Interaction between milk proteins and micronutrients

C H A P T E R

14 Interaction between milk proteins and micronutrients ` se Considinea, John Flanaganb, Simon M. Lovedayc,d, Th
ere Ashling Ellisd a

Fonterra Co-operative Group Ltd, Palmerston North, New Zealand bNaturex S.A., Avignon, France cAgResearch, Palmerston North, New Zealand dThe Riddet Institute, Massey University, Palmerston North, New Zealand

Introduction The existence of a three-dimensional, folded protein structure is dependent on several forces. These include hydrogen bonding, hydrophobic interactions, van der Waals’ forces, and electrostatic interactions. Some amino acid residues exhibit a hydrophobic character, and electrostatic forces are based on interactions between charged residues. Thus, the conformation of a protein is dependent upon the presence of particular amino acids and the variation of residues within the primary structure. Although a protein may be in its native state, interactions through hydrophobic, electrostatic, van der Waals’, and other forces can occur at exposed regions on its surface or in cavities and pockets. It is through these mechanisms that interactions between milk proteins and various micronutrients, such as vitamins, fatty acids, minerals, and surfactants, can occur. Protein structures can be readily destabilized from their native state by relatively minor changes in the environmental conditions. Variations in pH, temperature, and pressure, for example, can all induce structural transitions in proteins. In some cases, the objective of processing is to induce changes in protein structure, for example, the heating of whey proteins to form gels. In other cases, however, changes in the environmental conditions can elicit changes in protein structure that result in undesirable functional properties, for example, the loss of solubility or biological activity. The more recent studies into the interactions of proteins with micronutrients have changed tack somewhat, with the focus being more on how these interactions can be exploited so that proteins can be used as carriers for micronutrients in

Milk Proteins https://doi.org/10.1016/B978-0-12-815251-5.00014-1

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functional foods. The impact of these interactions on the bioaccessibility and bioavailability of the micronutrients in vivo is at the forefront of much of this research; however, it has also shed some new light on the interactions themselves and how the systems can be manipulated to increase binding. In addition to pH-, temperature-, and pressure-induced changes in protein structure, the presence of micronutrients can affect how the protein structure reacts to variations in pH, temperature, or pressure. By interacting with specific sites within the protein’s three-dimensional structure, micronutrients can render a protein more, or less, susceptible to denaturation.

Interaction between milk proteins and micronutrients Micronutrients, such as vitamins, minerals, fatty acids, and sugars, may interact with milk proteins through a variety of mechanisms. The main mechanism is through hydrophobic interaction, and the majority of studies on the interactions between milk proteins and micronutrients have focused on globular whey proteins, which have hydrophobic cavities and extensive secondary and tertiary structure. In contrast, the interactions between caseins and micronutrients are mostly based on electrostatic driving forces. Interactions between milk proteins and micronutrients can have appreciable effects on the physical and chemical properties of proteins during processing, and these effects are discussed in the next section. It should be noted that one significant advancement in this area in recent years has been the use of molecular docking studies to examine the interactions between food proteins and micronutrients. Although such studies have commonly been utilized in the medicinal chemistry area for some time, their application to food systems has been limited.

Vitamin A “Vitamin A” refers to a group of molecules that are structurally related to retinol, which consists of a β-ionone ring and an isoprenoid “tail” containing four conjugated double bonds and a terminal hydroxyl group. These “retinoids” may have carboxylic acid, aldehyde, or esterified fatty acid terminal groups, but the all trans-conjugated double bond structure is needed for biological activity (Weiser and Somorjai, 1992). Cis-trans isomerization, which leads to the loss of biological activity, is catalyzed by heat, transition metal ions, free radicals, and light, especially at wavelengths below 500 nm (Loveday and Singh, 2008). In foods, retinoids are often dissolved in the fat matrix, where they are protected from the oxidizing action of atmospheric oxygen by vitamin E and other antioxidants (Ball, 1988). They may also be dispersed in various colloidal carrier systems (Loveday and Singh, 2008). β-Lactoglobulin (β-LG) would seem to have a clear biological role as a ligand carrier, given its ability to bind a range of fatty acids, triglycerides, and vitamins. Nonetheless, no definite biological function has been attributed to β-LG (Creamer et al., 2011). It is part of the lipocalin family and has the three-dimensional structure that is common to all lipocalins. Each monomer consists of eight stranded antiparallel β-sheets bordered by an α-helix, which forms an inner hydrophobic area (Domı´nguez-Ramı´rez et al., 2013; Mensi et al., 2013). Papiz et al. (1986) identified that the structure of β-LG was remarkably similar to the structure of

Interaction between milk proteins and micronutrients

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retinol-binding protein (RBP). Two binding sites for hydrophobic ligands such as retinoid have been identified; one high affinity site is located inside the calyx, and there is some evidence for an additional low affinity binding site on the outside of the molecule between the α-helix and the β-barrel (Qin et al., 1998; Wu et al., 1999; Domı´nguez-Ramı´rez et al., 2013). The structural changes induced in different environments have been determined by X-ray crystallography (Qin et al., 1998). Further information about the structure and binding of β-LG can be found in Chapter 7. Different researchers have used a variety of methods to determine binding constants for the β-LG-retinoid complexation, thus making comparison between studies difficult. The range of retinoid binding constants reported for β-LG and the factors affecting their measurement are discussed in detail by Kontopidis et al. (2004). For example, Muresan et al. (2001) compared fluorimetry with equilibrium dialysis and found that the former yielded higher binding affinities than the latter. The pH, the genetic variant, and the source of the protein all contribute to the discrepancies in the literature. Free retinol is a rather unstable compound, especially in an aqueous environment, but its stability is greatly improved when it is bound to an RBP (Futterman and Heller, 1972). However, little endogenous retinol is found bound to β-LG when it is first purified, and the ligand most closely associated with the protein is palmitate (P
erez et al., 1989). Futterman and Heller (1972) used fluorescence measurements to deduce that bovine β-LG, like RBP, forms water-soluble complexes with retinol. Complexation with β-LG can make vitamin A more resistant to heat and UV light (Loveday and Singh, 2008). The β-LG-retinoid complex is slightly more resistant to tryptic hydrolysis than uncomplexed β-LG (Shimoyamada et al., 1996). Dufour et al. (1991) monitored the binding of retinol, retinyl acetate, retinoic acid, and β-carotene to native, esterified, and alkylated β-LG by the quenching of tryptophan fluorescence. The retinoids were bound to native or modified β-LG in a 1:1 M ratio with apparent dissociation constants in the range 10
8 M, whereas the molar ratio was 1:2 (ligand/protein) for β-carotene. A similar study using the same methods examined the difference in binding caused by genetic variation (Mensi et al., 2013). It was found that the dissociation constants were similar to those previously measured (10
8 M range), with the variant (β-LG A and β-LG B) not having a significant difference. Chemical modification of β-LG by methods such as methylation, ethylation (Dufour and Haertle, 1990), and alkylation (Dufour et al., 1991) have been shown to enhance the binding affinity for retinol, by opening up a second binding site. It may therefore be assumed that the partial change in the β-LG secondary structure that is produced by these treatments does not destroy the structure of the retinol-binding pocket. Similarly, moderate amounts of surfactants appear not to affect the retinol-binding site of β-LG (Taheri-Kafrani et al., 2008; Sahihi et al., 2012). In terms of the other whey proteins, very few studies have been carried out with ligand binding to α-lactalbumin (α-LA), bovine serum albumin (BSA), or indeed the immunoglobulins, in comparison with the vast range of studies with β-LG. The potential of ligands binding to α-LA was studied by Puyol et al. (1991); the binding of retinol and palmitic acid was assessed in a whey protein mixture. From this study, α-LA was shown to bind retinol more strongly than β-LG, but a much lower percentage of palmitic acid was bound to α-LA in comparison with β-LG. Futterman and Heller (1972) showed that, as with β-LG, BSA formed a strong fluorescent water-soluble complex with retinol. They also postulated that,

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14. Interaction between milk proteins and micronutrients

although no detectable retinol is bound to BSA in vivo, the possibility exists that this protein could serve as an auxiliary carrier if excess free retinol were introduced into the circulation. BSA can inhibit the photo-catalyzed oxidation of retinol but not retinoic acid (Shimoyamada et al., 1996). BSA is sometimes used in cell culture to reduce nonspecific binding of retinoids to plastic surfaces, but the binding of retinoids to BSA can affect their bioavailability (Klaassen et al., 1999). A recent study examined the molecular binding of β-carotene to whey protein isolate (WPI) (which contains all four of the major whey protein fractions) under a range of environmental conditions (temperature, pH, and salt concentration). The results from the docking studies revealed that β-carotene was bound to the residues of aromatic cluster II in α-LA, whereas it was bound to subdomain IIA of BSA (Allahdad et al., 2019). Raica et al. (1959) reported that retinol can also be bound to casein, and Mohan et al. (2013) found that a significant proportion of the vitamin A in commercial skim milk was bound to unmodified casein micelles. The insolubilization of the casein micelles in the acid casein and rennet casein forms promotes nutritional activity in retinol, which is not observed for the milk proteins in the native state (Adrian et al., 1984). Reassembled casein micelles have been shown to afford protection against high humidity and heat to β-carotene during storage (Sa´iz-Abajo et al., 2013; Loewen et al., 2018). The succinylation of casein has been explored as another method to increase the binding of vitamin A in the presence of this milk protein (Gupta et al., 2017).

Vitamin C Few studies have explored the interactions between vitamin C and milk proteins. A single binding site on BSA for ascorbic acid was recorded by Tukamoto et al. (1974). Oelrichs et al. (1984) also investigated the interactions between ascorbate and BSA. They suggested an intrinsic association constant of 2600 M
1 at 20°C. Dai-Dong et al. (1990) observed an increased stability of ascorbic acid in the presence of β-LG compared with in pure water, and vitamin C was more thermoresistant when heated in the presence of β-LG. In contrast to these studies, Puyol et al. (1994) reported a lack of interaction of ascorbic acid with β-LG or indeed with any of the other whey proteins. Puyol et al. (1994) suggested that the discrepancy between their work and that of Dai-Dong et al. (1990) may have been related to the methods used. Monitoring the reducing ability of ascorbic acid may not provide sufficient allowance for the effects of ascorbate losses through autoxidation. Puyol et al. (1994) also suggested that the antioxidant effect of the reductive thiols in β-LG and serum albumin may have a protective effect. Given the discrepancies among previous studies, another group recently sought to identify a precise binding site for vitamin C on β-LG (Shahraki et al., 2018). Spectroscopic and molecular docking methods were used with the researchers also investigating binding of vitamin K3 and folic acid at the same time. The results showed that vitamin C quenched the fluorescence through both static and dynamic mechanisms and that hydrogen bonds and van der Waals’ forces were the main interactions. Vitamin C was found to be surrounded by numerous amino acids of hydrophobic and hydrophilic nature, with the process seeming to occur automatically. The aforementioned amino acids, where binding occurred, were within subdomain B of the protein (Ala118, Asn88, Asn109, Glu108, Leu31, Leu39, Leu117, Lys91, Met107, Pro38, and Ser115) (Shahraki et al., 2018).

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Vitamin D The affinity of β-LG for vitamin D2 is about 10-fold greater than that for vitamin A and other retinoids (Dufour and Haertle, 1990; Cho et al., 1994; Wang et al., 1997b). For vitamin D2, 2 mol/mol of protein is bound, but only 1 mol of the various retinoids binds to β-LG (Dufour and Haertle, 1990; Cho et al., 1994; Wang et al., 1997a). Ergosterol and vitamin D3 will bind to β-LG at 2 mol/mol, and the binding affinity appears to be significantly greater than for vitamin D2. Wang et al. (1997b) showed that both vitamin D and cholesterol can bind to β-LG. However, they reported that only one molecule of vitamin D or cholesterol would bind in the calyx and suggested that the other molecule could be bound on an external site, as postulated by Monaco et al. (1987) and later confirmed using X-ray crystallography and fluorescence quenching studies (Yang et al., 2008; Liang and Subirade, 2012). Fig. 14.1 illustrates the sites at which vitamin D molecules will bind to β-LG, as identified crystallographically. The external site is located in a hydrophobic cleft between the entrance of the β-barrel and the α-helix, where hydrophobic ligands are stabilized by interactions with hydrophobic residues near the C-terminus (residues 136–149) (Yang et al., 2008). The binding of retinal, vitamin D2, and retinyl palmitate by β-LG was studied by Wang et al. (1999). Competitive binding experiments with palmitate indicated that retinal and palmitate did not compete for the same site; however, vitamin D2 appeared to displace palmitate at higher concentrations. Retinoids and vitamin D2 were bound more tightly than palmitate. Fogolari et al. (2000) demonstrated the importance of pH when binding ligands to β-LG, in some cases because of the effect of pH on the monomer-dimer-oligomer equilibria (Mercadante et al., 2012). Forrest et al. (2005) reported on the interactions of vitamin D3 with β-LG A under a range of environmental conditions (i.e., pH and ionic strength). They showed that binding depended greatly on the solution conditions. For example, at low pH [2.5 (ionic strength ¼ 0.15 M)], the EF loop (gate) is closed, and thus, vitamin D3 was probably weakly bound in the external hydrophobic surface. Upon lowering the ionic strength to 0.08 M,

FIG. 14.1 Structure of β-LG (gray) complexed with two molecules of vitamin D3 (white). Drawn using PyMOL (the PyMOL Molecular Graphics System, Version 1.3, Schr€ odinger, LLC) and PDB entry 2GJ5.

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14. Interaction between milk proteins and micronutrients

binding increased. It has been suggested that lowering the salt concentration allows more surface binding (Aymard et al., 1996). A dissociation constant of 0.02–0.29 μM was reported for β-LG A, with the apparent mole ratios of vitamin D3 bound per mole of β-LG A ranging from 0.51 to 2.04 (Forrest et al., 2005). Forrest et al. (2005) also studied the binding of vitamin D3 to β-casein, and reported on interactions under a range of environmental conditions. The binding constants of vitamin D3 to β-casein were dependent on pH and ionic strength. In agreement with the study of Lietaer et al. (1991), an increase in binding as a function of ionic strength was apparent at pH 6.6. This was attributed to enhanced hydrophobic interactions, creating more surface area for binding (Lietaer et al., 1991). Increased binding was associated with a weaker affinity, compared with lower ionic strength where the binding was stronger. Although the stronger interactions at low ionic strength were attributed to fewer protein interactions, Forrest et al. (2005) could not identify a reason for decreased binding at pH 8. A dissociation constant of 0.06–0.26 μM was reported for β-casein, with the apparent mole ratios of vitamin D3 bound per mole of β-casein ranging from 1.16 to 2.05. It was suggested by Forrest et al. (2005) that the rheomorphic nature of β-casein allowed the hydrophobic area to bind strongly with vitamin D3, in the most thermodynamically stable conformation. The hydrophobic interactions were aligned with the perturbation of phenylalanine and the quenching of tryptophan, both of which are located in the hydrophobic core. Haham et al. (2012) reported that encapsulating vitamin D in reassembled casein micelles improves its stability during thermal treatment or prolonged storage and maintains its bioavailability. The concept of delivering hydrophobic compounds in casein micelles is claimed in a patent from this group (Livney and Dalgleish, 2007). The potential of β-LG as a carrier for vitamin D3 in fortified foods has recently been explored (Diarrassouba et al., 2013). Finally, recent work in this area has examined the interactions of α-LA with vitamin D3. According to the results, α-LA has one binding site for the vitamin (assessed by molecular docking studies) and complexation occurring through hydrophobic intramolecular interactions reduced the heat stability of the protein (Delavari et al., 2015). Fluorescence quenching studies showed that there was a conformational change in α-LA upon interaction with the vitamin.

Other vitamins Milk also contains an array of vitamin-binding proteins, including vitamin B12-binding protein, folate-binding protein, vitamin D-binding protein, and riboflavin-binding protein. These proteins occur at low concentrations but may play a significant role in the uptake of vitamins from the diet (Salter and Mowlem, 1983). Folate-binding proteins are specifically involved in the uptake of folate in the intestine. They also reduce the availability of folate to bacteria in the gut and hence may have antibacterial properties (Ford, 1974). Raw bovine milk contains a riboflavin-binding protein (Kanno et al., 1991), and riboflavin bound to this milk protein has similar antioxidant activities to riboflavin bound to egg white riboflavin-binding protein (Toyosaki and Mineshita, 1988). This area, including the binding of trace elements, has been reviewed in detail by Vegarud et al. (2000). Since that review, Nixon et al. (2004) investigated the source of the cooperativity between folate-binding protein and folate, and their results suggested stoichiometric interactions.

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543

Minerals The abilities of certain milk proteins, in particular the caseins, to bind calcium are extremely well known. The extent of binding of the caseins is directly related to the number of phosphoserine residues and thus follows the order αS2- > αS1- > β- > κ-casein (see Chapter 6). Increased binding of calcium to the caseins results in reduced negative charges on the casein molecule, resulting in diminished electrostatic repulsion and consequently inducing precipitation. Caseins with high numbers of phosphoserine residues, such as αS1-casein B, αS1-casein C, and the αS2-caseins, are insoluble at Ca2+ concentrations above about 4 mM (Singh and Flanagan, 2005). However, β-casein is soluble at high Ca2+ concentrations (0.4 M) at temperatures below 18°C but is very insoluble above 18°C, even in the presence of low Ca2+ concentrations (4 mM). κ-Casein, with only one phosphoserine, binds little calcium and remains soluble at all Ca2+ concentrations. Although κ-casein does not bind calcium to any great extent, its ability to stabilize αS1-, αS2-, and β-caseins against precipitation by Ca2+ is well known, and κ-casein plays a large part in the stabilization of the casein micelle. This is discussed in more detail in Chapter 6. It is worth noting the use of more advanced techniques to probe the casein micelle. Recent activity in this area has seen the application of small angle neutron and X-ray scattering to further probe the casein micelle and in particular the interaction between the casein moieties and the minerals in milk (Smialowska et al., 2017). Small angle X-ray scattering analysis found that three scattering objects on a range of length scales were present and depended on the level of calcium present in the system. Overall, this showed the significant effect that the interaction of calcium had on the size of the casein aggregates formed and the overall structuring of caseinate. Sugiarto et al. (2009) tested whether sodium caseinate and/or WPI could bind and solubilize iron (ferrous sulfate) for food fortification. Caseinate had more binding sites than WPI, and iron was bound more strongly to caseinate, but caseinate was increasingly precipitated at >4 mM Fe. Caseinate-iron complexes with 2-mM Fe remained soluble as the pH was decreased from 7 to 5.5, whereas the solubility of WPI-iron complexes decreased with decreasing pH. Chelation of iron with milk proteins mitigated iron-catalyzed oxidation in emulsions, although some contribution from antioxidant amino acid side chains was also postulated (Sugiarto et al., 2010). More recent work in this area found that prior depletion of calcium from milk systems can dramatically improve the binding of iron to the casein proteins present, allowing much higher levels of iron to be stabilized for food fortification (Das et al., 2013; Mittal et al., 2015). Further studies by the same researchers found that, with a caseinate-based system, a three-way complex between casein, iron, and phosphate was formed (Fig. 14.2), with the ratio of the three being critical to the overall stability of the complex, as was confirmed by 31P-nuclear magnetic resonance (Mittal et al., 2016, 2018). By targeting hydrolyzates with iron-chelating abilities, it was found that the iron solubility could be enhanced by binding ferrous iron to whey protein hydrolyzates, which were fractionated using cascade membrane filtration (O’Loughlin et al., 2015). Intact casein will bind zinc and calcium, but tryptic hydrolyzates of αS1-, αS2-, β-, and κ-caseins also display mineral-binding properties. Termed caseinophosphopeptides (CPPs), these peptides can bind and solubilize high concentrations of calcium because of their highly polar acidic domain. Calcium-binding CPPs can have an anticariogenic effect,

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FIG. 14.2 Schematic diagram of the three-way iron-phosphate-casein complex, which imparts increased stability and solubility to the iron within the system.

in that they inhibit caries lesions through recalcification of the dental enamel (FitzGerald, 1998). This effect has been exploited in CPP-fortified chewing gums (Recaldent and Trident brands) using ingredients developed by CSIRO Australia. CPPs have also been reported to improve the intestinal absorption of zinc, as studied using an isolated perfused rat intestinal loop system (Peres et al., 1998). The amount of iron bound to CPPs produced with the enzyme Alcalase depends on the degree of hydrolysis and the temperature and pH during the binding reaction (Wang et al., 2011). Binding of iron to CPPs reduces iron-induced peroxidation in CaCo-2 cells, suggesting that CPPs could help to mitigate against unintended side effects of iron fortification (Kibangou et al., 2008). Other studies have also examined the calcium-binding affinity of CCPs, with the actual peptides responsible for the binding of calcium and magnesium purified from the CPP fractions. From this work, five phosphopeptides from CPPs were identified as having excellent mineral-binding properties (Cao et al., 2019). Enzymatic hydrolysis of β-LG dramatically increases its iron-binding capacity, which may be due to improved contact between iron and aromatic amino acids (Zhou et al., 2012). Lactoferrin has the ability to bind iron very strongly. In vivo, the ferric (III) form of iron is bound to lactoferrin (Anderson et al., 1989). Considerable interest has been expressed in supplementing bovine milk-based infant formulas with lactoferrin, as bovine milk contains much lower levels of lactoferrin than human milk and lactoferrin, isolated from human milk, can bind 2 mol of iron per mole of protein (Bezwoda and Mansoor, 1986). Nagasako et al. (1993) reported that lactoferrin can bind iron at sites other than its chelate-binding sites, probably on the surface of the molecule. The thermal stability of lactoferrin-iron complexes is enhanced by soluble soybean polysaccharide, which was apparently due to enhanced electrostatic repulsion (Ueno et al., 2012). Other studies involving the interactions of minerals/ions and milk proteins are listed in Table 14.1.

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TABLE 14.1 Interaction of milk proteins and minerals Milk protein

Mineral

Reference

β-LG A

Chromium

Divsalar et al. (2006b)

β-LG A and B

Lead

Divsalar and Saboury (2005)

Caseins and β-LG

Mercury

Mata et al. (1997)

Sodium caseinate

Iron

Shilpashree et al. (2015)

Casein phosphopeptides

Iron

Delshadian et al. (2018)

α-LA

Copper

Permyakov et al. (1988)

Whey protein concentrate

Iron; zinc

Shilpashree et al. (2016)

Whey protein hydrolyzates

Iron

Caetano-Silva et al. (2015)

Fatty acids Most of the fatty acids present in milk are found as triglycerides, which form the fat globule. P
erez et al. (1992) proposed that ruminant β-LG, because of its activity to bind fatty acids, might play a role in the activity of pregastric lipases. P
erez et al. (1989) demonstrated that two types of lipid, namely, free fatty acids and triglycerides, bound to β-LG. The total amount of fatty acids extracted from β-LG was 0.71 mol/mol of monomer protein. The predominant fatty acids were palmitic (31%–35%), oleic (22%–23%), and myristic (14%–17%) acids, which combined account for 66%–75% of the total fatty acids bound to β-LG. The unsaturated fatty acids extracted from β-LG were <31% of the total fatty acids and were mainly oleic and palmitoleic (4%–5%) acids. Although β-LG is often associated with fatty acids in milk at physiological pH, Frapin et al. (1993) showed that β-LG isolated at acid pH contained minimal bound fatty acids and delipidating had almost no effect on its fatty acid-binding affinities. As with retinol, there also seems to be controversy regarding the binding location of fatty acids. Narayan and Berliner (1998) suggested that fatty acids bind at the “external site” of β-LG. However, this conflicts with earlier studies by Puyol et al. (1991), which suggested competitive binding, and by Creamer (1995), which suggested an internal location as the primary binding site for fatty acids. Since then, several studies have shown ligands to bind in the internal cavity. Qin et al. (1998), using X-ray crystallography, showed 12-bromododecanoic acid binding inside the calyx, and Wu et al. (1999) revealed that palmitate binds in the central cavity (Fig. 14.3A and B) in a manner that was similar to the binding of retinol to the related lipocalin, serum RBP. Ragona et al. (2000) provided further evidence for cavity binding of β-LG and palmitic acid, as did Zsila et al. (2002) using circular dichroism spectroscopy, electronic absorption spectroscopy, and electrospray ionization mass spectrometry with cis-parinaric acid. Konuma et al. (2007) examined palmitic acid binding to a dimeric β-LG mutant A34C using heteronuclear nuclear magnetic resonance spectroscopy. Their results suggested a 1:1 binding stoichiometry. They indicated that the protein conformation should be complementary, at least in part, to the ligand’s structure if tight binding (dissociation constant of <10
7 M) is to

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Helix Cys119 Cys106

CysH121

I I A2 F G

H

G

H A2 Cys160 B

F

C E

C

2 Sheet 1 Sheet

She et She 2 et 1 Cys66

D D

A1 D

Lys60 Lys69

Palmitate

(A)

Helix CysH121

G F A1 G

A2

H E

D

Cys106 Cys119

B F

G

C E F

I

B

A2

H B

H G D

I

Cys160

C C

D

Cys66

Lys60

(B)

Palmitate

Lys69

FIG. 14.3 Diagram of the three-dimensional structure of β-LG that shows the relative positions of the five Cys residues, Lys60, Lys69, and the bound palmitate (A and B) (Wu et al., 1999). The helix and the strands that constitute sheets 1 and 2 are also labeled. The diagram was drawn from the PDB file 1GXA using RASMOL Version 2.6. Reproduced with the permission of Considine, T., Patel, H.A., Singh, H., Creamer, L.K., 2005a. Influence of binding of sodium dodecyl sulfate, all-trans-retinol, palmitate, and 8-anilino-1-naphthalenesulfonate on the heat-induced unfolding and aggregation of β-lactoglobulin B. J. Agric. Food Chem. 53, 3197–3205. Copyright 2005 Journal of Agricultural and Food Chemistry, American Chemical Society.

occur. They further highlighted the role of the flexible loops above the barrel in ligand binding. Konuma et al. (2007) hypothesized that the barrel’s entrance accommodates a variety of ligands, because of its plasticity, whereas the bottom of the cavity shows rigid and somewhat selective binding.

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Thus, it has been established that β-LG strongly binds 1 mol of long-chain fatty acids (myristic, palmitic, stearic acid, etc.) per mole of monomeric protein (Frapin et al., 1993; Dufour et al., 1994). The relative binding strengths for the saturated fatty acids from C8 to C20 have been measured with a variety of methods, including isothermal titration calorimetry, fluorescence spectroscopy, mass spectrometry, and equilibrium partition analysis (Loch et al., 2012), and, more recently, hybrid steered molecular dynamics have been employed (Yi and Wambo, 2015). There is remarkable agreement on both the order and the relative magnitude of the association constants, and the data clearly show increasing affinity with increasing chain length: C8  C10 < C12 < C14 < C16. The one exception to this rule is caprylic acid, which, although it has a chain length that is two carbons shorter than that of capric acid, shows a higher binding affinity. It is thought that this is due to strong electrostatic interactions between caprylic acid and two amino acid groups in β-LG, which overcome the van der Waals’ interactions. Binding is weaker with arachidonic acid (C20) than with palmitic acid (C16), and there is disagreement for stearic acid (C18), which is thought to bind either with a similar strength to palmitic acid or more strongly (Loch et al., 2012). Frapin et al. (1993) reported binding constants for several monounsaturated fatty acids, and Bello et al. (2011) measured the binding of lauric acid (C10) and sodium dodecyl sulfate (SDS) to β-LG genetic variants A and B. Fatty acid binding to β-LG is sensitive to changes in pH. Changes in binding constants are observed over the pH range 5.5–8.5 (P
erez and Calvo, 1995). This may be due to the electrostatic interactions; for example, as the pH increases, β-LG becomes negatively charged, thus making it less electrostatically inviting for a negatively charged fatty acid. The two lysine residues at the opening of β-LG’s ligand-binding cavity, Lys60 and Lys69, are likely to play a significant role in ligand affinity. The inability of porcine β-LG to bind fatty acids may be due to the substitution of Lys69 by glutamate, as suggested by Frapin et al. (1993) and P
erez et al. (1993). Creamer (1995) also hypothesized that lysine was involved in the binding process, whereby, at neutral pH, the carboxylate group of the fatty acid salt bridged to the positively charged ε-amino group. Loch et al. (2012) reported X-ray crystallography data suggesting that the head groups of fatty acids could form hydrogen bonds with Glu62 and Lys69. Puyol et al. (1991) studied the competition between the binding of retinol and the binding of free fatty acids to β-LG. They observed that, when the ratio between the concentrations of the total fatty acids (as palmitic acid) and retinol is similar to that found in milk, the fatty acids compete with retinol for binding to β-LG. Using intrinsic fluorescence studies, Frapin et al. (1993) and Dufour et al. (1994) suggested that an external, independent fatty acid binding site on the β-LG-retinol complex was in the groove between the α-helix and the β-sheets of the protein. Narayan and Berliner (1997) supported simultaneous binding of retinoids and fatty acids to β-LG. However, binding is more difficult to determine when several ligands are present. The organic anion binding sites of BSA are composed of two parts, a pocket that is lined with nonpolar amino acid chains and a cationic group that is located at or near the surface of the pocket (Swaney and Klotz, 1970). Most of the information available on the mechanism of binding to BSA has been obtained using organic dyes, anionic detergents, and fluorinated or spin-labeled derivatives. Free fatty acid binding involves hydrophobic interactions with the hydrocarbon chain and electrostatic interactions with the carboxylate anion of BSA

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14. Interaction between milk proteins and micronutrients

(Spector et al., 1969). Andersson et al. (1971) suggested that the fatty acid binding sites are located in clefts between the globular regions of the albumin polypeptide. One tryptophan is located deep inside the globular structure, whereas the other is located superficially, where it is fairly accessible to solvent (Futterman and Heller, 1972). Several of the strong fatty acid ˚ of the buried tryptophan residue (Spector, 1975). Spector binding sites are located within 10 A et al. (1969) reported that palmitate and palmitoleate were bound more tightly than oleate, linoleate, stearate, and myristate and much more tightly than laurate. Long-chain hydrocarbons that do not contain a free carboxyl group (methyl palmitate, cetyl alcohol, and hexane) were bound to a limited extent. P
erez et al. (1989) found that the amount of fatty acids bound to BSA in milk was 4.8 mol/ mol and that the predominant acids were oleic, palmitic, and stearic acids. Although the number of high affinity binding sites and the values of the apparent association constants for the binding of fatty acids to β-LG are lower than those for BSA (Anel et al., 1989), the molar concentration of β-LG in milk is much higher than that of BSA (about 30 times higher); therefore β-LG is considered to be the main fatty acid-binding protein in ruminant whey (P
erez et al., 1989, 1990), whereas α-LA binds no palmitic acid (P
erez et al., 1989). Barbana et al. (2006) reported that bovine holo-α-LA neither contains bound fatty acids in vivo nor has the ability to bind them in vitro. Cawthern et al. (1997) observed the lack of binding of stearic acid with bovine holo-Α-LA, using the fluorescent indicator acrylodated intestinal fatty acid-binding protein. However, these results were in contrast to their spin resonance and intrinsic protein fluorescence results (Cawthern et al., 1997), which showed that stearic acid was bound to holo-α-LA with a dissociation constant of 10–100  10
6 M. In contrast, interactions of apo-α-LA with fatty acids have been reported by Barbana et al. (2006). Bovine apo-α-LA displayed apparent affinity binding constants of 4.6  106 and 5.4  105 M
1 for oleic acid and palmitic acid, respectively, using partition equilibrium analysis; fluorescence spectroscopy showed a binding constant of 3.3  106 M
1 for oleic acid. The small fluorescence changes observed for palmitic acid made it difficult to obtain a binding constant. Complexes of α-LA with oleic acid have strong cytotoxic effects toward cancer cells, and the complex has been named human/bovine alpha-lactalbumin made lethal to tumor cells (HAMLET/BAMLET) (Brinkmann et al., 2011). Similar cytotoxic activity has recently been demonstrated with β-LG-oleate complexes (Lisˇkova´ et al., 2011). Further investigation into the molecular interaction between α-LA and oleic acid showed that the binding or complexation could be regarded as being weak and was very pH dependent, therefore indicating the importance of electrostatic interactions (Park et al., 2015). Protein confirmation (holo- or apostate) was also important. Overall, the study showed that the apostate and a pH in the region of pH 4.0 resulted in optimal conditions for binding.

Surfactants Some interesting amphiphilic surfactants, including cationic, anionic, and nonionic surfactants, have been widely used to study ligand binding to β-LG. These ligands provide useful information regarding the structure of the molecule and the way in which it unfolds in response to chemical and physical stimuli. Ionic surfactants are thought to bind in the

Interaction between milk proteins and micronutrients

549

hydrophobic calyx of β-LG at low concentration, whereas they unfold the protein at higher concentration (Hansted et al., 2011). SDS is an anionic surfactant that binds strongly to a small number of sites on β-LG at low concentration ( Jones and Wilkinson, 1976; Lamiot et al., 1994). Creamer (1995) demonstrated that SDS had a profound effect on the equilibrium unfolding of bovine β-LG by maintaining β-LG in the native confirmation despite high concentrations of urea. Busti et al. (2005) examined the interaction of alkyl sulfonates with β-LG and demonstrated one binding site per molecule. The efficiency of alkyl sulfonates in stabilizing native β-LG was related to the length of the hydrocarbon tail: longer alkyl sulfonates generally stabilized β-LG better against denaturation by urea (Busti et al., 2005). Alkyl sulfonates also retarded the thermal denaturation of β-LG to an extent that depended on the length of the alkyl group—longer tails were again more effective (Busti et al., 2006). This mirrors the link between alkyl group length and binding affinity that occurs with fatty acids (see earlier). Waninge et al. (1998) showed a substantial increase in the unfolding temperature of the β-LG-SDS complex at a molar ratio of 1:1. In contrast, a decrease in the unfolding temperature was observed with the addition of the cationic surfactant dodecyl trimethyl ammonium chloride (DTAC) at a similar ratio. Whereas DTAC was easily removed via dialysis, it was impossible to remove SDS by dialysis. Viseu et al. (2007) showed that DTAC not only disrupted the tertiary structure of β-LG but also drove an increase in the amount of α-helical secondary structure, in contrast to the total disruption of the structure that is caused by guanidine-HCl. Lu et al. (2006) showed that the anionic surfactant sodium perfluorooctanoate was a strong denaturant of β-LG. However, the denaturing ability of sodium perfluorooctanoate could be tempered with cationic surfactants, such as alkyl trimethyl ammonium bromide. Maulik et al. (1996) observed the binding of cetyl trimethyl ammonium bromide with β-LG and reported a two-stage interaction by first-order kinetics. At concentrations below 2 mM, tetradecyl trimethyl ammonium bromide was also shown to interact with α-LA and cause protein unfolding (Housaindokht et al., 2001). The interactions of nonionic sucrose esters with the casein micelle (Fontecha and Swaisgood, 1995) and β-casein (Clark et al., 1992) have also been studied. Creamer (1980) examined the effect of SDS on the self-association of β-casein. The results indicated that SDS binds on an external site of β-casein, such that the hydrophobic tail of SDS becomes involved in the casein self-association. This is supported by the lack of displacement of 8-anilino-1-naphthalene sulfonate (ANS) by SDS. It was also postulated that SDS binds to sites in or on the protein such that the amino acid residues involved in the self-association reaction can interact more favorably with one another. At low concentrations, SDS is thought to bind to a limited number of sites. Despite the increase in the negative charge of the protein when low concentrations of SDS are added, the normal monomer-polymer equilibrium moves predominantly to the polymer in solution; however, only protein monomers are present at high concentrations of SDS. The most recent advances in this area have used molecular docking studies to gain further insight into the interactions between proteins (mainly β-LG) and a range of surfactants such as cationic cetylpyridinium (Chavoshpour-Natanzi et al., 2018). The results indicated that cationic cetylpyridinium binds on the surface of β-LG, with hydrophobic and hydrogen bonds being the main interactions.

550

14. Interaction between milk proteins and micronutrients

Sugars and polyols Sugars and polyols belong to a family of small hydrophilic molecules that stabilize proteins and can be referred to as “osmolytes,” “cosolvents,” “compatible solutes,” or “cosolutes” (cosolutes is used here). The effect of sugars and polyols on the unfolding and denaturation of proteins is generally attributed to preferential exclusion of these solutes from the protein surface or equivalently “preferential hydration” of the protein. Two main elements contribute to this effect (Timasheff, 1998; McClements, 2002), as illustrated in Fig. 14.4. The first element is the nonspecific steric exclusion that arises because sugars and polyols are larger than water molecules. This effect is common across a wide range of cosolutes and depends more on the size of the cosolute molecules than on their chemical nature (Ebel et al., 2000; R€ osgen, 2007). Secondly, as hydroxyl-rich polyols and sugars have a strong affinity for hydrogen-bonded water molecule networks and a strong phobia toward the nonpolar groups of proteins, they are drawn to the aqueous environment in preference to the surface of the protein. This leads to the strengthening of hydrophobic interactions in cosolute solutions (Timasheff, 1998; Kamiyama et al., 1999). A study of the effects of trehalose, maltose, and sucrose on the structure of water found that trehalose binds to a larger number of water molecules than do maltose or sucrose, thus affecting the structure of water to a greater extent (Lerbret et al., 2005). The effect of cosolutes on the surface tension at the protein-solvent interface may also contribute to preferential exclusion, but these effects are very complex and somewhat controversial (Lin and Timasheff, 1996; Kaushik and Bhat, 1998; Chanasattru et al., 2007c). As water is able to penetrate into the layer immediately adjacent to the protein but cosolutes are sterically excluded, a concentration gradient of sugar molecules between the inner layer and the outer solution arises. This is a thermodynamically unfavorable situation because of the free energy that is required to maintain this concentration gradient. Subsequent

Cosolvent Water

Rg Cosolvent Protein Protein

(A)

(B)

FIG. 14.4 Protein-cosolvent-solvent interactions as a result of (A) steric interaction or (B) differential interaction effects. In (B), the exclusion of the cosolvent from the region surrounding the protein is clearly shown. Taken from McClements, D.J., 2002. Modulation of globular protein functionality by weakly interacting cosolvents. Crit. Rev. Food Sci. Nutr. 42, 417–471.

Interaction between milk proteins and micronutrients

551

movement of water molecules from the area surrounding the protein to outer parts leads to a dehydration of the protein molecule. This dehydration can result in tighter folding of the protein molecules. Unfolding of a compact protein structure increases the area of contact between the protein and the solvent and reveals nonpolar groups that were buried in the interior in the native protein. For these reasons, preferential exclusion is greater in the denatured state than in the native state, and it follows that denaturation is accompanied by a net increase in the degree of preferential exclusion (i.e., a net increase in cosolute activity in the aqueous phase). As this adds to the energetic cost of unfolding, denaturation is inhibited (Timasheff, 1998). Semenova et al. (2002) proposed an alternative explanation for the effect of sugars on the unfolding and denaturation of proteins, that is, direct hydrogen bonding between sugars and proteins, which results in additional hydration. However, the exclusion of sugars from the protein domain (Hammou et al., 1998; Timasheff, 2002) is not fully explained by this hypothesis. Higher concentrations of polyols and sugars increase the viscosity substantially, and this may inhibit diffusion-limited reactions such as protein aggregation (Kulmyrzaev et al., 2000). However, the more compact protein conformation that is induced by preferential exclusion may diffuse faster, overcoming viscosity effects (Rodrı´guez Nin˜o and Rodrı´guez Patino, 2002). The stabilizing effect of polyols increases with more hydroxyl groups on the polyols (Tiwari and Bhat, 2006; Romero et al., 2007; Politi and Harries, 2010) and with lower pH (Singh et al., 2011) and lower temperature (Xie and Timasheff, 1997b). Timasheff’s group has been dominant in research into the interactions of proteins and sugars or cosolvents as they describe them (Timasheff, 1993). Xie and Timasheff (1997a) reported the exclusion of trehalose from the domain of ribonuclease A at low temperatures. However, there was preferential binding of trehalose in both the native state and the unfolded state at 52°C (pH 5.5 and pH 2.8, respectively). Binding was stronger in the native state than in the unfolded state, indicating that denaturation in the presence of trehalose was still accompanied by a net increase in preferential exclusion (or, in other words, a net decrease in binding), and therefore denaturation was inhibited. The same group conducted a lot of earlier research that showed the exclusion of water from the domains of a range of globular proteins, in the presence of sucrose (Lee and Timasheff, 1981), lactose, and glucose (Arakawa and Timasheff, 1982). In all cases, they argued that the exclusion of sugars from the protein domain made unfolding of the protein less thermodynamically favorable. Timasheff’s group has measured the preferential exclusion of various cosolutes from BSA and β-LG in a number of studies (Timasheff, 1998). In a rare study involving sugars and casein, Mora-Gutierrez and Farrell (2000) also proposed preferential exclusion of sugar molecules from the casein domain, resulting in preferential hydration of the caseins. The ability of sugars to alter the heat- and pressure-induced denaturation of milk proteins is discussed later in this chapter.

Flavors The interaction of milk proteins and volatile flavor compounds has been reviewed in detail by K€ uhn et al. (2006); the reader should refer to this review for a more in-depth discussion of

552

14. Interaction between milk proteins and micronutrients

protein-flavor interactions. However, this section covers the area briefly. A number of flavor compounds are known to bind to milk proteins. Despite this knowledge, there are large discrepancies in the binding data because of the use of different methodologies, which appears to be a common feature of determining binding constants. β-LG is known to interact with a variety of flavor compounds including ionones ( Jouenne and Crouzet, 2000; Jung and Ebeler, 2003), lactones (Sostmann and Guichard, 1998; Guth and Fritzler, 2004), alkanes (Mohammadzadeh et al., 1969), aldehydes (van Ruth et al., 2002), esters, and ketones (Guichard and Langourieux, 2000; Jouenne and Crouzet, 2000). In contrast, very few studies have explored flavor binding to α-LA, with an exception being the binding of aldehydes and methyl ketones (Franzen and Kinsella, 1974), and 2-nonanone and 2-nonanal ( Jasinski and Kilara, 1985). BSA has been shown to bind alkanes (Mohammadzadeh et al., 1969), Damodaran and Kinsella (1980, 1981) studied the interactions between 2-nonanone and BSA, and Jasinski and Kilara (1985) compared the binding to BSA of 2-nonanone and nonanal. The binding of flavors to caseins or sodium caseinate has also received some attention, including the binding of diacetyl (Reineccius and Coulter, 1969), vanillin (McNeill and Schmidt, 1993), β-ionone, n-hexanol, ethylhexanoate, and isoamyl acetate (Voilley et al., 1991) to sodium caseinate. As the most extensively studied flavor compound is 2-nonanone, the binding strengths of this flavor to the whey proteins can be compared. Although different authors have reported different affinity constants, the trend BSA > β-LG > α-LA is consistent. Table 14.2 illustrates the interactions between 2-nonanone and various milk proteins (K€ uhn et al., 2006).

Other micronutrients Some of the milk proteins, most particularly the whey proteins, have been used as model proteins in studies involving a range of other micronutrients. The interaction of small heatshock proteins, such as alpha-crystallin, prevents the precipitation of α-LA when in the molten globule state (Lindner et al., 1997). This finding was confirmed by Sreelakshmi and Sharma (2001), who found that the active site of alpha-crystallin by itself can maintain a significantly denatured and unfolded protein in soluble form. Zhang et al. (2005) reported on the chaperone-like activity of β- and α-caseins. β-Casein was able to suppress the thermal and chemical aggregation of insulin, lysozyme, and catalase. A similar chaperone-like effect is seen with β-LG, α-LA, and BSA (Kehoe and Foegeding, 2011). The use of milk proteins as chaperones for drugs has also been studied. The interaction of chlorpromazine with β-LG and αs-casein affected the proteins in different ways. Far UV circular dichroism studies revealed that chlorpromazine increased the secondary structure of β-LG, whereas the structure of casein became further disordered (Bhattacharyya and Das, 2001). Divsalar et al. (2006a) also reported on the interaction between genetic variants of β-LG and an anticancer component. A number of the most recent studies that have examined the interactions between milk proteins and various bioactive compounds are listed in Table 14.3. Most studies were carried out with a view to using milk proteins as carrier molecules or particles for protecting and/or delivering bioactive compounds, thereby increasing their bioaccessibility to the body. The potential application of milk proteins as delivery systems or carriers for a range of

TABLE 14.2 Binding data for the interactions between 2-nonanone and milk proteins (25°C): n, number of binding sites per monomer; K, intrinsic binding constant Protein a

WPC

b

n

K (M21)

Method

Reference

61

1,920,000

Equilibrium dialysis

Jasinski and Kilara (1985)

0.2

53,000,000

Fluorescence spectroscopy

Liu et al. (2005)

c

WPI

1

2059

Headspace SPME

Zhu (2003)

Sodium caseinate

0.3

1858

Headspace SPME

Zhu (2003)

β-LG

1

2439

Equilibrium dialysis

O’Neill and Kinsella (1987)

0.2 0.5

6250 (40 ppm) 1667 (45 ppm)

Static headspace analysis

Charles et al. (1996)

14

122

Equilibrium dialysis

Jasinski and Kilara (1985)

α-LA

33

11

Equilibrium dialysis

Jasinski and Kilara (1985)

BSA

5–6

1800

Liquid-liquid partitioning

Damodaran and Kinsella (1980)

15

14,100

Equilibrium dialysis

Jasinski and Kilara (1985)

7

833

d

PFG-NMR spectroscopy

Jung et al. (2002)

a

WPC, whey protein concentrate. WPI, whey protein isolate. c SPME, solid-phase microextraction. d PFG-NMR, pulsed-field gradient nuclear magnetic resonance. € J., Considine, T., Singh, H., 2006. Interactions of milk proteins and volatile compounds: implications in Reproduced with the permission of Kuhn, the development of protein foods. J. Food Sci. 71, R72–R82; copyright 2006 Journal of Food Science, Institute of Food Technologists. b

TABLE 14.3 Recent studies of milk protein interactions with miscellaneous biologically active compounds Protein

Active agent

Notes

Reference

Epigallocatechin

Binding study

Wu et al. (2011)

Epigallocatechin-3-gallate

Binding study

Wu et al. (2013)

Epigallocatechin-3-gallate, chlorogenic acid, ferulic acid

Binding study

Jia et al. (2017)

Tea polyphenols

Binding study

Kanakis et al. (2011)

Polyphenol extracts (tea, coffee, cocoa)

Binding study

Stojadinovic et al. (2013)

Curcumin

Binding; encapsulation

Sneharani et al. (2010)

Apigenin, naringenin kaempferol, genistein

Binding study

Li et al. (2018)

Trans-resveratrol; curcumin

Binding study

Mohammadi and Moeeni (2015)

Epigallocatechin-3-gallate

Binding study

Al-Hanish et al. (2016); Radibratovic et al. (2019)

Polyphenolic compounds β-LG

α-LA

Continued

554

14. Interaction between milk proteins and micronutrients

TABLE 14.3 Recent studies of milk protein interactions with miscellaneous biologically active compounds—cont’d Protein

Active agent

Notes

Reference

Casein

Quercetin

Chitosan-casein nanoparticles

Ha et al. (2013)

Polymethoxyflavones

Binding study

He et al. (2013)

Curcumin (turmeric)

Binding study

Benzaria et al. (2013) Rahimi Yazdi and Corredig (2012)

Tannic acid

Binding study

Zhan et al. (2018)

Naringenin

Binding study

Moeiniafshari et al. (2015)

Eriocitrin

Binding study

Cao et al. (2019)

Flavonoids

β-Casein showed strongest interactions

Bohin et al. (2012)

Pelargonidin

Strongest interaction with casein and β-LG

Arroyo-Maya et al. (2016)

Polyphenols Polyphenols

Protein sequence influence on noncovalent binding Binding studies and structural changes

Nagy et al. (2012) Yildirim-Elikoglu and Erdem (2018)

Green tea catechins

Effect of milk proteins on bioaccessibility

Xie et al. (2013)

Cyanidin-3-O-glucoside

Binding study

Cheng et al. (2017)

Trans- and cis-resveratrol Tea polyphenols

Complexation study Review article

Cheng et al. (2018) Chanphai et al. (2018)

β-Casein

Various milk proteins

Various bioactive compounds α- and β-Caseins

Folic acid

Various milk proteins

Norbixin

Reassembled casein micelles

n-3 Polyunsaturated fatty acids

β-LG

Piperine (pepper alkaloid)

Bourassa and Tajmir-Riahi (2012) Cheese coloring agent

Zhang and Zhong (2013a,b) Zimet et al. (2011)

Binding study

Zsila et al. (2005)

Pharmaceutical compounds β-LG A

Bioactive peptides

Antihypertensive peptide

Roufik et al. (2006)

β-LG

Doxorubicin

Antibiotic

Agudelo et al. (2012)

Oxaliplatin

Anticancer drug

Ghalandari et al. (2015)

Casein

Alfuzosin

Prostate cancer drug in genipincrosslinked casein nanoparticles

Elzoghby et al. (2013)

β-Casein nanoparticles

Paclitaxel, vinblastine, mitoxantrone

Antitumor drugs

Shapira et al. (2010)

Lactoferrin

Gambogic acid

Antitumor compound

Zhang et al. (2013)

Effect of processing on milk protein structure

555

compounds has been examined in several in-depth reviews (Livney, 2010; Elzoghby et al., 2011; Abd El-Salam and El-Shibiny, 2012; Chanphai et al., 2018). The number of studies into the binding of polyphenols by milk proteins is especially notable. This area of research has seen an explosion of studies over the past decade and is by far the area of most significant development. A comprehensive review on the advances in the methods used to investigate these protein-phenolic compound interactions that were also carried out recently (Czubinski and Dwiecki, 2017).

Effect of processing on milk protein structure Heat has been used extensively in food processing for centuries and is a widely applied treatment in food production, primarily for the control of microbial populations. Fields of application are pasteurization under mild temperatures and sterilization under higher temperatures. However, heating may also affect texture and taste development and may result in flavor and color changes. The latter effects are often described as disadvantages of heat treatment. Changes in the organoleptic properties are generally as a result of structural changes occurring within the constituents of the food, namely, the proteins, polysaccharides, or fats. Another technology that is similar in its control of the microbial population of food products is high-pressure treatment. In contrast to heat processing, foods are preserved with minor changes in texture, flavor, or color, and high pressure can be considered to be a cold preservation technology. High pressure is a long-used technique in Japan and has become increasingly popular worldwide. However, high-pressure treatment may cause some conformational and structural changes to the individual constituents of the food, possibly resulting in altered functional and organoleptic properties. Both heat treatment and high-pressure treatment may cause the denaturation of globular whey proteins such as β-LG; although there may be differences in the mechanisms behind the denaturation process, the general process appears to be similar. Nonthermal processing is examined in detail in Chapter 8. The denaturation of whey proteins during the heat treatment of milk, the interactions of the denatured whey proteins with other milk components, and the effect of these reactions on the physical and functional properties of milk products have been extensively reported and reviewed in great detail (O’Connell and Fox, 2003; Singh and Havea, 2003). Studies have shown that heat-induced aggregation and gelation occur along detailed pathways and are influenced by the types of proteins and forces (disulfide bonding and hydrophobic interactions) present (Schokker et al., 1999; Havea et al., 2001; Abbasi and Dickinson, 2002). The use of heat to induce self-assembly and coassembly of milk proteins into micro-/nanoparticles is discussed in Loveday et al. (2012). The effect of high pressure on whole milk and individual constituents has become a subject of much recent activity, particularly regarding the effect of pressure treatment on the physical and functional properties of milk products (Anema, 2010) and the pressureinduced changes to individual proteins (Anema, 2012). Interested readers are referred to the reviews of Huppertz et al. (2006) and Considine et al. (2007a). The mechanistic effects of high-pressure processing and several other novel processing technologies were reviewed recently in the context of the industrial potential of these technologies in yogurt

556

14. Interaction between milk proteins and micronutrients

manufacture (Loveday et al., 2013). The use of high pressure in other dairy systems, such as whey or casein gels, has also been reviewed (Devi et al., 2013).

Protein denaturation by thermal and pressure treatments and effect of micronutrients The caseins have not been suitable candidates for observing changes in protein denaturation, because of their lack of defined secondary and tertiary structure. In contrast, the whey proteins have been studied widely as model globular proteins because of their well-defined secondary and tertiary structures, as outlined earlier. The interactions between whey proteins and other species that are induced by either heat treatment or pressure treatment may be divided into two separate classes: covalent interactions and noncovalent interactions. The most important covalent interaction involving whey proteins upon storage is their reaction with reducing sugars via the Maillard reaction to form discolored protein powders, which also have reduced solubilities and diminished nutritional properties. Noncovalent interactions can also occur; these may also lead to a loss of protein solubility after association of the proteins with polysaccharides, and these noncovalent interactions are driven primarily by reversible electrostatic interactions. In this chapter, the effects of noninteracting species on the unfolding and structural transitions of whey proteins are of specific interest. The marked increase in the thermal and conformational stability of globular proteins in aqueous media in the presence of sugars is well known and has been extensively studied.

Processing treatments involving ligands Several studies have shown that ligands can retard the heat or pressure denaturation of β-LG, and the type of ligand has an impact on this process. For example, during the heat denaturation of β-LG, both SDS and palmitate stabilized the native structure of β-LG against heat-induced structural flexibility, subsequent unfolding, and denaturation up to approximately 70°C, whereas both retinol and ANS provided very little stabilization (Considine et al., 2005a). When a similar range of ligands was used during pressure denaturation, a similar effect was noted, that is, higher pressures were required to cause unfolding of β-LG when a ligand was present (Considine et al., 2005b). It was noted in these studies and in the comparison study of heat and pressure using myristate and conjugated linoleic acid as ligands that β-LG unfolds slightly differently with respect to the type of treatment (Fig. 14.5) (Considine et al., 2007b). Barbiroli et al. (2011) have shown that endogenous ligands (mostly palmitic acid and stearic acid) bound to β-LG stabilize the tertiary structure against denaturation by urea or heat. They reported evidence that the binding of palmitic acid in the calyx enhanced the thermal stability of both the calyx region and the helix held against the outside of the β-barrel (the helix conceals the free thiol at Cys121). They believed that fatty acid binding in the calyx made the whole structure “tighter,” and inhibited the movement of the helix region and the exposure of Cys121, which is crucially involved in disulfide-bonded aggregation. In related work with synthetic ligands, Busti et al. (2006) reported that alkyl sulfonates with a chain length of >10 increased the denaturation temperature of β-LG at pH 6.8 by up to 13°C. In a recent study,

557

Effect of processing on milk protein structure

0.1 MPa

150 MPa

Native Stage I P SDS

Stage I P

ANS

Stage I P

Retinol

Stage I P

800 MPa

450 MPa

Stage II P

Stage III P Stage II P

Stage II P Stage II P

Stage III P Stage III P Stage III P

FIG. 14.5 Proposed three-stage model of the pressure denaturation of β-LG B and of β-LG B with added ANS, retinol, or SDS. Reproduced with the permission of Considine, T., Singh, H., Patel, H.A., Creamer, L.K., 2005b. Influence of binding of sodium dodecyl sulfate, all-trans-retinol, and 8-anilino-1-naphthalenesulfonate on the high-pressure-induced unfolding and aggregation of β-lactoglobulin B. J. Agric. Food Chem. 53, 8010–8018. Copyright 2005 Journal of Agricultural and Food Chemistry, American Chemical Society.

the conjugation of WPI with sugar beet pectin was found to increase the thermal stability of the secondary and tertiary structures of the protein. The conjugation was brought about through controlled dry heating (60°C, 79% relative humidity). It was surmised that the interactions (studied using circular dichroism and UV-visible spectroscopy alongside steady-state fluorescence spectroscopy) were covalent and noncovalent in nature (Qi et al., 2017). Hansted et al. (2011) conducted a detailed investigation of how surfactants affect thermally induced unfolding and aggregation of β-LG, using homologous series of cationic (alky trimethyl ammonium chlorides, xTAC), anionic (sodium alkyl sulfates, SxS), and nonionic (alkyl maltopyranosides, xM) surfactants. SxS inhibited thermal unfolding and aggregation at concentrations well below the critical micelle concentration, indicating that surfactant monomers were responsible for the effect. xM also inhibited aggregation, although only at above the critical micelle concentration, and smaller xM promoted unfolding at such concentrations. xTAC strongly promoted aggregation at subcritical micelle concentrations. The findings highlight the effect of the surfactant charge on aggregation at pH 6.5: anionic SxS and nonionic xM reduced aggregation, whereas cationic xTAC promoted aggregation. Hansted et al. (2011) also showed how the concentration of surfactants strongly modifies their effects, and they postulated surface interactions between β-LG and micelles of nonionic or cationic surfactants. Celej et al. (2005) compared the effects of the binding of two ANS derivatives, namely, 1,8-ANS and 2,6-ANS, on the thermostability of BSA. They reported that 1,8-ANS had a stronger effect on the thermal stability of BSA and that the binding parameters of the two ANS derivatives were quite different. This was thought to indicate that stereochemistry is an important factor in determining protein-ligand interactions. Thus, electrostatic interactions should also be considered, along with hydrophobic interactions. The authors emphasized the importance of the free ligand concentration rather than the ligand-to-protein mole ratio when determining protein stability. As discussed earlier, the binding of retinol to casein is through hydrophobic interactions (Poiffait and Adrian, 1991). β-Casein is the most hydrophobic casein and has a highly charged N-terminal domain, containing an anionic phosphoserine cluster that is clearly distinct from a very hydrophobic C-terminal domain (Swaisgood, 2003). There has been little work on the ability of the caseins to bind retinol, although Poiffait and Adrian (1991) reported that casein

558

14. Interaction between milk proteins and micronutrients

plays an important role in stabilizing retinol over time or during heat treatment. However, information in this area is limited.

Processing treatments involving sugars or polyols The effect of up to 70% (w/w) glycerol or sorbitol on the properties and functionality of β-LG was examined in several studies by Chanasattru and coworkers. Sorbitol strongly increased the thermal denaturation temperature of β-LG at pH 7, whereas glycerol had a very minor effect (Chanasattru et al., 2007b). This translated too much stronger gels with glycerol when 10% β-LG solutions were heated to 90°C for 70 min. Both polyols increased the complex modulus (G*) relative to controls, which was attributed to the strengthening of proteinprotein interactions, but the inhibitory effect of sorbitol on denaturation was thought to explain the low G* with this polyol. Later studies noted that glycerol decreases the surface tension at hexadecane-water interfaces, whereas sorbitol slightly increases it (Chanasattru et al., 2007c). The authors proposed that glycerol could interact with nonpolar regions on the surface of proteins in a way that counterbalanced steric exclusion effects, leading to small net effects on the denaturation temperature (Chanasattru et al., 2008). More recently, it was shown that, although these cosolutes tended to impart similar stabilization to the protein overall, they were actually stabilizing specific regions on β-LG. This finding could be of significant importance, given that some of these cosolutes interact with residues that may be important for the final functionality of the protein (Barbiroli et al., 2017). This group also studied the effects of polyols in β-LG-stabilized emulsions (Chanasattru et al., 2007a). Glycerol and sorbitol improved the emulsion stability against salt-induced flocculation to an approximately equal extent on a % (w/w) basis. This effect was attributed partly not only to increased viscosity (especially for sorbitol) but also to a predicted reduction in attractive van der Waals’ and hydrophobic interactions that was large enough to overcome a slight weakening of electrostatic repulsion. Similar studies on β-LG- and casein-stabilized emulsions were discussed by Dickinson (2010). The effect of small mono- and polyhydroxy alcohols on the thermal stability of β-LG at pH 5.5 was examined in more detail by Romero et al. (2007), using a homologous series of four-carbon alcohols with from one to four hydroxyl groups. All alcohols destabilized β-LG but to an extent that decreased as the number of hydroxyl groups increased. The authors proposed that 1-butanol was hydrophobic enough to interact with nonpolar regions on the surface of β-LG, whereas more hydroxylated (and therefore more hydrophilic) alcohols interacted preferentially with water instead of protein, and thereby had less destabilizing effect. This theory aligns well with the proposal from Chanasattru et al. (2008) that glycerol (1,2,3-propanetriol) interacts with nonpolar regions on the surface of the protein. Boye and Alli (2000) reported on the thermal denaturation of 1:1 mixtures of α-LA and β-LG in the presence of a range of sugars, using differential scanning calorimetry (DSC). Sugars protected against heat-induced denaturation and the protection offered (i.e., the size of the increase in the thermal transition temperature of β-LG) were in the order galactose ¼ glucose > fructose ¼ lactose > sucrose > sugar-free control. No significant effects of sugar were observed with apo-α-LA. Interestingly, an earlier study by the same authors solely on α-LA found an increase in the thermal transition temperature of both the apo form and the holo form of α-LA when either 50% sucrose or 50% glucose was added

Effect of processing on milk protein structure

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(Boye et al., 1997). This increase was fully reversible in the holo form but only partly reversible in the apo form. The thermal transition temperature of β-LG was found to be increased in the presence of sucrose, lactose, and glucose at 10%–50% (Boye et al., 1996b). Jou and Harper (1996) found an increase in the DSC thermal transition temperature of whey protein concentrates following the addition of sugars, and the protection offered by the sugars was in the order maltose > trehalose > sucrose. Lactose was also found to provide some protection against heat-induced denaturation. Dierckx and Huyghebaert (2002) followed the heat-induced gelation of a WPI solution using DSC and small amplitude oscillatory rheometry. They found that, by adding increasing concentrations of sucrose or sorbitol, both the thermal transition temperature of the protein denaturation process and the gelation temperature were increased, with a linear relationship existing between the transition and gelation temperatures. They suggested that, because of the differences in the gelation mechanisms observed at different pH values, sucrose and sorbitol affected protein-protein interactions in gels through the enhancement of hydrophobic interactions. Kulmyrzaev et al. (2000) had previously conducted a study on the effect of sucrose on the thermal denaturation, gelation, and emulsion stabilization of WPI. They also observed increases in the thermal transition temperatures on the addition of increasing concentrations of sucrose and improved gel formation and enhanced emulsification flocculation. They postulated that sucrose played different roles in a predenatured (improved heat stability) and a postdenatured (enhanced protein-protein interactions) whey protein solution system. In a study on the effects of different lactose concentrations (within a naturally occurring range) on the formation of whey protein microparticulates, Spiegel (1999) put forward a two-stage process in the aggregation of whey proteins: up to approximately 85°C, the aggregation of whey proteins is limited by the slow unfolding of the individual proteins; above 100° C, aggregation is the rate-limiting step, as the rate of unfolding is high. Lactose (at 500 mM) was also found to increase the temperature of the denaturation of WPI at pH 9.0 by approximately 3°C. However, the authors realized the effect that the Maillard reaction was having in these systems a factor that some reports seem to ignore. Baier and McClements (2001) found that increased concentrations of sucrose (up to 40%) could increase the thermal stability of BSA. These systems had a higher gelation temperature and produced gels with a lower complex shear modulus. Similar effects were found in a subsequent study (Baier and McClements, 2003). A further study by the same group (Baier et al., 2004) showed that 40% glycerol increased the temperature of gelation of BSA, but no change in the temperature of denaturation of BSA with an increasing concentration of glycerol was detected. Some early DSC work (Dumay et al., 1994) showed that the presence of 5% sucrose was sufficient to reduce the extent of β-LG unfolding by 22% following high-pressure treatment at 450 MPa for 15 min. In a later study, Dumay et al. (1998) found that adding sucrose to β-LG solutions prior to pressure-induced gelation resulted in gels with decreased pore size and strand thickness. They attributed this to a reduction in the number of protein-protein interactions occurring under the influence of pressure. Keenan et al. (2001) reported that low concentrations of sucrose aided in the pressureinduced gel formation of a range of milk protein-containing systems, but that gel formation was reduced at higher sucrose concentrations. In another group of studies, the pressureinduced gelation properties of skim milk powder were found to be improved by adding

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low concentrations of sucrose, glucose, or fructose, whereas high (45%–50%) sugar concentrations inhibited gel formation (Abbasi and Dickinson, 2001). Boye et al. (1996a) described how lactose, sucrose, and glucose increased the temperature of denaturation of BSA, with 50% glucose having a greater stabilizing effect than 50% sucrose. Wendorf et al. (2004) studied the ability of different proteins (ribonuclease A, BSA, and egg white lysozyme) to adsorb to a liquid-solid interface in the presence of a range of sugars. They found that the ability of sugars to reduce protein adsorption followed the trend trisaccharides > disaccharides > six-carbon polyols > monosaccharides, and this was explained by the stabilization of the protein in the native state in solution. Other studies have also shown the beneficial effects of sugars in protecting against denaturation induced by freeze drying, spray drying, and chemicals. At low temperatures, high concentrations of sugar cause a substantial increase in the solution viscosity and can thus affect protein denaturation. Tang and Pikal (2005) showed that, by negating the thermal stabilizing effects of sucrose by adding denaturants, the increased stability of β-LG in the freeze drying process could be directly attributed to a viscosity effect. Murray and Liang (1999) explored the addition of sucrose, trehalose, lactose, and lactitol to whey protein concentrate solutions prior to spray drying and found that the foaming properties of the spray-dried powders were dramatically decreased when sugars were absent. Trehalose was particularly successful in retaining the original foaming properties of both whey protein concentrate and β-LG but did not perform as well in spray-dried BSA powders (Murray and Liang, 1999).

Conclusions The interaction of milk proteins with various micronutrients is primarily governed by the physicochemical properties of the proteins. The whey proteins, with extensive secondary and tertiary structure and significant hydrophobicity (albeit largely shielded in the native form), tend toward hydrophobic interactions with ligands. Preferential exclusion effects govern the interaction of sugars and polyols with proteins, thus affecting their denaturing properties in the presence of pressure or heat. Electrostatic interactions drive the association of minerals and proteins. In the food industry, an increasing emphasis is being placed on foods that will have a physiologically functional benefit, in addition to the nutritional benefit of the food. This is being driven by consumers who are becoming increasingly more health aware and health responsible. The challenge now for the food scientist is to deliver the required physiologically functional activities into the final food product, while retaining product quality and shelf life. Knowledge of the interactions of these micronutrients with milk proteins, a major component in many food products, is necessary to achieve this aim. Relevant examples of this concept are detailed in patents concerning the delivery of micronutrients in complexes with β-LG (Swaisgood, 2001) or casein micelles (Livney and Dalgleish, 2007). An explosion of studies in the area of polyphenol-protein interactions in particular has been evident over the past decade. Although the concept of using milk proteins as nutrient carriers has been explored in a range of protein-nutrient combinations, there is still relatively little knowledge about how

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nutrient interactions can be used to manipulate the functionality of proteins during processing. The binding of ligands to certain whey proteins increases their resistance to thermal denaturation, and noninteracting solutes such as sugars can also stabilize proteins against heat processing. Mineral binding to caseins affects their solubility, which has obvious implications for beverage products. Greater knowledge of protein-micronutrient interactions will enable the use of milk proteins as nutrient carriers and will allow the use of micronutrients as processing aids; perhaps both objectives could even be achieved simultaneously.

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