Effects of solvent and alkaline earth metals on the heat-induced precipitation process of sodium caseinate Francesco Lopez a,⇑ , Francesca Cuomo a , Pierandrea Lo Nostro b , Andrea Ceglie a a Dipartimento di Agricoltura, Ambiente ed Alimenti (DIAAA) and CSGI, University of Molise, Campobasso I-86100, Italy b Department of Chemistry and CSGI, University of Florence, Sesto Fiorentino 50019, Florence, Italy article info Article history: Received 4 May 2012 Received in revised form 27 July 2012 Accepted 30 July 2012 Available online 8 August 2012 Keywords: Caseinate Fluorescence Precipitation temperature Deuterium oxide Divalent ions abstract The precipitation temperatures of sodium caseinate in H 2 O and D 2 O in the presence of Mg 2+ , Ca 2+ , Sr 2+ and Ba 2+ were investigated through fluorescence, turbidity and conductivity experiments. As for the ability of the divalent cations (1–17.5 mM) to induce the precipitation process in H 2 O, the sequence Ba 2+ P Ca 2+ > Mg 2+ > Sr 2+ was found. Remarkably, while at low salt concentrations (<10 mM) precipitation temperatures (T Ps ) were found to change significantly depending on the specific cation, at higher concentrations (>10 mM) the differences among the different cations were greatly reduced. By fit- ting these results with a modified Jones–Dole equation, we confirmed that the less hydrated ions possess a greater capacity to induce precipitation. In D 2 O, the order of ion ability to induce caseinate precipitation was Ba 2+ > Ca 2+ > Sr 2+ > Mg 2+ . The dif- ferent hydrophobicity between D 2 O and H 2 O was shown to affect significantly the T Ps of caseinate in the presence of calcium, strontium and barium. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The functionality of a protein depends on the specific features of both the polypeptide and of the solvent. Proteins fold into charac- teristic and functional three-dimensional structures that are deter- mined by the specific interactions between the chemical functionalities of their amino acid sequence and the surrounding solvent. Positively and negatively charged amino acids can attract each other and contribute to the overall protein conformation, while the non polar amino acids have a strong tendency to associ- ate with one another. Such an effect in proteins gives rise to the formation of hydrophobic cavities and contributes to the stabiliza- tion of the protein structure. The extent of these interactions de- pends on the protein type, pH and ionic strength of the dispersion, on the nature and concentration of the ionic species that are dissolved in the medium, as well as on the solvent characteristics. The influence of salts on macromolecular aggregates has been reported in several papers (Baldwin, 1996; Cuomo, Palazzo, Ceglie, & Lopez, 2009; Curtis, Prausnitz, & Blanch, 1998; Kunz, Lo Nostro, & Ninham, 2004; Lawal, Afolabi, Adebowale, Ogunsanwo, & Bankole, 2005; Lo Nostro, Peruzzi, Severi, Ninham, & Baglioni, 2010; Lo Nos- tro et al., 2006; Voinescu et al., 2006; Zhao, 2005). While the origin of many specific ion effects is still debated, it is widely accepted that the mechanism by which salts destabilize proteins is relevant to the suppression of aggregation. A fundamental parameter for the formation of ion pairs in solution is ion hydration, which in turn depends on the ions’ charge density (Collins, 1997). In sum- mary, as demonstrated by Collins, small ions of high charge density are strongly hydrated (kosmotropes) whereas large monovalent ions of low charge density are weakly hydrated (chaotropes). On the other hand, it is well known that the affinity of ions for proteins depends on the presence of the binding sites that can be made more or less accessible by modulating the hydrophobic– hydrophilic balance of the protein interactions. A suitable strategy for altering this intricate equilibrium is the use of deuterium oxide (D 2 O). In fact, it has been demonstrated for a number of proteins that aggregation phenomena are greatly influenced by replacing H 2 O with D 2 O. For example, the deuterated solvent stabilized the oligomeric form of halophilic malate dehydrogenase, (Bonnete, Madern, & Zaccai, 1994) and it also affects the stability of some proteins such as b-lactoglobulin (Verheul, Roefs, & de Kruif, 1998). D 2 O is able to exert the above mentioned effects by enhanc- ing the self-association of several proteins (Chakrabarti, Kim, Gup- ta, Barton, & Himes, 1999). Although the mechanism of protein assembly in D 2 O is not well understood, it is thought to be due to the enhancement of hydrophobic interactions and hydrogen bonding which are much stronger in D 2 O than in H 2 O. Presumably, in deuterium oxide there is a greater entropic effect and conse- quently an enhancement of the hydrophobic interactions (Kres- heck, Schneider, & Scheraga, 1965; Parker & Clarke, 1997). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.07.117 ⇑ Corresponding author. Tel.: +39 0874404632; fax: +39 0874404652. E-mail address: lopez@unimol.it (F. Lopez). Food Chemistry 136 (2013) 266–272 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem