The effect of pH on the structure and phosphate mobility of casein micelles in aqueous solution

Highlights

• The large majority of phosphoserines is immobilized in the CCP of casein micelles.

• With decreasing pH inorganic and organic phosphate are protonated differently.

• Inorganic and organic phosphate are progressively mobilized with decreasing pH.

• Organic phosphate is mobile in flocculated casein at pH < 5.0.

• CCP nanoclusters dissolved at pH 4.8 are reformed when the pH is increased to 6.7.

Abstract

The mobility of phosphate groups in aqueous solutions of casein micelles and sodium caseinate from bovine milk was determined with magic angle spinning (MAS) ³¹P NMR as a function of the pH between pH 4 and pH 8. The chemical shifts and the relative amplitudes of the signals from mobile inorganic phosphate (orthophosphate) and mobile organic phosphate (phosphorylated serines) as well as that of immobile phosphate (colloidal calcium phosphate, and immobile phosphorylated serines) were determined. Sodium caseinate contained very little orthophosphate and all phosphates were mobile over the whole pH range. In micellar casein solutions most of the phosphate was immobile at pH > 6.0, but the fraction of mobile organic and inorganic phosphate increased sharply between pH 5.5 and pH 4.5, showing the disintegration of the CCP nanoclusters. Protonation of the phosphates with decreasing pH was determined from the chemical shift and was related to their mobility. The signal of mobile organic phosphate was different for micellar casein solutions and sodium caseinate demonstrating the influence of calcium phosphate in the former. The microscopic structure of protein solutions was investigated with confocal laser scanning microscopy. Large protein clusters were observed below pH 5.2 with a density that increased with decreasing pH down to pH 3.9. The mobility of either organic or inorganic phosphate at pH 6.8 was not significantly different after the pH had been reduced to 4.8 and subsequently increased to 6.8, but the microstructure was strongly influenced by the pH-cycling.

Introduction

Casein is the major protein component of cow's milk, representing about 80% of its protein content. Its structure and behaviour in aqueous solution have been investigated intensively for decades, see (Dalgleish, 2011, De Kruif, 2014, De Kruif et al., 2012, Holt et al., 2013, Horne, 2009) for recent reviews, but are still not fully understood. Bovine casein consists of mainly four types: α_s1, α_s2, β and κ, and in milk the majority of casein is assembled into spherical aggregates, so-called casein micelles, with an average radius of about 100 nm. Currently, the generally accepted view is that the casein micelles contain nanoclusters of calcium orthophosphate, so-called colloidal calcium phosphate (CCP), that are distributed throughout the micelles. Phosphorylated serines (SerP) in the caseins bind to the nanoclusters or even become an integrated part of the CCP, which maintains the integrity of the micelles together with attractive interactions between the casein chains (Holt et al., 2013). The size of the casein micelles is limited by κ-casein that forms a polyelectrolyte brush at the surface of the micelle. Steric hindrance of the κ-casein layer together with electrostatic repulsion inhibits aggregation of the micelles in milk.

The net charge of the casein micelles decreases with decreasing pH from the value in milk (pH 6.7) towards its iso-electric point (pI ≈ 4.6). Acidification also leads to progressive protonation of organic and inorganic phosphate and causes progressive dissolution of the CCP until all phosphate is solubilized at pH < 5.3 (Dalgleish and Law, 1989, Famelart et al., 1996, Le Graet and Brulé, 1993, Marchin et al., 2007). It was observed that the size of casein micelles in unheated milk decreases only very little with decreasing pH until pH 5.0 (Anema et al., 2004, Dalgleish et al., 2004, De Kruif, 1997, Moitzi et al., 2010). At lower pH, the proteins aggregate leading to precipitation or gelation, which is the basis for yoghurt formation. Even though the casein micelles remain largely intact, at least down to pH 5.0, their internal structure and the interaction between the caseins change during acidification as the CCP progressively dissolves. It is clear that in order to understand this process fully it is necessary to investigate the state of both the organic and inorganic phosphate as a function of the pH.

³¹P NMR spectroscopy is a non-destructive technique that can be used to quantify the degree of protonation of the phosphoserine of casein as a function of the pH (Belton et al., 1985, Humphrey and Jolley, 1982, Sleigh et al., 1983). It was shown with this technique that the majority of organic (P_o) and inorganic (P_i) phosphorus in micellar casein solutions is immobile at pH 6.7 and that it can be characterized by magic angle spinning (MAS) NMR (Bak et al., 2001, Rasmussen et al., 1997, Thomsen et al., 1995). However, as far as we are aware, no systematic investigation has been made so far of the mobility of P_o and P_i in aqueous solutions of casein micelles as a function of the pH.

Here we report on a MAS ³¹P NMR investigation of aqueous solutions of casein micelles in the form of native phosphocaseinate over a wide pH-range (4–8). Our aim was to determine quantitatively the fraction of mobile P_o and P_i as a function of the pH in order to trace the dissolution of the CCP. We also determined the degree of protonation of the mobile phophoserine and inorganic phosphate and compared it with that of caseins without CCP, i.e. sodium caseinate (NaCas). The effect of decreasing the pH on the microscopic structure of the casein solutions was visualized by confocal laser scanning microscopy (CLSM). Finally, we investigated the effect of pH-cycling on the mobility of P_i and P_o and the microscopic structure for a micellar casein solution at pH 6.8 before and after acidification. In this investigation we have studied the effect of acidification on the state of organic and inorganic phosphate for casein in pure water. The influence of the presence of minerals on the effect of acidification will be addressed in a future investigation.