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Milk and dairy products are complex matrices rich in diverse macronutrients and micronutrients. Lactose, a key component, interacts with milk proteins primarily through hydrogen bonding, while proteins interact via hydrogen bonds, hydrophobic interactions, and electrostatic forces. These interactions, along with mineral-protein interactions, significantly influence the functionality and stability of dairy products. The physical state of lactose and the nature of mineral interactions—shaped by the type, concentration, and processing conditions—can trigger reactions that alter the physicochemical properties of the system. Additionally, the stability of these systems is affected by the specific types and concentrations of proteins and minerals involved. Processing steps such as thermal treatment, concentration, fermentation, and drying, as well as non-thermal technologies like high-intensity ultrasound, further modify these interactions, impacting product quality and storage stability. Understanding these intricate relationships is crucial for optimizing the design and formulation of dairy products. This review examines the mechanisms of lactose-protein, lactose-mineral, and protein-mineral interactions in both liquid and solid systems, highlighting the significant implications these interactions have on processing and product stability.
School of Science, RMIT University, Bundoora, Australia
Juhi Saxena
Spraying Systems Co., PathoSans, Truganina, Australia
Tuyen Truong
School of Science, RMIT University, Bundoora, Australia
School of Science, Engineering and Technology, RMIT Vietnam, Ho Chi Minh City, Vietnam
Jayani Chandrapala*
School of Science, RMIT University, Bundoora, Australia
*Address all correspondence to: jayani.chandrapala@rmit.edu.au
1. Introduction
Milk and other dairy products are essential components of human diet as they are rich sources of high-quality proteins, lactose, vitamins, and minerals [1, 2]. However, technological and product quality challenges are often encountered with both liquid and solid-state dairy systems. These challenges can arise from the complex interactions between milk components.
Lactose, the primary sugar source in milk, is a commonly used ingredient or additive in the food and dairy industry due to its unique physiochemical properties [3, 4, 5, 6]. However, the behavior of lactose in dairy products can significantly affect the storage stability and quality of certain powder products, such as milk protein powders and infant formula [3, 5, 6, 7, 8]. For instance, the degree of lactose crystallization causes stickiness, caking, collapse, and accelerations of the Maillard reaction in lactose-containing milk powders during storage [7] as amorphous lactose is metastable, thermodynamically unstable, and hygroscopic [9].
The mineral content of milk, although constituting a relatively small fraction at approximately 8–9 g/L, includes essential cations like calcium, magnesium, sodium, and potassium, as well as critical anions including inorganic phosphate, citrate, and chloride. Of these, calcium is gaining particular attention for its enrichment in food and dairy products, aligning with the increasing recognition of high calcium intake [10, 11, 12]. However, fortifying milk with calcium can be challenging, especially if the product needs to be heat-treated [10]. The added calcium ions can cause significant protein instability by decreasing the net charge of the proteins, resulting in protein aggregation during processing and storage [13, 14]. This instability can ultimately compromise the quality and shelf life of calcium-fortified milk products.
In addition to being an excellent source of nutrition, milk proteins and their interactions with other components play an important role in conferring desirable functional properties to final dairy products [15, 16]. It is widely known that the processing of milk proteins through heat or high pressure can result in a modification to protein structure, resulting in altered interactions between the proteins and other milk components [17, 18, 19]. However, these interactions are influenced by various factors, including pH, temperature, moisture content, milk protein composition, pressure, drying procedures, processing parameters, and the presence of other ingredients [4]. For example, low pH can cause caseins to lose their steric stability and induce coagulation, while high temperature can cause whey proteins to denature and facilitate aggregation. The extent of these changes in functionality is a function of the presence of lactose and minerals [15, 20, 21]. Another instance, dairy products are highly susceptible to Maillard reaction based on their composition (lactose and lysine in caseins and whey proteins) and under low relative humidity conditions [22, 23, 24, 25]. On the other hand, studies have shown that the presence of lactose has a protective effect on the thermal denaturation of whey proteins [26]. A typical example of a solid-state interaction between lactose and protein is that the amorphous phase of lactose stabilizes the structure of proteins during spray drying and freeze drying [7]. Lactose maintains or increases the hydration of protein molecules, therefore contributing to their stability. Meanwhile, the presence of milk proteins can inhibit or delay lactose crystallization in milk powders due to the development of interactions in the solid state between proteins and amorphous sugars [27, 28]. Besides, minerals as impurities in lactose solutions would influence lactose crystallization in a direction that was depended on the type and the concentration of the salt present [29]. For example, the presence of calcium can directly affect the behavior of lactose, hindering the removal of water surrounding lactose molecules and inhibiting the overall crystallization process [30, 31].
Furthermore, emerging dairy processing technologies such as ultrasound have great influence on these component interactions. For instance, physical shear forces generated through acoustic cavitation with the application of high intensity ultrasound can disrupt heat-induced protein aggregates by breaking intermolecular and intramolecular protein bonds. This disruption prevents the formation of aggregates during subsequent heating, reducing heat-induced viscosity increase, and enhancing heat stability [32]. However, these effects depend on the system’s composition, pH, temperature, sonication time, amplitude, and the presence or absence of applied pulses during treatment.
Although the factors controlling the physical attributes of dairy products are well documented with various processing techniques, further exploration of the interactions between milk proteins, lactose, and minerals that occur during the dairy manufacturing processes is needed. The current body of knowledge on structure-function relationships and interactions in lactose-protein-mineral systems is limited, particularly in understanding the precise molecular mechanisms and the impact of various processing methods. Thus, it is important to expand on this topic to better understand how these interactions affect the functionality, stability, and nutritional quality of the final dairy products. This review covers the fundamental properties of individual dairy components and their behavior in milk systems. Specifically, it focuses on the interactions between milk proteins, lactose, and minerals, particularly calcium, along with the major implications of these interactions in both liquid and solid-state dairy systems. Liquid-state protein-protein interactions through covalent, hydrophobic, and electrostatic interactions, as well as lactose-protein interactions through hydrophobic interactions and milk protein, lactose, and mineral complexations, will be discussed in detail. Furthermore, lactose crystallization and minor reactions associated with lactose, as influenced by the presence of minerals and proteins in the solid state, will be examined. By studying these interactions in detail, a comprehensive understanding of the factors that affect the physical and functional properties of dairy products can be gained. This knowledge can be used to develop more effective manufacturing processes and improve the overall quality of dairy products.
1.1 Milk components and their applications
1.1.1 Lactose
Lactose, a disaccharide made up of glucose and galactose molecules, is the primary carbohydrate found in milk. Lactose (C12H22O11) is naturally present in milk of most mammals, albeit in varying concentrations depending on the species. For instance, human milk contains approximately 7% lactose, while cow and goat milks contain around 4.5–5% lactose [2, 5]. In milk, lactose exists in two basic configurations, 𝛼- and 𝛽-lactose, which are continuously converted into each other through mutarotation [2]. These two forms differ from the position of the hydroxyl group on the carbon atom of the glucose molecule. There are marked differences between 𝛼- and 𝛽-lactose, especially in solubility and crystallization behavior. For instance, 𝛼-lactose is much less soluble and crystallizes as a monohydrate, while 𝛽-lactose is more soluble and forms anhydrous crystals [33].
Physicochemical behavior of lactose is found to have a significant impact on various dairy products and the properties of ingredients during processing and storage [3, 4, 29]. Lactose is less soluble than other sugars, such as sucrose and glucose [3, 34]. In general, lactose solubility is lower in dairy systems compared to pure lactose solutions. This is because the presence of components with high water binding capacity in dairy systems (e.g., proteins and polysaccharides) increases the competition for water, thereby reducing the solubility of lactose [35]. Moreover, the presence of salts influences the solubility of lactose by changing the structure of surrounding water molecules. For instance, adding sodium phosphate can reduce lactose solubility [36], while the addition of potassium phosphate can increase the solubility of lactose [37].
Like other sugars, lactose molecules nucleate and crystallize when the concentration of this sugar overcomes its maximum solubility at a specific temperature. The dairy industry applies this principle to crystallize lactose from whey, a by-product of cheese-making and yoghurt production [30]. Lactose crystallization consists of a set of complex reactions that strongly depend on the experimental conditions used (temperature, agitation, and supersaturation) and, most importantly, the composition of the samples (water, minerals, soluble proteins) [5]. Various lactose crystal forms can theoretically be formed; but 𝛼-lactose monohydrate is the main crystalline form produced from dairy products under normal industrial conditions [5]. These 𝛼-lactose monohydrate crystals are very hard, slighly hygroscopic, and dissolve slowly, rendering it as the most stable form [3]. However, during spray drying, the rapid removal of water causes lactose to dry under its saturation point, thus leading to a rapid increase in its viscosity. Hence, amorphous lactose in a glassy state is formed, which also contains some moisture. For instance, lactose in infant formula (IF) powders is present in amorphous or crystalline forms, with the former being predominant [38]. Amorphous lactose is hygroscopic in nature, which makes it prone to plasticization by water [39]. The instability of amorphous lactose in dairy powders is strongly related to many storage and handling problems of these powders. As a result, control of processing and storage conditions are crucial to control the lactose behavior in ingredients.
1.1.2 Milk proteins
Milk proteins comprise two main groups: caseins and whey proteins. They are widely used in the food industry as functional ingredients due to their high nutritional and functional properties, such as water binding, foaming, emulsification, and gelation [40].
1.1.2.1 Casein
Casein constitutes approximately 80% of the total protein in bovine milk. They are present as macromolecular aggregates of proteins and minerals forming colloidal particles named micelles with a mean size of 150 nm [18, 21, 41]. A micelle contains about 94% protein and 6% colloidal calcium phosphate (CCP, which include calcium, magnesium, phosphate, and citrate). They are proline-rich, open-structured rheomorphic proteins, which have distinct hydrophobic and hydrophilic domains [42, 43]. Table 1 summarizes key physicochemical features of caseins against whey proteins, highlighting their unique structure and properties. Casein micelles have a unique structure that allows them to interact with other milk components, such as whey proteins, lactose, and minerals, and contribute to the texture and stability of dairy products [40]. Casein molecules can hardly be denatured because they have little secondary and tertiary structure [35]. There are four individual types of casein molecules, the αs1-, αs2-, β-, and κ-CN in the approximate ratio of 4:1:3.5:1.5, respectively. Casein proteins can be divided into two groups such as the calcium-sensitive and the non-calcium-sensitive. αs1-, αs2-, and β-CN are classified as calcium-sensitive caseins, whereas κ-CN is highly phosphorylated and thus calcium-sensitive [41, 45].
Properties
Whey proteins
Caseins
Structure
Well-defined tertiary and quaternary structure
Lack well-defined secondary, tertiary, and quaternary structure; possess random coiled structure
Amino acid composition
Relatively high in sulfur-containing amino acids; low in proline
Low in sulfur-containing amino acids; high in proline
Physical state
Exist as globular proteins in the form of monomer-octamers, depending on pH
Exist as large colloidal aggregates called casein micelles
Solubility at pH 4.6
Soluble at pH 4.6
Insoluble at pH 4.6
Heat stability
Heat-labile (can be completely denatured, particularly when heating at 90°C or high)
Very heat-stable (can withstand severe heat treatment such as sterilization, ultrahigh temperature, or retort processing)
Table 1.
Comparison of selected physiochemical properties of casein and whey proteins based on information from [44].
1.1.2.2 Whey proteins
Whey proteins have been widely used as an ingredient for many food and dietary supplement products, such as nutritional bars, infant formula, and protein and/or energy drinks (Table 2). Whey proteins are globular proteins with high levels of α-helix structure. The acidic-basic and hydrophobic-hydrophilic amino acids are distributed in a balanced form [46]. Whey proteins are characterized with a well-defined tertiary structure, and they are heat labile. During food processing and storage, whey proteins are liable to denaturation followed by aggregation. The aggregation of proteins in a food matrix can result in dramatic changes in the global structure and texture.
Functionality
Description
Main mechanism
Applications
Solubility
Ability to dissolve in a solvent
Hydrophilicity
Recombined, UHT and sterilized milk, soups and sauces, infant and clinical nutrition, sports beverages, protein-fortified beverages
Water holding capacity
Interaction with product components to provide high water-holding capacity
Functional properties and underlying mechanisms for the applications of proteins in food products. Reprinted with permission from [19].
Whey proteins include β-lactoglobulin (β-Lg), α-lactalbumin (α-Lac), immunoglobulins (IG), bovine serum albumin (BSA), bovine lactoferrin (BLF), and lactoperoxidase (LP), together with other minor components [47, 48]. β-Lg is the major whey protein in bovine milk, representing about 50% of total whey protein or 12% of the total protein of milk. When in isolated form, it exhibits a low solubility and a low ionic strength. α-Lac represents about 20–25% of the proteins of bovine whey and is a calcium-binding protein [40, 49, 50].
1.1.3 Minerals
Milk minerals are crucial for human health and development, and they play a critical role in dairy processing, particularly in cheese-making and other processes that involve salt-protein interactions [51]. In milk, these ions are associated between themselves and with proteins. For instance, Schuck et al. [52] reported that adding mineral salts to milk changed the casein micelle structure and micellar mineral composition, causing significant variations in water transfer during the drying process. Thus, the mineral content has a pronounced effect on the technological properties of milk, as it affects its susceptibility to renneting, fouling of heat exchanges, gelation, and sedimentation [53]. Minerals are present in two primary forms in milk, soluble (or diffusible), and micellar (Table 3). Soluble minerals are present in a highly dynamic equilibrium between ionic and associate forms, mainly represented by citrate, phosphate, sulfate, and chloride salts [55]. The mineral balance, mainly the divalent cations of Ca and Mg and phosphate, is affected by changes in composition and milk processing parameters such as pH and temperature [55].
Minerals
Total concentration (mM)
Concentration (mM)
Calcium
29.4–30
Micellar
19–20.2
Soluble
9–10
Ionic
1.9–3.0
Magnesium
3.42–5.43
Micellar
1.23–1.95
Soluble
2.18–3.47
Ionic
0.54–0.86
Inorganic phosphate
21
Micellar
9.7
Micellar Ca:P
2.08
Soluble
11.2
Ionic
0.82–0.85
Citrate
9.32
Micellar
0.9–1
Soluble
8.2
Soluble sodium
24.2
Soluble potassium
34.7
Soluble chloride
30.2
Table 3.
Minerals distribution in bovine milk. Reprinted with permission from [54].
1.1.3.1 Calcium
Calcium (Ca) is an essential mineral abundant in milk and dairy products. It plays a crucial role in various physiological functions in the body, including the development and maintenance of strong bones and teeth, as well as nerve and muscle function [56, 57]. In dairy production, Ca is often used as a coagulant in cheese-making. It helps to give the cheese its firm texture and plays a vital role in the development of the cheese curd. Ca can also be added to dairy products as a nutrient supplement to improve their nutritional profile [53, 58].
On average, the total Ca content in bovine milk is 30 mM, of which approximately 66% is bound to the casein micelles, while 33% is in the serum phase [56, 59, 60]. Ca in the serum phase is largely soluble and associated with serum phosphate and citrate, and only a small portion is ionic (∼ 10% of total Ca). Micellar Ca can locate both on the surface and inside the casein micelles. Ca located on the surface helps micelle aggregation during the formation of the Para casein reticulum during cheese-making [61]. At the same time, Ca is present in the internal part of casein micelles as Ca phosphate, also defined as colloidal Ca (CCP; Ca3(PO4)2), having an average diameter of around 2.5 nm. Colloidal Ca fraction is essential in the formation and stabilization of casein micelles [62, 63], influencing the milk coagulation ability [64, 65]. It is known that adding Ca salts to milk decreases rennet clotting time and increases curd firmness [40, 51, 64, 66, 67]. Beyond dairy products, the fortification forms of Ca in other food products are also available, typically as inorganic (e.g., calcium carbonate, calcium hydroxide, calcium chloride, and calcium gluconate) and organic (e.g., calcium citrate, calcium malate, calcium lactate and calcium gluconate).
1.1.3.2 Magnesium
Magnesium (Mg) is the second-most important divalent mineral in milk, with an average concentration of ∼4.6 mM. The distribution of Mg in milk is about two-thirds in the serum phase and one-third in the colloidal phase, with ∼16% of the soluble Mg being in ionic form (Mg2+) [68, 69]. In dairy production, magnesium is often used as a coagulant in cheese-making. It helps to increase the firmness and texture of the cheese and improve flavor development [70]. In addition to its use as a coagulant in cheese-making, magnesium can also be added to milk-based beverages to increase their magnesium content and provide additional health benefits. The effects of ionic Mg on physicochemical instability of milk protein are very similar to Ca2+, but less pronounced than Ca2+ [70]. The binding of magnesium to caseins is pH-dependent, with optimal binding occurring at a pH of around 6.0–7.0. At lower pH levels, binding to caseins is reduced, while at higher pH levels, the protein becomes denatured and loses its ability to bind magnesium.
1.1.3.3 Phosphate
Phosphate is a naturally occurring mineral that can be found in milk. In milk, phosphates play an important role in maintaining the pH of milk and contributing to the buffering capacity of milk [71]. It is present in milk in the form of various compounds, including calcium phosphate, magnesium phosphate, and potassium phosphate. They are commonly used in the dairy industry due to their functional properties, such as their ability to bind calcium ions and stabilize proteins [72, 73]. The most used phosphate in dairy products is sodium phosphate, which is used as an emulsifier, thickener, and pH regulator [52]. Inorganic phosphate is present in milk in various forms, and from a total concentration of about 20 mM, approximately 10 mM are in the colloidal state and 10 mM are diffusible [71]. In the diffusible fraction, inorganic phosphate can be free or combined with calcium to form a calcium phosphate salt. In the colloidal state, inorganic phosphate and organic phosphate can be combined with calcium to form micellar calcium phosphate, which greatly influences the structure and stability of casein micelles [42, 68].
The interactions between lactose, milk proteins, and minerals are crucial to produce various dairy ingredients and products with unique textures, stabilities, flavors, and nutritional profiles. The extent of these interactions is a function of many processing and storage parameters including the composition of the system (Figure 1). Interactions affect the structure of each component and thereby exert both positive and negative effects of the final product. Thus, understanding these interactions helps the dairy industry create wide range of liquid and solid-state products.
Figure 1.
Schematic representation of the interactions between lactose, milk proteins, and minerals in dairy systems, and how these interactions are influenced by various processing and storage conditions.
2.1 Lactose-protein interactions
Lactose-protein interactions are fundamental in determining the structure, stability, and functionality of dairy systems. These interactions predominantly occur through hydrophobic interactions, hydrogen bonding, and electrostatic forces [74]. Hydrophobic interactions occur between the nonpolar regions of proteins and lactose molecules, influencing the solubility and stability of the proteins and affecting the texture and viscosity of liquid-state dairy products. In addition, the hydroxyl (OH) groups of lactose can form hydrogen bonds with the amine and carboxyl groups of milk proteins, stabilizing the proteins and affecting their interactions, which influences the overall texture of the products. Electrostatic bonding happens between the positively and negatively charged groups of the molecules. This interaction can be direct or indirect, with lactose complexing with charged groups of proteins. These types of interactions or complexations can lead to changes in protein solubility, stability, and even aggregation within the product. In some instances, excessive hydrogen and electrostatic interactions can lead to texture issues, while balanced hydrophobic interactions can result in desirable textural attributes in some dairy products.
During processing and storage of milk protein products, lactose might be involved in various chemical modifications such as early Maillard reaction or physical modifications such as lactose crystallization where lactose interacts with itself to induce nucleation and crystal formation [75]. Furthermore, the lactose-protein interaction can vary significantly based on environmental factors such as pH, temperature, and the presence of other solutes. In addition, processing conditions, time, and processing methods, such as thermal or non-thermal processing, affect these interactions, thereby changing the ultimate final product consequences. The state of lactose (crystalline or amorphous) and the specific type of proteins involved (e.g., casein, whey proteins) are additional variables that define these interactions. In practical applications, understanding lactose-protein interactions is essential for optimizing the processing and storage of dairy products.
2.1.1 Maillard reaction
Heat treatment has been a cornerstone technology in the food and dairy industries, primarily employed to sterilize and prolong the shelf life of milk products [76, 77, 78]. The Maillard reaction, also known as nonenzymatic glycation, is a complex process that occurs between amino acids and reducing sugars during heat treatment. It impacts product quality by causing undesirable flavor, texture, and color changes, nutritional losses, and potentially harmful compounds such as melanoidins [25, 79, 80]. In the case of milk, lactose reacts with the free amino acid side chains of milk proteins (mainly ε-amino groups of lysine residues) to proceed to early, intermediate, and advanced stages of Maillard reaction and forms enormous kinds of Maillard reaction products [25, 81]. The rate of Maillard reaction is affected by temperature, time, water activity (aw), pH, active reactants, and the type of ratio of reducing sugar to lysine in dairy systems [82]. The Maillard reaction can take place during heat processing of dairy products with a low relative humidity, such as spray drying of milk, and during the processing of liquid products, including sterilization/pasteurization of beverages and liquid foods. Notably, once initiated during the processing phase, the Maillard reaction continues to evolve, affecting the product throughout its shelf life [5, 83].
It is well known that the Maillard reaction is closely associated with the conditions of heat processing, such as temperature and duration of heating [80, 84]. This relationship is attributed to the fact that varying levels of heat can modify the glycation processes and the binding sites of glycoproteins in bovine milk. In a recent study by Zhang et al. [80], raw milk samples heated at 75, 90, 105, 120, or 135°C for durations of 5, 15, or 30s showed increased levels of fluorescence intensity, furosine, lactulose, and 5-hydroxymethylfurfural, indicating a positive correlation with heating temperature and duration. Besides, the number of glycated proteins in milk heated at 135°C for 5 seconds rose from 14 to 49, and binding sites increased from 47 to 166 compared to raw milk. In the Maillard reaction of proteins and peptides, the amino groups of lysine and arginine residues exhibit varying reactivity levels. While lysine stands out as one of the most reactive amino groups, the arginine residue, in comparison, demonstrates considerably lower reactivity [24]. Additionally, lysine residues in caseins seem to be more reactive than in serum proteins, while κ-casein seems to be the most reactive casein [85]. In a recent study by Nielsen et al. [86], the effect of lactose on the Maillard reaction was explored in six milk protein mixtures, including pure whey protein isolate (WPI), pure casein micellar isolate (MCI), MCI + WPI combination with and without lactose (4.7% w/v), under heat treatment at 121°C for 0, 15, or 30 minutes. They discovered that the initiation of Maillard reaction is highly influenced by the presence of lactose. Notably, the concentration of furosine (a marker of the early-stage Maillard reaction) was significantly (P < 0.05) higher in MCI compared to WPI samples before heating, which was likely due to the higher content of lactose in MCI as compared with the level in WPI. Upon heating MCI with lactose for 15 or 30 min, the concentration of furosine increased from an initial 0.015 to 0.112 and 0.109 mol, respectively. A parallel increase in furosine levels was observed in WPI and the MCI/WPI combination when heated in the presence of lactose. Similarly, Paul et al. [87] found that lactose content which is crucial for the level of initial process induced browning of the powder, while additional browning reaction that occurs during storage is mostly independent of lactose content but more by proteins already involved in advanced Maillard reaction steps prior to storage [87]. As a result, they suggested that the levels of lactosylated proteins in the fresh product should be carefully monitored as they could evolve later into melanoidins during storage.
In addition to temperature, the rate of deteriorative reactions and storage stability are related to water activity and water content of dairy systems [75, 88, 89]. Miao and Roos [88] found that the crystallization of lactose enhances Maillard reaction, due in part of the release of water. In addition to this, they also noted that the type of crystals formed plays a critical role. The α-lactose monohydrate is the most commercial solid state, and it has the most stable crystalline form, which are harder, more stable, and also being less hygroscopic, while β-anhydrous crystals have greater hygroscopicity and a much higher solubility [3, 90]. Amorphous lactose is very hygroscopic, and under stressful storage conditions, such as high relative humidity and/or increased temperatures, the metastable amorphous lactose will proceed through an irreversible transition to form stable crystalline forms [91]. Furthermore, the crystallization of amorphous lactose to α-lactose monohydrate releases water. When this occurs in a closed environment (e.g., a bag or can of powder), the relative humidity increases, further promoting reactions that are enhanced at a higher water activity (aw) [5]. In a study by Ceylan Sahin et al. [89], different water activity was found in spray-dried cheese powder samples stored using three different types of packaging materials for 12 months. Samples with higher water activity were found to have a greater extent of Maillard reaction. Linear correlations were detected between water activity and non-enzymatic browning (P < 0.01).
Numerous studies demonstrated that the rate of Maillard reaction increases when the water activity values exceed 0.3 [92, 93]. Morgan et al. [75] found that high temperatures and increased water activity increase Maillard reaction, although lactose crystallization in the system was not directly influenced by the Maillard reaction. Similarly, Yan et al., [94] investigated the relationship between Maillard reaction and lactose crystallization at different water activities. They found that, with the increase in aw, whey protein-lactose model formed more browning and crystallization products than casein-whey-lactose model, indicating that the presence of micelle macromolecules and the interaction between casein and whey proteins limited the browning and crystallization in casein-whey-lactose models [94].
Moisture increase in dairy products also results in changes in glass transition temperature (Tg). Acevedo et al. [95] found Maillard reaction progresses extremely slow at temperature below Tg and increases with water content at low relative humidity (RH). Glass transition affects the mobility of the molecules in the spray-dried powders. When the materials are in a glassy state, the molecular mobility is somewhat limited, while in the rubbery state, greater molecular mobility was observed [96, 97, 98]. Different molecular mobilities may affect the reaction kinetics of the Maillard reaction. An increase in the reaction rate of Maillard reaction has been observed when the temperature is above the glass transition temperature, and the glass transition temperature of the sample (Tg) is depressed significantly due to water production via Maillard reaction [96, 97, 98]. They concluded that this behavior is diffusion-controlled and favored by water plasticization.
Furthermore, Maillard reaction is governed by solid water interactions and structural changes within the system. Amorphous materials undergo structural changes at Tg, which is a function of relative proportion of glass-forming components and water content. Increased water content leads to matrix plasticization, a decrease in Tg, and various chemical and physical changes. These factors can cause the matrix to collapse, affecting the Maillard reaction. So, they highlighted the importance of the system structure. In a study by Miao and Roos [99], a critical value of difference between the particle and its glass transition temperature (T-Tg) was observed. When the temperature differences were above this critical value, a significant increase in the rate of Maillard reactions was observed. At lower temperatures close to Tg, the Maillard reaction rate was low and more water content dependent [99]. A later study by Gómez-Narváez et al. [1] on whey powders also suggested that lactose in the rubbery-like state promotes Maillard reactions. During the spray-drying process, the moisture contents of the particles change rapidly, and thus the glass transition temperatures of the particles also change [1]. The results of Miao and Roos [99] suggested that the change in the glass transition temperature during the spray drying process is also an important factor that needs to be considered when studying Maillard reactions in spray dryers.
The Maillard reaction can influence the solubility of powders. In a related study, Le et al. [81] investigated the protein changes in relation to solubility, Maillard reaction, and protein cross-linking of whole milk power (WMP), skim milk power (SMP), and whey protein concentrate (WPC) when stored at different relative humidities (RHs). They found that an increase in certain Maillard reaction indicators (furosine) correlated with a drop in solubility for MPC, WMP, and SMP, a pattern absent in WPC. The amount of furosine in WMP increased from around 159 mg/100 g of protein to almost 3000 mg/100 g of protein after 12 weeks of storage at 66% RH. This is about twice the amount reached in MPC power under the same conditions. This could be explained by the higher lactose/protein ratio in WMP [81].
The physical and functional properties of powders are affected by the degree of Maillard reaction by enhancing protein interactions and aggregation. Maillard reaction can induce specific protein cross-linking (e.g., GOLD-glyoxal lysine dimer, MOLD-methyglyoxal lysine dimer) [100] through heat and storage, leading to aggregation [81, 101]. Covalent cross-links include intermolecular disulphide bridges, lysinoalanine (LAL) and lanthionine (LAN) [21, 102]. The amount and ratio of covalent/non-covalent aggregation might change when heating in the presence of saccharides, due to the Millard reaction. Supporting this hypothesis, Fan et al. [74] verified that the Maillard reaction promotes protein cross-linking and, in turn, is influenced by protein cross-linking. Specifically, they found that the Maillard reaction was slower when the degree of protein cross-linking was greater in modified milk protein concentrate powders.
2.1.1.1 Glycation
The Maillard reaction is also known to lead to protein glycation, a process in which proteins become modified by the reaction of their amino groups with reducing sugars [103], and thus significantly influences the physicochemical and functional attributes of proteins such as solubility, hydrophobicity, and interactions within the food matrix. The dynamics of the glycation process are determined by various parameters including the nature of the protein and saccharide involved, as well as the environmental conditions such as temperature and pH [104, 105]. An interesting observation by Milkovska-Stamenova and Hoffmann [83] reveals that lactose-free milk exhibits higher glycation levels compared to regular milk. This is attributed to the enhanced reactivity of monosaccharides like D-glucose and D-galactose relative to the disaccharide lactose. Furthermore, the degree of glycation in whey proteins escalates notably when UHT milk is stored at elevated temperatures (28°C and 40°C) as opposed to cooler conditions (4°C), as demonstrated by Holland et al. [104]. This trend is consistent with findings by Guyomarc’h et al. [106], where β-lactoglobulin (β-LG) and β-casein (β-CN) in skim milk powder showed increased glycation at storage temperatures above 4°C. Storage duration is another pivotal factor impacting protein glycation levels in milk products. For instance, Rauh et al. [107] documented an increase in the glycation level of β-lactoglobulin, α-lactalbumin, and caseins in UHT milk after a six-month storage period at room temperature, a discovery made possible through liquid chromatography-mass spectrometry (LC-MS) analysis. Given these insights, it is advisable to store dried milk and whey solids under dry and cool conditions and to practice stock rotation to mitigate browning and other undesirable changes during storage.
2.1.1.2 Lactosylation
Lactosylation is a specific type of glycation reaction that occurs between lactose and proteins or amino acids, and it is an integral component of the Maillard reaction. The occurrence of lactosylation may not cause browning, but can lessen the nutritional quality of milk due to the blockage of lysine, which is no longer available for digestion [92]. This reaction is mainly induced by heat treatment. Nevertheless, some authors have demonstrated that lactosylation also occurs during storage of milk powder [106]. Whey proteins, in particular, undergo lactosylation within a temperature range of 25 to 45°C, with the rate intensifying and peaking at around 65°C. Interestingly, lactosylation progresses more rapidly in a dry state than in a liquid state at elevated temperatures, binding typically 4 to 11 lactose molecules to each protein, although studies have reported varying numbers of lactose molecules per protein [6, 75, 108].
Environmental conditions such as water activity and pH remarkably influence lactosylation. Optimal water activity ranges between 0.4 and 0.8, facilitating the reaction by enhancing component mobility. Moreover, pH levels significantly dictate the extent of lactosylation; for instance, lower pH levels decrease reactive unprotonated amine groups, whereas higher pH levels amplify protein negative charge, leading to increased electrostatic repulsions and, consequently, heightened lactosylation [109]. The rate of lactosylation escalates further with a rise in temperature and relative humidity. However, the presence of the Maillard reaction over time consumes lactosylated proteins, thereby reducing their prevalence in the final product [109].
Lactosylation also leads to the formation of reactive amino acid residues, instigating intra- and intermolecular crosslinks and thereby affecting the protein structure [108]. The interaction between proteins and lactose, and the subsequent Maillard reaction, can cause a loss of protein tertiary structure, inducing steric stress and prompting protein unfolding [86]. In some cases, lactosylation results in a shift in protein structure, such as the β-sheet structure of β-lactoglobulin (BLG), suggesting the formation of hydrogen bonds between BLG and lactose, especially at low relative humidities [7].
The presence of lactose can modify the structure and functionality of milk proteins, leading to changes in properties such as solubility, viscosity, and heat stability [5, 19, 110]. Conversely, milk proteins can also influence the crystallization of lactose in dairy products, thereby affecting properties such as texture and shelf life [111, 112, 113]. Thus, knowledge of the mechanisms occurring in protein-lactose systems is important to develop desirable properties in dairy products. In the case of dairy powders, the state of lactose significantly impacts shelf life, particularly in the meta-stable state in whey powders produced by spray-drying or freeze-drying. Under harsh storage conditions, these powders may experience a glass transition zone of lactose, leading to increased molecular mobility and subsequent physio-chemical changes like lactose crystallization, particle caking, and lipid oxidation [114, 115, 116]. Therefore, controlling the amorphous phase of lactose or managing the crystallization process becomes a pivotal consideration for the dairy industry to mitigate issues like caking, especially in high-temperature and high-humidity environments [117].
2.1.2 Denaturation, coagulation, and protein stabilization
Lactose significantly influences the conformation and aggregation of milk proteins, playing a key role in protein stabilization [118]. Research focusing on the thermal stabilities of whey proteins has demonstrated that lactose, like other sugars, can stabilize protein aggregation under thermal stress [119, 120]. This stabilization effect is further supported by findings showing varying adhesive strengths of lactose in whey deposits on surfaces [121]. Differential scanning calorimetry (DSC) has been employed to illustrate how lactose presence elevates the thermal denaturation temperature (Td) and denaturation enthalpy of proteins such as β-lactoglobulin (β-Lg) and Bovine Serum Albumin (BSA) [75, 81, 122].
The stabilization of protein conformation by lactose is often discussed in terms of preferential hydration, where lactose is excluded from the nonpolar surfaces of proteins, contributing to stability by maintaining or enhancing protein hydration [27, 48, 123]. In dry states, lactose stabilizes proteins by forming hydrogen bonds with protein molecules, replacing those between water and the protein, thereby preserving the protein’s native conformation [124, 125]. Additionally, lactose can form amorphous matrices that encapsulate protein molecules, inhibiting structural changes by progressively removing water [126, 127]. This concept of stabilizing proteins within an amorphous sugar matrix is supported (Figure 2) by Buera et al. [128].
Figure 2.
Schematic interpretation of the effect of sugar crystallization on (a) the acceleration of chemical reactions, (b) protein denaturation, (b) the membrane integrity, and (d) and release of encapsulated compounds. Reprinted with permission from [128].
Lactose also binds to proteins, maintaining their colloidal stability. The physical state of the sample, such as the formation of a glassy matrix by amorphous lactose, plays a crucial role in this process [129]. Hajihashemi et al. [7] emphasized that saccharides, including lactose, protect protein structure through mechanisms like the creation of an amorphous sugar network, which reduces protein mobility and reactivity, or by diluting proteins in a solid state to reduce intermolecular contact. Furthermore, lactose’s hydrophilicity can disrupt the direct interaction of caseins, affecting the protein network [130]. The impact of residual water content on protein stability varies depending on the type of protein; for example, whey proteins remain unaffected by moisture changes, whereas casein proteins are more sensitive to hydrophilic alterations [131].
2.1.3 Lactose solubility and crystallinity
The solubility and crystallinity of lactose are significantly impacted by its molecular interactions with whey proteins, primarily through hydrogen bonding. Proteins have been recognized for their ability to delay the crystallization of lactose, as demonstrated in studies by Thomsen et al. [129] and Hajhashemi et al. [123]. These interactions hinder lactose mobility, potentially due to an elevation in the glass transition temperature (Tg) caused by preferential water sorption, as suggested by Maidannyk and Roos [132].
The crystallization process of lactose is influenced by a multitude of factors including relative humidity (RH), duration, water content, and temperature. Hajihashemi et al. [7] noted that the presence of proteins modifies the crystallization behavior of lactose by altering its Tg. This alteration, alongside molecular rearrangements and interactions within the new microenvironment, significantly impacts crystallization dynamics. During lyophilization, water molecules are replaced by lactose in the β-lactoglobulin (BLG) structure. This substitution, coupled with the intermolecular hydrogen bonding between lactose and proteins, results in elongated chain lengths and retards the crystallization of lactose. Seville et al. [133] observed that, during the drying process, hydrophobic proteins tend to migrate through the aqueous phase and accumulate at the powder surface. Conversely, hydrophilic lactose relocates to the core of the particle. Rapid cooling and water removal during this process often leave lactose in a hygroscopic, unstable, amorphous state. Under conditions of low RH and temperature, lactose molecules fail to arrange efficiently, leading to a structure that is more open and porous. Conversely, an increase in temperature and RH enhances molecular mobility and reduces viscosity, causing lactose to transition into a syrup-like supercooled liquid state. Peleg [134] discussed how, at higher moisture levels, lactose is released from the particle surface due to capillary forces and surface tension. This release leads to the merging of lactose with adjacent particles at contact points, forming viscous liquid bridges. These bridges, initially leading to onset sticking and caking issues, can solidify over time to form robust connections, ultimately affecting the flowability and crystallization behavior of lactose.
Furthermore, the type of protein present is important. They found that crystallization was easily induced in heated skim milk powder (SMP) but delayed or even inhibited by the presence of whey protein concentrates (WPC). WPC35 (with 35% protein and 51% lactose) reacted faster than WPC60 (with 60% protein and 23% lactose). Consequently, the influence of the Maillard reaction on crystallization depended on the protein/lactose ratio in the milk systems studied.
2.2 Lactose-minerals interactions
The interaction between lactose and minerals within milk and dairy products is crucial, markedly impacting their physicochemical and structural characteristics. The nature of these interactions is largely determined by the specific type of mineral present, its concentration, and the ambient conditions of the system, particularly the pH and temperature. These variables collectively influence how lactose interacts with minerals, thereby shaping the overall quality, stability, and functionality of dairy products. Understanding these interactions is paramount for optimizing processing techniques and enhancing the nutritional and sensory profiles of dairy products.
2.2.1 Lactose crystallization
Minerals as impurities in lactose solutions would govern lactose crystallization in a direction that was dependent on the concentration and the type of salt present [30, 135]. Minerals can act as nucleating agents, thus accelerating the crystallization of lactose. Furthermore, minerals can sometimes integrate into the crystal lattice of lactose, altering its growth rate and morphology. Some minerals can also block the active sites on growing crystals and inhibit the crystal growth, either by forming complexes with calcium or by reducing the availability of lactose for crystallization.
2.2.1.1 Influence of Ca on lactose crystallization
Calcium is one of the most important minerals in milk and dairy products, and it plays a crucial role in the interaction with lactose. Jelen and Cloture [135] demonstrated that impurity levels greater than 10% with CaCl2 could retard the crystal growth rate of lactose, although can lead to formation of more stable lactose crystals. Conversely, the addition of NaH2PO4 in deproteinated whey increased the growth rate of lactose crystals by 30%. Wijayasinghe et al. [30] concluded that the presence of 0.12 and 0.072% (w/w) Ca significantly increased the enthalpy of water evaporation (P < 0·05) in comparison with pure lactose. The energy required to evaporate water from the pure lactose solution was ∼679 J/g, while it increased to 932 and 946.9 J/g in the presence of 0.12 and 0.072% Ca, respectively. Moreover, the enthalpy of water evaporation decreased to 813.7 J/g with the reduction in Ca concentration (0.035%). The presence of Ca affects the behavior of lactose, hindering the removal of water surrounding lactose molecules by forming a strong hydration layer and thus inhibiting the overall crystallization [30, 31, 136]. The divalent Ca2+ ion exerts a strong electric field that promotes the structuring of water; thereby, Ca2+ can organize the structure of water [37, 137]. Salts have been shown to affect both nucleation and growth stages of lactose crystallization through differential binding to lactose, depending on the radius/charge ratio of the cation present in the salt and its hydrophilic and hydroscopic nature [29]. Ca, with its high radius/charge ratios of cations, has a low ability to bind lactose [29], although it may be incorporated into lactose crystals, hindering the growth rate and crystal size. These structural changes in water molecules can directly affect the solubility of lactose and subsequent supersaturation of the lactose solution, leading to different behavior during crystallization [29]. Additionally, Ca2+ ions strongly interact with four to six layers of water molecules via ion-dipole interactions, further restricting the mobility of water molecules [138] and densely packing them in these hydration layers compared to water molecules in pure lactose solutions, resulting in restricted mobilities. Mimouni et al. [113] suggested that the binding of Ca with water could create local supersaturation spots, promoting the production of small crystals and retarding crystal growth. Moreover, the formation of calcium-lactose complexes is facilitated through the interaction of the hydroxyl groups of lactose molecules with the water molecules residing in the hydration layer of calcium. This interaction has been substantiated through the application of Fourier-transform infrared spectroscopy (FTIR). Figure 3 depicts the intensity of two prominent FTIR peaks, specifically observed around 1022 and 1065 cm−1 in lactose solutions containing calcium. These peaks have been demonstrated to exhibit an inverse relationship with the concentration of calcium in the solution [30, 136]. This observation underscores the complex nature of calcium-lactose interactions and their potential influence on the physicochemical properties of dairy products.
Figure 3.
FTIR spectra for lactose model solutions with the presence of lactic acid (LA) (1% or 0.05% w/w) or Ca (0.12% or 0.035%), control pure lactose (L). Reprinted with permission from [30].
2.2.1.2 Influence of other minerals on lactose crystallization
Other minerals such as magnesium, sodium, potassium, and anions such as phosphate and chlorides, along with trace minerals, can influence lactose crystallization. Magnesium can form less stable complexes with lactose. These complexes may alter the solubility characteristics of lactose and inhibit its crystallization under certain conditions. The formation of magnesium-lactose complexes can reduce the availability of lactose molecules for crystallization, thereby affecting the size, shape, and distribution of lactose crystals in dairy products. Magnesium ions can compete with calcium ions for binding sites in milk. Since calcium is a primary promoter of lactose crystallization, higher concentrations of magnesium can inhibit calcium’s ability to facilitate the formation of stable lactose crystals. This competition can lead to slower crystallization kinetics or the formation of less stable crystalline structures [135].
Sodium and potassium influence lactose crystallization to a lesser extent compared to calcium. These ions can change the properties of the medium, such as ionic strength, and thereby alter the solubility characteristics of the lactose solution, influencing crystallization kinetics [136, 138]. Phosphate anions can stabilize calcium-lactose complexes or directly inhibit crystallization in some cases. Chloride ions enhance lactose crystallization kinetics by stabilizing calcium ions or influencing the solubility of lactose. Trace minerals such as iron (Fe), zinc (Zn), and copper (Cu) may act as catalysts or inhibitors based on their concentrations and the presence of specific structures [30]. While calcium is the primary mineral influencing lactose crystallization due to its interactions with lactose and proteins, other minerals also play significant roles depending on their concentrations and chemical interactions in dairy systems. The interactions between lactose and minerals through various mechanisms are of utmost importance for the physico-chemical and functional aspects of dairy products. Therefore, this topic warrants further in-depth research.
2.3 Protein-mineral interactions
The significance of minerals in milk and dairy products is well acknowledged, particularly concerning their role in modulating the thermal stability of milk proteins and milk-based ingredients and their functionality. This modulation is primarily achieved through their influence on protein conformation and structure [139, 140]. The production process of milk protein-based ingredients encompasses several critical stages, each of which can impact calcium partitioning. Calcium ions (Ca2+) are known to induce protein instability by reducing the net superficial charge, which facilitates protein aggregation and enhances hydrophobic interactions. These stages include temperature adjustments (heating and cooling), concentration processes (evaporation and drying treatments), and the separation of milk components, such as through membrane filtration [141, 142, 143, 144]. Temperature variations significantly influence protein conformation and stability, as well as the solubility of minerals during both thermal and cold processing. The addition of soluble calcium salts, such as calcium chloride, calcium hydroxide, and calcium gluconate, elevates the concentration of Ca2+ ions, potentially leading to protein instability in Ca fortified milk systems. This disruption has profound implications for the solubility, stability, and texture of dairy products, including calcium-fortified milks and various dairy-based ingredients and formulated products. This instability tends to affect whey proteins more than caseins, as documented in several studies [145, 146, 147, 148, 149]. The monovalent sodium and potassium ions do not involve in direct interactions, although they influence the ionic strength of the system and the buffering capacity of the milk, leading to protein instability. However, phosphates play a major role in terms of anions. These effects are not only critical during the processing phase but also play a pivotal role in determining the quality and storage stability of the final products [13, 150, 151, 152, 153]. The degree to which these changes are permanent or temporary largely depends on the specific conditions of processing and the nature of the changes observed [21]. Thus, understanding the interactions between minerals and milk proteins is vital for producing high-quality dairy products.
2.3.1 Temperature and pH-protein aggregation
Chemical changes occurring within milk during heating also include alterations in the solubility of calcium phosphates, leading to a consequential shift in the pH of milk. This change is part of an equilibrium process involving the formation of insoluble calcium phosphates, as depicted in Eq. (1). Interestingly, most pH-induced denaturation of proteins and alterations in mineral balance are found to be reversible upon cooling the milk [154]. For instance, research has shown that approximately 60% of calcium and 40% of phosphate in the serum phase migrate to the colloidal phase when heated to 90°C for 40 minutes. Upon subsequent cooling to 4°C for 20 hours, between 75 and 90% of the heat-precipitated calcium phosphate is resolubilized. These observations indicate that heating milk to temperatures below 90°C does not significantly impact the composition of calcium phosphate, its dissolution behavior, or the quantity of calcium directly bound to casein, provided the heating is followed by an extended period of cooling [154].
3Ca2++2HPO42−⇌Ca3(PO4)2+2H+E1
In the dairy industry, a significant challenge is the formation of milk solid deposits on the surfaces of processing equipment during ultra-high temperature (UHT) processing of milk products, such as high-protein solutions and creams, a phenomenon known as fouling. Fouling results in product loss, diminished efficiency, and increased energy expenditures [155, 156]. The main culprits for component fouling during thermal processing are the heat-induced destabilization of proteins and the precipitation of calcium phosphate [156, 157]. Moreover, whey proteins in milk, especially β-lactoglobulin (β-LG), are prone to heat-induced denaturation and aggregation. The susceptibility of β-LG stems from two main reasons: its abundance in bovine and most mammalian milk and the presence of a free thiol group that can engage in -SH oxidation or -SH/-S-S- interchange reactions, impacting protein stability and aggregation [21, 158, 159]. In contrast, caseins are more resilient to heat-induced denaturation and aggregation, only affected at temperatures exceeding 100°C. However, calcium phosphate can contribute to fouling deposits even at temperatures above 50°C. Research has shown that calcium ions can promote the heat-induced aggregation of β-LG, with the structure of the aggregates depending on a critical molar ratio of calcium to protein [160, 161, 162, 163]. For instance, the addition of calcium to whey protein concentrate (WPC) solutions has been observed to intensify fouling [164]. Similarly, at equivalent protein content and pH, whey protein isolate (WPI) and pure β-LG exhibit less fouling compared to whey, a difference attributed to the higher mineral content of the latter [165]. These observations align with the findings of Petit et al. [166], who investigated the aggregation and fouling behavior of β-LG in relation to [Ca2+]. The study revealed that the overall rate of β-LG unfolding and subsequent aggregation escalated by 1.5 times with increasing [Ca2+], which acted as a catalyst, particularly during the aggregation phase.
Research by Barone et al. [13] indicates that augmenting levels of CaCl2 in infant milk formula reduces the net zeta-potential from −51.1 to −22.1 mV and increases ionic calcium concentration from 1.44 to 6.99 mM. Similarly, Philippe et al. [167] observed that the addition of 4.5 mM CaCl2 to skim milk increased Ca2+ levels from 1.56 to 2.86 mM. Moreover, they noted that calcium fortification led to approximately 80% of the mineral associating with the micelle, accompanied by a rise in inorganic phosphate and citrate levels [167]. The increase in Ca2+ concentration reduces the surface charge on whey proteins, diminishing the magnitude of electrostatic charge between proteins due to charge shielding. This phenomenon is believed to stem from calcium-mediated bridging between the carboxylic acid groups of aspartic and glutamic acids, fostering the crosslinking of whey protein molecules. Consequently, this process triggers protein aggregation and potentially gel formation [168, 169].
Calcium and phosphate, crucial components of the mineral fraction in milk, significantly influence the coagulation process. The levels of these ions are pivotal in determining the gelation properties of milk. Research has consistently demonstrated that elevated ionic calcium levels enhance the gelation properties of milk [61]. For instance, the introduction of calcium to preheated cow milk at 90°C for 10 minutes notably decreases the gelation time and amplifies the curd-firming rate. This effect arises because calcium addition lowers the pH of the milk, subsequently reducing the electrostatic repulsion between micelles. This reduction facilitates the formation of calcium bridges between casein particles and elevates the level of calcium colloidal phosphate, promoting coagulation. However, there is a threshold to the beneficial effects of calcium addition. Exceeding 10 mM of calcium can adversely affect curd formation. Excessive calcium enhances the positive charges on the micelle surface, leading to increased charge repulsion and, consequently, the formation of weaker gels or the complete inhibition of gelation [170, 171]. Conversely, introducing phosphate into milk results in delayed onset gelation during rennet-induced coagulation. This delay is attributed to the reduction of the ionic calcium amount, illustrating the delicate balance between minerals and their profound impact on the coagulation properties of milk.
2.3.2 Protein stabilization
Whey proteins, under normal conditions, exhibit considerable physicochemical stability in solution, especially at pH levels distant from their isoelectric point, due to their high charge-to-mass ratio. In the typical pH range of dairy products (6.5–7.0), whey proteins carry a negative charge because of their globular structure, which exposes numerous carboxylic acid residues on their surface [47]. Consequently, whey proteins demonstrate a zeta potential between −35 and − 25 mV, resulting in substantial intermolecular repulsive forces and, hence, a stable physicochemical state, as depicted in Figure 4.
Figure 4.
Schematic illustration of intermolecular attractive forces of proteins mediated by ionic calcium. (a) Innate repulsive forces of whey protein in solution; (b) reduced zeta potential of protein and increased protein intermolecular attractive forces caused by calcium ions (red dots); (c) intermolecular association of proteins mediated by calcium ions; (d) detail of protein-calcium interaction bridges between the carboxylic group of aspartic and glutamic amino acids and attractive interactions between hydrophobic domains of protein (gray) [13]. Reprinted with permission from [54].
The addition of calcium not only facilitates crosslinking between proteins but also induces significant conformational changes in them. This is predominantly due to the closer proximity of proteins caused by calcium-mediated bridges, which displace water molecules between proteins, enhancing hydrophobic interactions within the hydrophobic domains of whey protein. This increased interaction leads to the formation of more thermodynamically stable aggregates, characterized by higher entropy. However, the impact of Ca2+ on protein stability can vary. In certain cases, Ca2+ enhances the stability of specific proteins, particularly when it binds strongly to intramolecular sites. This effect is well-documented in whey protein α-lactalbumin (α-lac), and to a lesser extent in β-lactoglobulin (β-lg) [172]. α-Lac, in its apo-state (calcium-depleted), exhibits a high affinity for Ca2+, significantly more than in its holo-state (calcium-bound). Binding of Ca2+ to apo-α-lac induces conformational changes, increasing the protein’s resistance to denaturation under heat treatment by elevating its enthalpy level [173]. This property has been leveraged in the production of value-added whey protein concentrates, enriched in α-lac, which are extensively used in the formulation of nutritional dairy-based products, such as infant formula. Furthermore, the heat capacity of α-lac shows a linear correlation with increasing Ca2+ concentrations ranging from 0 to 10 mM, with corresponding ΔH (kJ/mol) values ranging from 217 to 313 [174]. Consequently, the denaturation temperature of α-lac is observed to be a function of the logarithm of [Ca2+]. On the other hand, recent studies indicate that calcium can reduce the activation energy and enthalpy required for protein unfolding and denaturation, leading to protein destabilization at lower temperatures [166]. Kaushik et al. [175] found that the thermal stability of Ca-enriched milk (500 ml/L Ca) using various calcium salts (such as Ca-chloride, Ca-acetate, Ca-hydroxide, and Ca-citrate) was lower compared to non-Ca-fortified milk, except for Ca-citrate, which exhibited better heat stability. However, the use of Ca-citrate was associated with increased viscosity, presenting a potential disadvantage in certain applications [175].
In conclusion, the interactions between milk proteins, lactose, and minerals in dairy products are complex and multifaceted. A comprehensive understanding of these interactions is crucial for the design, formulation, and processing of dairy products to achieve desirable outcomes. Among these interactions, protein-protein, protein-lactose, lactose-mineral, and protein-mineral interactions are particularly significant. Protein-protein interactions can lead to aggregation, affecting texture and solubility, while protein-lactose interactions, such as the Maillard reaction, can impact flavor and nutritional quality. Lactose-mineral interactions depend on mineral type and concentration and can influence solubility and crystallization patterns. Furthermore, protein-mineral interactions, especially with calcium (Ca), can destabilize proteins by reducing surface charge and enhancing hydrophobic interactions in a concentration- and protein type-dependent way. These interactions occur through various mechanisms, including electrostatic interactions, hydrogen bonding, hydrophobic bonding, covalent linking, and complexation. These interactions are influenced by various intrinsic factors (e.g., type of protein, lactose forms, and calcium salt chemistry), as well as extrinsic factors (e.g., pH, moisture, temperature, ionic strength, pressure, and shear force). To date, progress has been made in understanding interactions between protein, lactose, minerals, and other components in different dairy systems. However, more research is needed to thoroughly investigate ways to maximize the total nutritional value and product performance while avoiding physicochemical destabilization. Future research should focus on further understanding the mechanisms behind these interactions and developing new techniques and technologies to improve and optimize the quality, functional properties, and nutritional value of dairy products. Most of the above research is based on the interaction between two components, a better understanding of how each of these components present in various properties interacts with each other is vital in controlling the quality of dairy products. In addition, advanced processing technologies need to be explored further to understand any deviations in these interactions compared to the normal processing methods currently employed in the industry. By gaining a comprehensive understanding of these complex interactions, a wide range of value-added products that meet consumer demands for nutritious products could be produced in a sustainable manner.
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Written By
Yuanyuan Zhao, Juhi Saxena, Tuyen Truong and Jayani Chandrapala
Submitted: 13 June 2024Reviewed: 24 June 2024Published: 13 September 2024
Open access peer-reviewed chapter - ONLINE FIRST
