Distribution of Salts in Milk and Cheese: Critical Methodological Aspects

Distribution of Salts in Milk and Cheese: Critical Methodological Aspects: Comparison

Please note this is a comparison between Version 2 by Vicky Zhou and Version 1 by Thom Huppertz.

The salt fractions of milk consist of cations (e.g., Ca, Mg, and Na) and anions (e.g., phosphate, citrate, and chloride). These salts are present as free ions or in complexes with other ions or proteins, primarily the caseins. Furthermore, significant levels of Ca and phosphate are also found in insoluble form, inside the casein micelles. The distribution of salts between this micellar phase and the soluble phase is important for the stability and properties of milk and dairy products. Various processes, such as (ultra-)centrifugation, (ultra-)filtration, dialysis, and selective precipitation have been used to separate the micellar and soluble phases in milk and dairy products to allow for studying the salts’ distribution between these phases. These different methods can lead to different levels of soluble salts because the salts in the supernatant from centrifugation, the permeate from ultrafiltration, and the diffusate from dialysis can differ notably. Hence, understanding which components are fractionated with these techniques and how this affects the levels of the soluble salts determined is critical for milk and dairy products. Applying the aforementioned methods to cheese products is further challenging because these methods are primarily developed for fractionating the soluble and micellar phases of milk. Instead, methods that analyze salts in water-soluble extracts, or soluble phases expressed from cheese by pressing or centrifugation are typically used. This review focuses on the significance of salt distribution and variations in salt fractions obtained using different methodologies for both milk and cheese.

milk salts
cheese
ultracentrifugation
water-soluble extract
cheese press

The distribution of salts in different forms and states of solubility in milk and dairy products is referred to as the salt distribution in milk and dairy products. In this context, the term “salts” is preferred over either “minerals” or “inorganic constituents” because several constituent salts in milk are not inorganic and most of the inorganic constituents in milk are not natural components that can be mined. For example, citrate salts are neither inorganic nor a mineral but they play a key role in the milk salts system ^[1]. Salts in milk are present either as free ions or as ions complexed with other ions, proteins, and peptides; furthermore, they can also occur in undissolved form, within the casein micelles ^[2]. The undissolved salts consist primarily of calcium and phosphate as well as some magnesium and citrate and are referred to as micellar calcium phosphate (MCP) ^[3].

Micellar calcium in milk is the calcium that—together with inorganic (P_i) and organic (P_o) phosphate—forms the MCP nanoclusters in the casein micelles as well as the Ca associated directly with casein molecules in complexes that do not contain P_i. Soluble Ca in milk and dairy products is present either as ionic Ca²⁺ or complexed with anions, such as citrate and phosphate ^[4]. Various forms of P can be found in milk as well. P_i occurs as orthophosphate and is found in soluble and insoluble forms. Soluble P_i is found as free ions and complexes with cations, whereas insoluble P_i is found as part of the aforementioned MCP nanoclusters. Most P_o is found in the form of phosphorylated serine (SerP) residues in the caseins but is also found in phospholipids, nucleotides, and sugar phosphates ^[5]. Milk contains approximately 120 mg Ca per 100 g, of which ~31% is soluble Ca and ~69% is micellar Ca. Furthermore, 100 g of milk contains 102 mg P, of which 30% is soluble P_i, 33% is micellar P_i, 12% is soluble P_o, and 25% is insoluble P_o [2,6]^[2][6].

The concentrations of salts, such as Ca, K, Mg, P, Cu, Fe, and Mn, in milk and dairy products can be determined by digesting the sample and measuring the total amount of salt using atomic absorption spectroscopy (AAS) or inductively coupled plasma optical-emission spectroscopy (ICP-OES) ^[7]. Some other salts, such as Cl, citrate, and lactate, cannot be determined with by these techniques and require different methods.

In addition to knowing the total concentrations of the salts, it is often desirable to know the distribution of the salts between the micellar and soluble phases in milk and dairy products. This distribution strongly affects the stability of casein micelles and, in turn, often that of milk and dairy products [4,8]^[4][8]. This distribution of salts between the micellar and soluble phases is usually estimated from concentrations of salts in the whole sample and the soluble phase, determined using the analytical methods as described above. However, the separation of the micellar and soluble phases is a point of careful consideration because any separation method used should not alter the distribution of the salts between the micellar and soluble phases ^[9]. The soluble fraction should have the same composition as the aqueous phase of milk and, therefore, to obtain this, the equilibrium in milk should be maintained ^[10]. Preparation of soluble fractions that still contain notable amounts of insoluble components—due to an inappropriate choice of methodology for the specific product matrix—can lead to inaccurate conclusions, as described and exemplified in Section 3.2. Hence, appropriate separation methods for each product matrix type are crucial.

The first reported study for obtaining the soluble fraction of milk used a porous earthenware filter ^[11]. Since then there have been four main approaches to separate the soluble and micellar fractions, i.e., centrifugation, ultrafiltration (UF), dialysis, and by rennet-induced coagulation of casein micelles ^[10]. In these approaches, the centrifugal supernatant, UF permeate, dialysate, and the rennet whey, respectively, are considered to represent the soluble fraction of the product. However, some more recent studies, as outlined in Section 3.2, have shown that these methods are by no means interchangeable and can give vastly differing outcomes in some cases. Furthermore, these fractionation methods cannot be used for all the dairy matrices and may require matrix-specific adaptations in process parameters like centrifugal force, amount of sample, and degree of sample dilution ^[10]. Moreover, for cheese and processed cheese, methods like cheese juice extraction and water-soluble extract have also been utilized [12,13,14,15]^{[12][13][14][15]}.

This paper reviews the distribution of salts in milk and milk products between the soluble and micellar phases, with emphasis on the methodological approaches to fractionate these phases in milk and cheese. It will provide guidance on which methods should be the most suitable for measuring the salts’ distribution in different dairy products.

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