy proteins with calcium and in this case, less calcium may be accessible at the powder surface. Similarly to our study, Rautarey et al. [36] observed with EDX spectrum that synthesized vaterite crystals were stabilized by proteins secreted during the root growth from the chickpea seeds.
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3.2.2. X-ray diffraction (XRD) X-ray diffraction analysis was performed on spray-dried acid mineral powder, in the absence or in the presence of soy proteins. Both XRD diffractograms were characteristic of an amorphous phase (Fig. 5A). Hence the meta-stable solution of CaCO3 dissolved into citric acid, which was spray-dried, was specifically that of an amorphous phase (in accordance with SEM results, Fig. 3B). The addition of soy proteins at that step of the kinetic allowed the former solution to remain in an amorphous state. We can expect that whether the meta-stable phase of the kinetic would have been left, the XRD diffractogram would have been characteristic of a crystalline phase (presence of well-defined peaks). XRD diffractogram of CaCO3 precipitated from water shows a crystalline phase (Fig. 5B) contrary to the XRD diffractogram of CaCO3 dissolved into citric acid solution. These XRD results confirm the SEM interpretation on calcite crystal erosion in the presence of citric acid, leading to an amorphous phase. Yet, the transition of such a meta-stable amorphous phase into a crystalline form over the time would be naturally favoured while leading to a lower energetic level. Nevertheless, the presence of soy proteins prevents this transient amorphous phase from being recrystallised. As reported in literature [14,34,37], in biominerals, the crystalline calcium carbonate phase with uniform orientation, size and complex structures is transformed from a transient amorphous phase and this transformation process can be modified and tailored by various organic templates.
Fig. 4. EDX spectrum of acid mineral spray-dried powder in absence (A, up) or in presence (B, down) of soy proteins.
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L.-S. Canabady-Rochelle et al. / Journal of Colloid and Interface Science 345 (2010) 88–95 Table 1 Atomic concentration (%) of acid mineral spray-dried powder in absence or in presence of soy proteins.
With soy protein
2500
Without soy protein 2000
Counts
Peak 1500
O 1s N 1s Ca 2p C 1s S 2p
1000
500
0 0
20
40
60
80
2 theta
Atomic concentration (%) Acid mineral spray-dried powder no soy proteins
Acid mineral spray-dried powder soy proteins
36.40 0.00 3.74 59.85 0.00
20.46 9.26 0.59 69.48 0.20
In our study, soy proteins, rich in aspartic acid and glutamic acid residues, may prevent mineral phase to be recrystallised probably through potential electrostatic interactions.
60000 50000
Control CaCO3
Counts
40000 30000 20000 10000 0 0
20
40
60
80
2 theta Fig. 5. XRD diffractograms. (Up) Acid mineral spray-dried powder in the absence or in the presence of soy proteins. (Down) CaCO3 control spray-dried powder.
3.2.3. X-Photoelectron Spectroscopy (XPS) Global XPS spectra (Fig. 6) are presented for spray-dried acid mineral powders, in the absence (bottom spectrum) or in the presence of soy protein (top spectrum). The following peaks O1s, Ca2p and C1s were identified in the absence of proteins while in presence of protein, the spectrum allowed the identification of the following peaks: O1s, N1s, Ca2p, C1s and S2p. The peaks of O1s, N1s, Ca2p, C1s and S2p are, respectively, localized at 531 eV, 400 eV, 347 eV, 284.60 eV and 162.85 eV. In the presence of soy protein, the first peak of Ca set at 447 eV has almost disappeared and the second peak of Ca (347 eV) significantly decreased in intensity as well. The atomic concentration (%) corresponding to each peak was quantified (Table 1). In the presence of soy protein isolate, the intensity of the peak corresponding to Ca1s is almost disappeared (447 eV) or decreased (347 eV), which can be related to Ca–soy protein complexation. Calcium has disappeared from the extreme surface layer and may be probably stabilized inside soy proteins considering SEM results. XPS analysis also enables the determination of the nature of bonds involved at the surface of the powder through the deconvolution of each peak. In our study, each peak of a specific element (O1s, N1s, Ca2p, C1s, and S2p) was deconvoluted using the model published by Gerin et al. [38]. Whatever the spray-dried powders, each peak were decomposed as follows (results not shown). The peak O1s was decomposed into three peaks attributed to O@C, O–C, and O–C'O bounds. The peak N1s (specific to the presence of soy proteins) was deconvoluted into NH, NH2, N–(C@O) and C–NH3 bounds. Finally, the peak C1s was decomposed into four peaks attributed to C–(C, H), C–(O,N), C@O, and O–C@O. The absence of bonds involving Ca at the extreme surface of the spray-dried powder, would indicate that Ca might be rather absorbed inside soy proteins or would interact through electrostatic interactions rather than covalent interactions. Further investigations should be performed to support these hypotheses. 4. Conclusion In citric acid solution calcite crystals were submitted to erosion giving rise to smaller amorphous particles. In presence of soy proteins a complexation mechanism, exothermic in nature, occurred with mineral phase, which would prevent from CaC03 recrystallisation and kept the system in an amorphous state. SEM pictures performed on spray-dried powder showed soy proteins would bind to the mineral phase and XPS investigations on the same samples demonstrated that the calcium concentration decreased at the extreme surface of studied powder. Acknowledgment
Fig. 6. XPS spectra of acid mineral spray-dried powder in the absence (down) or in the presence (up) of soy proteins.
We would like to thank the laboratory of Mineral Chemistry at the University of Sciences and Techniques (LSCM laboratory, UHP
L.-S. Canabady-Rochelle et al. / Journal of Colloid and Interface Science 345 (2010) 88–95
Nancy 1, Vandoeuvre-Lès-Nancy), the laboratory of Mines French Engineering School in Nancy and the laboratory of Physical Chemistry and Microbiology for Environment (LCPME, UMR 7564 CNRS, Henry Poincaré University Nancy 1, Vandoeuvre-Lès-Nancy). We are indebted to Unilever Research and Development center (Vlaardingen, the Netherlands) for their financial support. The authors would like to thank Prof. Carole C. Perry for the final reading of the manuscript. References [1] L. Guéguen, Nutr. Clin. Metab. 14 (2000) 206. [2] J.L. Volatier, J. Maffre, A. Couvreur, Enquête Individuelle et Nationale Sur Les Consommations Alimentaires (INCA), TEC & DOC Lavoisier, Paris, 2000. [3] J.P. Bonjour, T. Chevalley, S. Ferrari, R. Rizzoli, Cah. Nutr. Diet. 40 (2005). [4] E. Renner, J. Dairy Sci. 77 (1994) 3498. [5] B.E.C. Nordin, Nutrition 13 (1997) 664. [6] R.P. Heaney, J. Am. Coll. Nutr. 19 (2000) 83S. [7] R.P. Heaney, J. Am. Coll. Nutr. 20 (2001) 192S. [8] M.B. O’Connell, P.L. Stamm, Clin. Rev. Bone Miner. Metab. 2 (2004) 357. [9] L.S. Canabady-Rochelle, C. Sanchez, M. Mellema, S. Banon, Dairy Sci. Technol. 89 (2009) 257. [10] B.M. Exl, R. Fritsché, Nutrition 17 (2001) 642. [11] D.L. Swagerty, A.D. Walling, Am. Fam. Phys. 65 (2002) 1845. [12] H.A. Lowenstam, S. Weiner, On Biomineralization, Oxford University Press, New York, 1989. [13] H. Herman, L. Addadi, S. Weiner, Nature 331 (1988) 546. [14] L. Addadi, S. Raz, S. Weiner, Adv. Mater. 15 (2003) 959. [15] T. Kato, T. Suzuki, T. Amamiya, T. Irie, M. Komiyama, Supramol. Sci. 5 (1998) 411.
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