The caseins: Structure, stability, and functionality

The caseins: Structure, stability, and functionality

3

T. Huppertz*, P.F. Fox†, A.L. Kelly† NIZO, Ede, The Netherlands, †University College, Cork, Ireland

*

3.1 Introduction There are two main classes of protein in milk, which can be separated based on their solubility at pH 4.6 at 20°C. Under these conditions, a high proportion of the proteins, called caseins, precipitate, while the proteins that remain soluble are known as serum proteins or whey proteins. Approximately 80% of the total nitrogen in bovine, ovine, caprine, and buffalo milk is casein; however, casein represents only ~40% of the nitrogen in human milk. Approximately 3% of the total nitrogen in bovine milk is soluble in 12% trichloroacetic acid (TCA) and is referred to as nonprotein nitrogen (NPN); its principal constituent is urea. The milk fat globule membrane contains several specific proteins, including many enzymes, at trace levels; these represent ~1% of the total protein in milk. Because of their ready availability and relative ease of separation and isolation, the milk proteins have been studied since the very beginning of protein chemistry. The first research paper on milk proteins (curd) appears to have been published by Berzelius in 1814. The term “casein” appears to have been used first in 1830 by Broconnet, that is, before the term “protein” was introduced in 1838 by Mulder, whose studies included work on milk proteins. The preparation of casein from milk by isoelectric precipitation was improved and standardized by Hammarsten (1883); isoelectric casein is still often referred to as casein nach Hammarsten. Isoelectric casein was initially considered to be homogeneous, but the first evidence that it is heterogeneous was published by Osborne and Wakeman (1918), followed by further evidence of heterogeneity, which suggested that isoelectric casein actually consists of three proteins, α-, β-, and γ-caseins. The α-casein fraction resolved by free boundary electrophoresis was later fractionated into calcium-sensitive (αs-) and calcium-insensitive (κ-) fractions, and the αs-casein fraction was resolved further into two distinct proteins, now known as αs1- and αs2-caseins. The caseins in raw milk assemble into rather unique structures known as casein micelles, which are essentially spherical colloidal entities which include all four casein species, and are held together and stabilized by unique physicochemical properties, such as the ability of phosphorylated serine residues to bind calcium, and the relatively amphiphilic character of the partially glycosylated κ-casein, which is present mainly as a stabilizing layer on the micelle surface. The relationship between the caseins and calcium is in fact critical from many perspectives, for example, differences in calcium sensitivity of the individual caseins, the role of calcium in the structure of the casein Proteins in Food Processing. https://doi.org/10.1016/B978-0-08-100722-8.00004-8 © 2018 Elsevier Ltd. All rights reserved.

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Proteins in Food Processing

micelle and dairy products, and the nutritional significance of the micelle being able to solubilize far higher levels of calcium in milk than would be expected. In addition, these properties of the micelles can be exploited to destabilize and recover the casein fraction of milk relatively simply, including by isoelectric precipitation as mentioned above, but also by limited enzymatic hydrolysis, for example, on renneting milk. The properties of the casein micelle are exploited in the production of a wide range of protein products based on the caseins, which have applications as food ingredients and in other areas. Caseins have been recovered by isoelectric (acid casein) or enzymatic (rennet casein) destabilization for decades, and further products with altered functionality through, for example, the reaction of acid casein with alkali to yield more soluble products such as sodium caseinate. In recent years, attention has focused on methods to recover casein micelles in a more native or functional state, for example, by membrane processing, or fractionation of caseins to yield valuable isolated or enriched casein fractions; the properties of β-casein, for example, make its isolation relatively straightforward by the manipulation of factors such as temperature and ionic strength. The historical development of the understanding of the chemistry and properties of caseins and the casein micelle was reviewed by Fox and Brodkorb (2008). The very extensive literature on various aspects of milk proteins has been reviewed at regular intervals, including textbooks by McKenzie (1970, 1971), Fox (1982, 1989), Walstra and Jenness (1984), McSweeney and Fox (2013), Thompson et al. (2014), and Fox et al. (2015). All the principal milk proteins have been isolated and characterized thoroughly at the molecular and physicochemical (functional) levels. However, the milk proteins are still an active and fertile subject for research: knowledge of the structure of the caseins is being refined, new biological functions are being identified, and the genetic control of milk protein synthesis is being elucidated, creating the possibility of altering the protein profile of milk and exploiting the mammary gland to synthesize exogenous, possibly pharmaceutically important, proteins. In this chapter, the heterogeneity, molecular and functional properties of the caseins, the structure and properties of the casein micelle, the role of caseins as food ingredients, and bioactive peptides derived from the caseins will be discussed.

3.2 Chemistry of caseins 3.2.1  αs1-Casein αs1-Casein represents ~40% of the total casein in bovine milk. The reference protein for αs1-CN is αs1-casein B-8P (ExPASy entry name and file number CAS1_Bovin and P02662, respectively), which contains 199 amino acids, including 16 Ser residues, 8 of which are phosphorylated (Ser45, Ser47, Ser64, Ser66, Ser67, Ser68, Ser75, and Ser115); in αs1-casein-9P, Ser41 is also phosphorylated (Manson et al., 1977). The key characteristics and amino acid sequence of αs1-casein B-8P are shown in Table  3.1 and Fig.  3.1, respectively. The protein has a molecular mass of ~23.0 kDa prior to phosphorylation, which increases to ~23.6 kDa as a result of the phosphorylation of 8

The caseins: Structure, stability, and functionality51

Table 3.1 

Amino acid composition and properties of αs1-CN B-8P

Amino acid

Number

Characteristic

Value

Ala Arg

9 6

199 25

Asn

8

Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp

7 0 14 25 9 5 11 17 14 5 8 17 16 5 2

Total residues:  Positively charged residues (Lys/Arg/His)  Negatively charged residues (Glu/Asp/SerP)   Aromatic residues (Tyr/Phe/Thr)

Tyr Val

10 11

40 20

Molecular mass:   Based on primary structure   Including phosphorylation

23.0 kDa 23.6 kDa

Isoelectric pH:   Based on primary structure   Including phosphorylation

4.91 4.42

Extinction coefficient at 280 nma

25,900 M−1 cm−1

Aliphatic indexa

75.43

Grand average of hydropathicity (GRAVY)a

−0.704

HΦave (kJ/residue)a

4.89

a

Values are based on the primary structures of the protein and do not take into account posttranslational modification of the structures.

Ser residues. Based on its primary sequence, an isoelectric point (pI) of ~4.9 would be expected for αs1-casein, but this decreases by ~0.5 pH units on phosphorylation of the 8 Ser residues, which is in agreement with its experimentally determined pI (4.4–4.8; Trieu-Cuot & Gripon, 1981; Eigel et al., 1984). There are two centers of phosphorylation in αs1-casein, that is, f41–51, containing Ser41 (only in the 9P variant), Ser45 and Ser47, and f61–70, containing residues Ser64, Ser66, Ser67, and Ser68; these centers of phosphorylation are crucial in the stabilization of the calcium phosphate nanoclusters in the casein micelles (de Kruif & Holt, 2003). The distribution of charged amino acids in αs1-casein B-8P is shown in Fig. 3.2. In addition to αs1-casein B, a number of other genetic variants have been identified, an overview of which is shown in Table 3.1. In αs1-CN A, amino acid residues 14–26 are missing as a result of exon skipping (Grosclaude et al., 1970); variant αs1-CN C contains Gly instead of Glu at position 192 (Grosclaude et al., 1969). In αs1-CN D, the Ala residue at position 53 is replaced by a phosphorylated Thr-residue (Grosclaude et  al., 1972), whereas the replacement of Gln by Lys at position 59 Glu by Gly at position 192 of Glu by Gly is found in αs1-CN E (Grosclaude et al., 1976). αs1-CN F contains Leu instead of SerP at position 66, whereas no amino acid sequence has been

1 10 20 Arg- Pro- Lys- His- Pro- Ile- Lys- His- Gln- Gly- Leu- Pro- Gln- Glu- Val- Leu- Asn- Glu- Ans- Leu21 30 40 Leu- Arg- Phe- Phe- Val- Ala- Pro- Phe- Pro- Glu- Val- Phe- Gly- Lys- Glu- Lys- Val- Asn- Glu- Leu41 50 60 Ser- Lys- Asp- Ile- Gly- SerP- Glu- SerP- Thr- Glu- Asp- Gln- Ala- Met- Glu- Asp- Ile- Lys- Gln- Met61 70 80 Glu- Ala- Glu- SerP- Ile- SerP- SerP- SerP- Glu- Glu- Ile- Val- Pro- Asn- SerP- Val- Glu- Gln- Lys- His81 90 100 Ile- Gln- Lys- Glu- Asp- Val- Pro- Ser- Glu- Arg- Tyr- Leu- Gly- Tyr- Leu- Glu- Gln- Leu- Leu- Arg101 110 120 Leu- Lys- Lys- Tyr- Lys- Val- Pro- Gln- Leu- Glu- Ile- Val- Pro- Asn- SerP- Ala- Glu- Glu- Arg- Leu121 130 140 His- Ser- Met- Lys- Glu- Gly- Ile- His- Ala- Gln- Gln- Lys- Glu- Pro- Met- Ile- Gly- Val- Asn- Gln141 150 160 Glu- Leu- Ala- Tyr- Phe- Tyr- Pro- Glu- Leu- Phe- Arg- Gln- Phe- Tyr- Gln- Leu- Asp- Ala- Tyr- Pro161 170 180 Ser- Gly- Ala- Trp- Tyr- Tyr- Val- Pro- Leu- Gly- Thr- Gln- Tyr- Thr- Asp- Ala- Pro- Ser- Phe- Ser181 190 200 Asp- Ile- Pro- Asn- Pro- Ile- Gly- Ser- Glu- Asn- Ser- Glu- Lys- Thr- Thr- Met- Pro- Leu- Trp

Fig. 3.1  Amino acid sequence of bovine αs1-CN B-8P. 1.75 1.25 0.75 0.25 −0.25 −0.75 −1.25 −1.75

1

21

41

61

81

101

121

141

161

181

Fig. 3.2  Distribution of charged amino acid residues (in black; Lys = +1, Arg = +1, His = +0.5, Glu = −1, Asp = −1, SerP = −1.5, N-terminus = +1, and C-terminus = −1), aromatic amino acid residues (in red; Phe, Tyr, and Thr) and hydrophobic nonaromatic amino acid residues (in green; Val, Leu, Ile, and Ala) along the amino acid chain of αs1-CN B-8P.

The caseins: Structure, stability, and functionality53

reported for αs1-CN G (Mariani et al., 1995) and αs1-CN H results from an 8-amino acid deletion at positions 51–58 (Mahe et al., 1999). Aliphatic index, grand average hydropathicity (GRAVY), and hydrophobicity all suggest that αs1-CN is a moderately hydrophobic protein. αs1-CN B-8P contains 25 amino acid residues capable of carrying a positive charge and 40 capable of carrying a negative charge; the protein has a positively charged N-terminus, followed by a high concentration of negative charges, including all but one of the SerP residues between positions 30 and 80. A moderate and even distribution of positive and negative charges occurs between residues 81 and 150, whereas the remainder of the protein, with the exception of the 10-amino acid C-terminus, is largely uncharged. Some distinct patches of significant hydrophobicity are observed between residues 20–35 and 160–175. The secondary structure of αs1-casein has been studied using a number of different approaches, including FT-IR and CD. The percentage of α-helix in αs1-casein has been estimated as 5%–15% (Herskovits, 1966), 8%–13% (Byler et al., 1988), 20% (Creamer et  al., 1981), or 13%–15% (Malin et  al., 2005). In terms of the β-sheet, values of 17%–20% have been reported (Byler et al., 1988; Creamer et al., 1981). Malin et al. (2005) reported 34%–46% extended β-sheet-like structures in αs1-CN, and Byler et al. (1988) reported 29%–35% turn structures. The presence of polyproline II structures in αs1-casein is evident from Raman optical activity spectra (Smyth et al., 2001).

3.2.2  αs2-Casein αs2-Casein (ExPASy entry name and file number CAS2_Bovin and P02663, respectively) constitutes up to 10% of total casein in bovine milk and exhibits heterogeneity in the level of phosphorylation (Swaisgood, 1992; Farrell et al., 2009) and intermolecular disulphide bonding (Rasmussen et al., 1992, 1994). αs2-Casein A-11P, the reference protein, contains 207 amino acids, including 11 SerP residues, resulting in a molar mass of ~24.3 kDa for the nonphosphorylated protein and 25.2 kDa for the 11P variant. The key characteristics and amino acid sequence of αs2-casein A-11P are shown in Table 3.2 and Fig. 3.3, respectively. Nonphosphorylated αs2-casein has a pI of ~8.3 but the phosphorylation of 11 Ser residues reduces the pI considerably, to ~4.9. Three centers of phosphorylation have been identified, that is, f8–16, containing the phosphorylated residues Ser8, Ser9, Ser10, and Ser16, f56–63, which contains the phosphorylated residues Ser56, Ser57, Ser58, and Ser61, and f126–133, which contains the phosphorylated residues Ser129 and Ser131 (de Kruif & Holt, 2003). In addition to the aforementioned 11P variant of αs2-CN A, 10P, 12P, and 13P forms of this protein were also observed by Brignon et al. (1976) while Fang et al. (2016) showed the existence of 9P, 14P, and 15P forms of αs2-casein. The distribution of charged amino acids in αs2-casein A-11P is shown in Fig. 3.4. In addition to αs2-casein A, variants B, C, and D have also been reported; the specific mutation for αs2-casein B is not known. αs2-Casein C differs from the A variant at positions 33 (Gly instead of Glu), 47 (Thr instead of Ala), and 130 (Ile instead of Thr; Mahe & Grosclaude, 1982). In αs2-casein D, residues 51–59 are absent (Grosclaude et al., 1978).

54

Table 3.2 

Proteins in Food Processing

Amino acid composition and properties of αs2-CN A-11P

Amino acid

Number

Characteristic

Value

Ala Arg

8 6

207 33

Asn

14

Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp

4 2 16 24 2 3 11 13 24 4 6 10 17 15 2

Total residues:  Positively charged residues (Lys/Arg/His)  Negatively charged residues (Glu/Asp/SerP)   Aromatic residues (Tyr/Phe/Thr)

20

Molecular mass:   Based on primary structure   Including phosphorylation

24.3 kDa 25.2 kDa

Isoelectric pH:   Based on primary structure   Including phosphorylation

8.34 4.95

Extinction coefficient at 280 nma

29,005 M−1 cm−1

Aliphatic indexa

68.7

Grand average of hydropathicity (GRAVY)a

−0.918

Tyr Val

12 14

HΦave (kJ/residue)a

4.64

39

a

Values are based on the primary structures of the protein and do not take into account posttranslational modification of the structures.

The two Cys residues in αs2-casein, that is, Cys36 and Cys40, can form intra- or intermolecular disulphide bonds with other αs2-casein molecules, with the former being more common, by a ratio of ~5:1 (Rasmussen et al., 1992, 1994). Sequence alignment showed homology for residues 42–122 and 124–207 (Farrell et al., 2009). According to Farrell et al. (2009), the αs2-CN molecule can be divided into five distinct regions, that is, two phosphopeptide regions of high charge and low hydrophobicity (residues 1–41 and 42–80), a hydrophobic region with a slight positive charge (residues 81– 125), a phosphopeptide analog (residues 126–170) with high negative charge, and a region with high hydrophobicity and strong positive charge (residues 171–207). Highly variable estimates of the level of the secondary structure of αs2-casein have been reported, that is, 54% α-helix, 15% β-sheet, 19% turns, and 13% unspecified structure (Garnier et  al., 1978), 24%–32% α-helix, 27%–37% β-sheet, 24%–31% turns, and 9%–22% unspecified structure (Hoagland et al., 2001), 45% α-helix, 6% β-sheet, and 49% unspecified structure (Tauzin et al., 2003), or 46% α-helix, 9% βsheet, 12% turns, 7% polyproline II, 19% noncontinuous α-helix or β-sheet, and 7% unspecified secondary structure (Farrell et al., 2009). The presence of polyproline II (15%) was suggested by Adzhubei and Sternberg (1993).

The caseins: Structure, stability, and functionality55 1 10 20 Lys- Asn- Thr- Met- Glu- His- Val- SerP-SerP-SerP- Glu- Glu- Ser- Ile- Ile- SerP- Gln- Glu- Thr- Tyr21 30 40 Lys- Gln- Glu- Lys- Asn- Met- Ala- Ile- Asn- Pro- Ser- Lys- Glu- Asn- Leu- Cys- Ser- Thr- Phe- Cys41 50 60 Lys- Glu- Val- Val- Arg- Asn- Ala- Asn- Glu- Glu- Glu- Tyr- Ser- Ile- Gly- SerP- SerP- SerP- Glu- Glu61 70 80 SerP- Ala- Glu- Val- Ala- Thr- Glu- Glu- Val- Lys- Ile- Thr- Val- Asp- Asp- Lys- His- Tyr- Gln- Lys81 90 100 Ala- Leu- Asn- Glu- Ile- Asn- Gln- Phe- Tyr- Gln- Lys- Phe- Pro- Gln- Tyr- Leu- Gln- Tyr- Leu- Tyr101 110 120 Gln- Gly- Pro- Ile- Val-Leu-Asn- Pro- Trp- Asn- Gln- Val- Lys- Arg- Asn- Ala- Val- Pro- Ile- Thr121 130 140 Pro- Thr- Leu- Asn- Arg- Glu- Gln- Leu- SerP- Thr- SerP- Glu- Glu- Asn- Ser- Lys- Lys- Thr- Val- Asp141 150 160 Met- Glu- Ser- Thr- Glu- Val- Phe- Thr- Lys- Lys- Thr- Lys- Leu- Thr- Glu- Glu- Glu- Lys- Asn- Arg161 170 180 Leu- Asn- Phe- Leu- Lys- Lys- Ile- Ser- Gln- Arg- Tyr- Gln- Lys- Phe- Ala- Leu- Pro- Gln- Tyr- Leu181 190 200 Lys- Thr- Val- Tyr- Gln- His- Gln- Lys- Ala- Met- Lys- Pro- Trp- Ile- Gln- Pro- Lys- Thr- Lys- Val201 210 Ile- Pro- Tyr- Val- Arg- Tyr- Leu-

Fig. 3.3  Amino acid sequence of αs2-CN A-11P.

1.75 1.25 0.75 0.25 −0.25 −0.75

201

191

181

171

161

151

141

131

121

111

91

101

81

71

61

51

41

31

21

11

−1.75

1

−1.25

Fig. 3.4  Distribution of charged amino acid residues (in black; Lys = +1, Arg = +1, His = +0.5, Glu = −1, Asp = −1, SerP = −1.5, N-terminus = +1, and C-terminus = −1), aromatic amino acid residues (in red; Phe, Tyr, and Thr) and hydrophobic nonaromatic amino acid residues (in green; Val, Leu, Ile, and Ala) along the amino acid chain of αs2-CN A-11P.

56

Proteins in Food Processing

3.2.3  β-Casein β-Casein A2-5P (209-amino acid residues, ExPASy entry name and file numbers are CASB_Bovin and P02666, respectively) is the reference protein for the β-casein family, which constitutes up to 35% of the caseins in bovine milk. The key characteristics and the amino acid sequence of β-casein A2-5P are shown in Table 3.3 and Fig. 3.5, respectively. This 209-amino acid protein has a molecular mass of 23.6 kDa for the primary structure prior to posttranslational phosphorylation, and 24.0 kDa following phosphorylation of 5 Ser residues, that is, Ser15, Ser17, Ser18, Ser19, and Ser35; of these, the first four form a center of phosphorylation (de Kruif & Holt, 2003). The pI of nonphosphorylated β-casein A2 is estimated at 5.1, decreasing to ~4.7 as a result of phosphorylation, which is somewhat lower than the experimental value of 4.8–5.0 observed by Trieu-Cuot and Gripon (1981). β-Casein is strongly amphipathic; the Nterminus residues 1–40 of β-CN contain all the net charge of the molecule, have low hydrophobicity, and contain only 2 Pro residues. The middle section of β-CN, that is, residues 41–135, contains little charge and has moderate hydrophobicity, whereas the C-terminal section, residues 136–209, contains many of the apolar residues and is characterized by little charge and high hydrophobicity. The distribution of charged amino acids in β-casein A2-5P is shown in Fig. 3.6. Table 3.3 

Amino acid composition and properties of β-CN A2-5p

Amino acid

Number

Characteristic

Value

Ala Arg

5 4

209 20

Asn

5

Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp

4 0 20 19 5 5 10 22 11 6 9 35 16 9 1

Total residues:  Positively charged residues (Lys/Arg/His)  Negatively charged residues (Glu/Asp/SerP)   Aromatic residues (Tyr/Phe/Thr)

Tyr Val

4 19

a

28 14

Molecular mass:   Based on primary sequence   Including phosphorylation

23.6 kDa 24.0 kDa

Isoelectric pH:   Based on primary sequence   Including phosphorylation

5.13 4.65

Extinction coefficient at 280 nma

11,460 M−1 cm−1

Aliphatic indexa

88.5

Grand average of hydropathicity (GRAVY)a

−0.355

HΦave (kJ/residue)a

5.58

Values are based on the primary structures of the protein and do not take into account posttranslational modification of the structures.

The caseins: Structure, stability, and functionality57

20 1 10 Arg- Glu- Leu- Glu- Glu- Leu-Asn- Val- Pro- Gly- Glu- Ile- Val- Glu- SerP- Leu- SerP-SerP-SerP- Glu21 30 40 Glu- Ser- Ile- Thr- Arg- Ile- Asn- Lys- Lys- Ile- Glu- Lys- Phe- Gln- SerP- Glu- Glu- Gln- Gln- Gln41 50 60 Thr- Glu-Asp- Glu- Leu- Gln- Asp- Lys- Ile- His- Pro- Phe- Ala- Gln- Thr- Gln- Ser- Leu- Val- Tyr61 70 80 Pro- Phe- Pro- Gly- Pro- Ile- Pro- Asn- Ser- Leu- Pro- Gln- Asn- Ile- Pro- Pro- Leu- Thr- Gln- Thr81 90 100 Pro- Val- Val- Val- Pro- Pro- Phe- Leu- Gln- Pro- Glu- Val- Met- Gly- Val- Ser- Lys- Val- Lys- Glu101 110 120 Ala- Met- Ala- Pro-Lys- His- Lys- Glu- Met- Pro- Phe- Pro- Lys- Tyr- Pro- Val- Glu- Pro- Phe- Thr121 130 140 Glu- Ser- Gln- Ser-Leu- Thr- Leu- Thr- Asp- Val- Glu- Asn- Leu- His- Leu- Pro- Leu- Pro- Leu- Leu141 150 160 Gln- Ser- Trp-Met- His- Gln- Pro- His- Gln- Pro- Leu- Pro- Pro- Thr- Val- Met- Phe- Pro- Pro- Gln161 170 180 Ser- Val- Leu- Ser-Leu- Ser- Gln- Ser- Lys- Val- Leu- Pro- Val- Pro- Gln- Lys- Ala- Val- Pro- Tyr181 190 200 Pro- Gln- Arg- Asp- Met- Pro- Ile- Gln- Ala- Phe- Leu- Leu- Tyr- Gln- Glu- Pro- Val- Leu- Gly- Pro201 Val- Arg- Gly- Pro- Phe- Pro- Ile- Ile- Val

Fig. 3.5  Amino acid sequence of β-CN A2-5P.

1.75 1.25 0.75 0.25 –0.25 –0.75

201

191

181

171

161

151

141

131

121

111

101

91

81

71

61

51

41

31

21

1

–1.75

11

–1.25

Fig. 3.6  Distribution of charged amino acid residues (in black; Lys = +1, Arg = +1, His = +0.5, Glu = −1, Asp = −1, SerP = −1.5, N-terminus = +1, and C-terminus = −1), aromatic amino acid residues (in red; Phe, Tyr, and Thr) and hydrophobic nonaromatic amino acid residues (in green; Val, Leu, Ile, and Ala) along the amino acid chain of β-CN A2-5P.

58

Proteins in Food Processing

In addition to the aforementioned A2 variant of β-CN, a number of other genetic variants have been observed, that is, β-casein A1 (His for Pro at position 67; Bonsing et al., 1988), β-casein A3 (Gln for His at position 106; Ribadeau-Dumas et al., 1970), β-casein B (His for Pro at position 67 and Arg for Ser at position 122; Grosclaude et al., 1974a), β-casein C (no phosphorylation on Ser35, Lys for Glu at position 37 and His for Pro at position 67), β-CN D (Lys for SerP at position 18), β-casein E (Lys instead of Glu at position 36; Grosclaude et  al., 1974b), β-casein F (His for Pro at position 67 and Leu for Pro at position 152; Visser et al., 1995), β-casein G (His for Pro at position 67, Leu for Pro at position 137 or 138; Dong & Ng-Kwai-Hang, 1998), β-casein H1 (Cys for Arg at position 25 and Ile for Leu at position 88; Han et al., 2000, His for Pro at position 67), β-casein H2 (Glu for Gln at position 72, Leu for Met at position 93, and Gln for Glu somewhere in the region 114–169; Senocq et al., 2002), and β-casein I (Leu for Met at position 93; Jann et al., 2002). The presence of 7%–25% α-helix structure in β-CN was reported by Herskovits (1966), Noelken and Reibstein (1968), Creamer et al. (1981), Graham et al. (1984), Caessens et al. (1999), and Farrell et al. (2001) and Qi et al. (2004, 2005); the presence of 15%–33% β-sheet and 20%–30% turn structure have been reported for this protein (Creamer et al., 1981; Graham et al., 1984; Farrell et al., 2001; Qi et al., 2004, 2005). Using optical rotary dispersion analysis, Garnier (1966) suggest that polyproline II could be an important feature in β-casein structure. Subsequent studies have confirmed the presence of 20%–25% polyproline II structure in β-CN (Farrell et al., 2001; Syme et al., 2002; Qi et al., 2004).

3.2.4  κ-Casein Compared with the other caseins, κ-CN displays some unique features; it is the smallest of the caseins, is only very lightly phosphorylated, has low sensitivity to calcium, and is the only casein that may be glycosylated. The primary sequence of the 169-amino acid κ-CN A 1P (ExPASy entry name CASK_Bovin, file accession number P02668) is shown in Fig. 3.7. Variable degrees of phosphorylation have also been found for κ-CN. κ-CN 1P is phosphorylated on the Ser149, while κ-casein 2P is phosphorylated at Ser121 also (Mercier, 1981; Minkiewicz et al., 1996; Talbo et al., 2001; Holland et al., 2006). κ-Casein 3P is also phosphorylated at Thr145 and is thus the only casein with phosphorylation at Thr rather than Ser residues (Holland et al., 2006). The large distances between the SerP and ThrP residues means that κ-casein does not have a center of phosphorylation. The key characteristics and the amino acid sequence of κ-casein A-1P are shown in Table 3.4 and Fig. 3.7, respectively, while the distribution of charged amino acids in κ-casein A-1P are shown in Fig. 3.8. Almost half of the κ-casein molecules in bulk bovine milk are not glycosylated (Vreeman et al., 1986) but the remainder can contain up to 6 glycans, at Thr-residues 121, 131, 133, 142, 145, and 165 (Pisano et al., 1994; Molle & Leonil, 1995; Minkiewicz et al., 1996). The glycans consist of galactose (Gal), N-acetylglucosamine (GalNAc), and neuraminic acid (NeuAc). The monosaccharide GalNac, the disaccharide Galβ(1–3)GalNac, the trisaccharide NeuAcα(2–3)Galβ(1–3)GalNAc, and Galβ(1–3)[NeuAcα(2–6)]GalNac, and the tetrasaccharide NeuAcα(2–3)Galβ(1–3)[NeuAcα(2–6)]GalNac have been found

The caseins: Structure, stability, and functionality59 1 10 20 Gln- Glu- Gln- Asn- Gln- Glu- Gln- Pro- Ile- Arg- Cys- Glu- Lys- Asp- Glu- Arg- Phe- Phe- Ser- Asp21 30 40 Lys- Ile- Ala- Lys- Tyr- Ile- Pro- Ile- Gln- Tyr- Val- Leu- Ser- Arg- Tyr- Pro- Ser- Tyr- Gly- Leu41 50 60 Asn- Tyr- Tyr- Gln-Gln- Lys- Pro- Val- Ala- Leu Ile- Asn-Asn- Gln- Phe- Leu- Pro- Tyr- Pro- Tyr61 70 80 Tyr- Ala- Lys- Pro- Ala- Ala- Val- Arg- Ser- Pro Ala- Gln- Ile- Leu- Gln- Trp- Gln- Val- Leu- Ser81 90 100 Asn- Thr- Val- Pro- Ala- Lys- Ser- Cys- Gln- Ala Gln- Pro- Thr- Thr- Met- Ala- Arg- His- Pro- His101 110 120 Pro- His- Leu- Ser- Phe- Met- Ala- Ile- Pro- Pro Lys- Lys- Asn- Gln- Asp- Lys- Thr- Glu- Ile- Pro121 130 140 Thr- Ile- Asn- Thr- Ile- Ala- Ser- Gly- Glu- Pro Thr- Ser- Thr- Pro- Thr- Thr- Glu- Ala- Val- Glu141 150 160 Ser- Thr- Val- Ala- Thr- Leu- Glu- Asp- SerP- Pro Glu- Val- Ile- Glu- Ser- Pro- Pro- Glu-Ile- Asn161 Thr- Val- Gln- Val- Thr- Ser- Thr- Ala- Val

Fig. 3.7  Primary amino acid sequence of κ-CN A-1P.

Table 3.4 

Amino acid composition and properties of κ-CN A-1P

Amino acid

Number

Characteristic

Value

Ala Arg

14 5

169 17

Asn

8

Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val

4 2 15 12 2 3 12 8 9 2 4 20 13 15 1 9 11

Total residues:  Positively charged residues (Lys/Arg/His)  Negatively charged residues (Glu/Asp/SerP)   Aromatic residues (Tyr/Phe/Thr)

14

Molecular mass:   Based in primary sequence   Including phosphorylation

19.0 kDa 19.1 kDa

Isoelectric pH:   Based on primary sequence   Including phosphorylation

5.93 5.60

Extinction coefficient at 280 nma

19,035 M−1 cm−1

Aliphatic indexa

73.3

Grand average of hydropathicitya

−0.557

HΦave (kJ/residue)a

5.12

a

28

Values are based on the primary structures of the protein and do not take into account posttranslational modification of the structures.

161

151

141

131

121

111

101

91

81

71

61

51

41

31

21

11

1.75

Proteins in Food Processing

1

60

1.25 0.75 0.25 −0.25 −0.75 −1.25 −1.75

Fig. 3.8  Distribution of charged amino acid residues (in black; Lys = +1, Arg = +1, His = +0.5, Glu = −1, Asp = −1, SerP = −1.5, N-terminus = +1, and C-terminus = −1), aromatic amino acid residues (in red; Phe, Tyr, and Thr) and hydrophobic nonaromatic amino acid residues (in green; Val, Leu, Ile, and Ala) along the amino acid chain of κ-CN A-1P.

attached to κ-CN. Saito and Itoh (1992) estimated the typical glycan content of κ-casein to comprise of 56.0% tetrasaccharide, 18.5% branched trisaccharide, 18.4% linear trisaccharide, 6.3% disaccharide, and 0.8% monosaccharide. The different glycoforms of κ-CN can be separated on the basis of the isoelectric point and molecular mass, resulting in forms of κ-casein with an isoelectric point as low as ~3.5 (Holland et al., 2004, 2005, 2006). The monoglycoform of κ-CN is glycosylated exclusively at Thr131, the diglycoform also at Thr142, and the triglycoform at Thr133 also (Holland et al., 2005). Additional glycosylation of the tetra-­glycoform of κ-CN occurs at Thr145 (Holland et al., 2006). The remaining two glycosylation sites of κ-CN are most likely Thr121 and Thr165 (Pisano et al., 1994; Minkiewicz et al., 1996). In general, κ-CN B appears to be more highly glycosylated than κ-CN A, and displays a more complex and variable glycosylation pattern (Coolbear et al., 1996). The presence of residues Cys11 and Cys88 creates a complex disulphide bonding pattern among κ-CN molecules, with all possible combinations (Cys11–Cys11, Cys11–Cys88, and Cys88–Cys88) being observed. In addition, some monomeric κ-casein may also exist due to intramolecular disulphide bonding, but this amounts to no more than 10% of κ-casein (Farrell et al., 1996). The remaining κ-casein occurs in multimeric structures, starting from trimers (Swaisgood et al., 1964), but possibly reaching octamers and higher (Talbot & Waugh, 1970; Pepper & Farrell, 1982; Farrell et al., 1998; Groves et al., 1998; Farrell et al., 1996). Of the 169-amino acids of κ-casein, 17 can be positively charged, 28 can be negatively charged, and there are 14 aromatic residues. Negative charges occur only in the N-terminal fragment from residues 1–20 and the C-terminal fragment of residues

The caseins: Structure, stability, and functionality61

115–169. Additional negative charges arising from the phosphorylation and glycosylation are also found in the C-terminal segment residues 115–169. Positive charges are found only in the N-terminal segment 1–116. Most hydrophobic patches are also found in this segment, between residues 21 and 110; segments 1–20 and 110–169 show predominantly hydrophilic behavior. Phosphorylation and glycosylation in the C-terminal part increase hydrophilicity further. From a technological perspective, the Phe105–Met106 bond of κ-casein is extremely important, as its hydrolysis by chymosin or other proteinases initiates the gelation of milk during cheese making. The N-terminal segment, residues 1–105, arising from the chymosin-induced hydrolysis of κ-CN is called para-κ-CN, whereas the Cterminal fragment from residues 106–169 is called the caseinomacropeptide (CMP); glycosylated CMP is usually referred to as glycomacropeptide (GMP). The molecular mass of κ-casein A without posttranslational modification is ~19.0 kDa and a pI of ~5.9 is expected. As a result of phosphorylation and glycosylation, the molecular mass increases and the pI decreases, to values as low as ~3.5 (Holland et al., 2006). For the unglycosylated monophosphorylated variants of κ-CN A and B, pI values of 5.56 and 5.81 were found by two-dimensional electrophoresis (Holland et  al., 2004). Para-κ-casein, which remains associated with the casein micelles after rennet-induced hydrolysis of κ-casein, actually carries a net positive charge at neutral pH values. In addition to κ-CN A, several other genetic variants of κ-casein have been observed. The other major variant is κ-casein B, which has substitutions at position 136 (Ile for Thr) and 148 (Ala for Asp; Mercier et al., 1973). The C variant of κ-CN has a substitution of His for Arg at position 97 (Miranda et al., 1993), whereas κ-casein E results from a Gly for Ser substitution at position 155 (Miranda et al., 1993) and κ-CN F1 contains Val instead of Asp at position 148 (Sulimova et al., 1992). κ-CN F2 is a variant of κ-CN B, containing His instead of Arg at position 10 (Prinzenberg et al., 1996), as is κ-casein G1, containing Cys instead of Arg at position 97 (Erhardt, 1996). κ-CN G2 contains Ala instead of Asp at position 148, whereas κ-casein H contains Ile for Thr at position 135 and κ-casein I contains Ala instead of Ser at position 104 (Prinzenberg et  al., 1999). Mahe et  al. (1999) described the occurrence of κ-CN J, which arises from an Arg for Ser substitution at position 155. With respect to the secondary structure of κ-CN, NMR studies suggest a high degree of flexibility in the macropeptide part of the molecule (Rollema et al., 1988), but FT-IR and CD measurements suggest 10%–20% α-helix, 20%–30% β-­structure, and 15%–25% β-turns (Byler & Susi, 1986; Griffin et al., 1986; Ono et al., 1987; Kumosinski et  al., 1991, 1993; Sawyer & Holt, 1993; Farrell et  al., 1996; Farrell et  al., 2002). The proportion of α-helical structure in κ-casein increases with increasing temperature (10–70°C), at the expense of β-structure and turns, which decrease with temperature (Farrell et al., 2002). Antiparallel and parallel β-sheets or βαβ structural motifs have been suggested in the hydrophobic domain of κ-casein (Raap et al., 1983), as has a β-turn-β-strand-β-turn motif centered on the ­chymosin-sensitive Phe105–Met106 region (Creamer et  al., 1998). Using Raman optical activity measurements, Syme et al. (2002) identified the presence of polyproline II helical confirmation in κ-CN.

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3.3 Casein interactions 3.3.1 Self-association of caseins All caseins show a tendency to self-association, although the degree and nature differ between caseins, and such associations are strongly influenced by environmental conditions such as pH, ionic strength, and temperature. Self-association of αs1-casein occurs as consecutive self-association to dimers, tetramers, hexamers, etc., which is strongly dependent on pH and ionic strength (Ho & Waugh, 1965; Payens & Schmidt, 1965, 1966; Schmidt & van Markwijk, 1968; Swaisgood & Timasheff, 1968; Schmidt, 1970a,b). Increasing ionic strength at pH 6.6 results in less αs1-casein monomers and larger oligomers (Ho & Waugh, 1965; Schmidt & van Markwijk, 1968; Schmidt, 1970b), while increasing pH to >6.6 increases electrostatic repulsion and thus reduces association (Swaisgood & Timasheff, 1968). Differences also occur as a result of genetic variation, with αs1-casein C showing considerably stronger self-association than variants B and D (Schmidt, 1970a). The notable temperature dependence of the self-association of αs1-casein means that, at 37°C, virtually only dimers are observed but, at lower or higher temperatures, higher order structures are observed also (Malin et al., 2005). Hence, it is likely that, during casein micelle synthesis in the mammary gland, αs1-casein occurs as dimers. Like αs1-casein, αs2-caseins also shows consecutive self-association which is strongly dependent on ionic strength. However, the association of αs2-casein is less extensive than that of αs1-casein. The association of αs2-casein reaches a maximum at 20°C and an ionic strength of 0.2–0.3, and decreases at higher and lower ionic strengths (Snoeren et  al., 1980). Under these conditions, αs2-casein forms spherical particles (Snoeren et al., 1980; Thorn et al., 2008). However, the incubation of αs2-­casein at, for example, 37 or 50°C, results in ribbon-like fibrils with a diameter of ~12 nm and length >1 μm, which occasionally form loop structures (Thorn et al., 2008). The presence of distinct polar and hydrophobic domains results in temperature-­ dependent micellization behavior of β-casein, with the C-terminal part forming the core of the micelles. Below <5°C, β-casein exists primarily as monomers, but some polymers are also present (Payens & van Markwijk, 1963; Farrell et al., 2001). As the temperature is increased, β-casein undergoes self-association reactions, yielding micelles with a narrow size distribution (Payens & van Markwijk, 1963; Payens & Heremans, 1969; Payens et al., 1969; Schmidt & Payens, 1972; Niki et al., 1977; Andrews et al., 1979; Arima et al., 1979; Buchheim & Schmidt, 1979; Evans & Phillips, 1979; Takase et al., 1980; Schmidt, 1982; Thurn et al., 1987; Kajiwara et al., 1988; Leclerc & Calmettes, 1997a,b, 1998; Farrell et al., 2001; De Kruif & Grinberg, 2002; O'Connell et al., 2003; Qi et al., 2004, 2005; Gagnard et al., 2007). There appears to be a critical concentration above which micelles are formed, ranging from <0.5 mg/mL to about 2 mg/mL (Schmidt & Payens, 1972; Niki et al., 1977; Evans & Phillips, 1979), depending on temperature, ionic strength, and pH. The number of monomers in the micelles varies from 15 to 60 (Schmidt & Payens, 1972; Buchheim & Schmidt, 1979; Takase et al., 1980; Thurn et al., 1987; Kajiwara et al., 1988; Farrell et al., 2001) and the radius has been variously reported to be 7.3–13.5 nm (radius of gyration; Andrews et al., 1979;

The caseins: Structure, stability, and functionality63

Thurn et al., 1987; Kajiwara et al., 1988), 15 nm (Stokes radius; Niki et al., 1977; Thurn et al., 1987), or 8–17 nm (based on electron microscopy; Arima et al., 1979; Buchheim & Schmidt, 1979). Increasing ionic strength shifts the monomer-polymer equilibrium to polymers but has little effect on the number of monomers in the micelle (Schmidt & Payens, 1972; Takase et  al., 1980), whereas increasing temperature also shifts the equilibrium to polymers and increases the number of monomers in the micelle (Takase et al., 1980). The properties for this monomer-polymer equilibrium can be treated using a shell model for the polymer micelle, with a continuous distribution of intermediates between the monomer and largest polymer micelle (Tai & Kegeles, 1984; De Kruif & Grinberg, 2002; O'Connell et al., 2003; Mikheeva et al., 2003). κ-Casein isolated from milk occurs in the form of multimeric complexes with an average molecular mass of ~1180 and ~1550 Da at 25 and at 37°C, respectively (Groves et al., 1998) and a radius of 5–10 nm (Buchheim and Schmidt, 1979; Parry & Carroll, 1969; Pepper & Farrell, 1982; Farrell et al., 1996; Thurn et al., 1987; de Kruif et al., 2002). These values are independent of protein concentration (de Kruif et al., 2002). Reduction of the disulphide bridges between κ-CN molecules leads to amphipathic monomers which can associate into micellar structures, as seen for β-casein. However, micellization of reduced-κ-CN shows no strong temperature dependence (Swaisgood et al., 1964; Vreeman et al., 1981), indicating that the association of reduced κ-casein is dominated less by hydrophobic interactions than the association of β-casein. The critical micelle concentration is ~0.53 mg/mL at an ionic strength of 0.1, but only 0.24 mg/mL at an ionic strength of 1.0 (Vreeman, 1979; Vreeman et al., 1977, 1981). Micelles of reduced κ-CN have been estimated to contain ~30 κ-CN molecules, a molecular mass of ~570–600 kDa (Vreeman, 1979; Vreeman et al., 1981, 1986), and a diameter of 21– 23 nm (Vreeman et  al., 1981; De Kruif & May, 1991). At 37°C and above, reduced κ-casein can also form fibrillary structures with a diameter of 10–12 nm and lengths up to 600 nm (Farrell et al., 2003a,b; Thorn et al., 2005; Ecroyd et al., 2008, 2010; Leonil et al., 2008). When native κ-CN is used, it is the dissociated form that is involved in fibril formation (Ecroyd et al., 2010). Fibril formation, which has been shown to result in an increased proportion of β-sheet structure (Ecroyd et al., 2008; Leonil et al., 2008) is more extensive at higher temperatures (Thorn et al., 2005) and is more extensive for nonglycosylated κ-CN than for its glycosylated counterpart (Leonil et al., 2008).

3.3.2 Interactions with other caseins In addition to self-association, all caseins also show the propensity to associate with other caseins. In fact, it is these interactions that are crucial for the formation of casein particles, as described later. Detailed studies and reviews on the topic are provided by Farrell et al. (2003a,b, 2006, 2013). As outlined previously, under physiological conditions at 37°C, αs1-casein forms only dimers and no monomers appear to be present (Malin et al., 2005). β-Casein, as outlined above, forms rather large micelles, containing, on average, ~20 molecules. However, in 1:1 mixtures of β-casein and αs1-casein, that is, at the ratio at which they occur in milk, considerably smaller particles are observed (Farrell et al., 2006), suggesting the formation of mixed complexes and indicating that

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Proteins in Food Processing

the formation of αs1-casein:β-casein complexes is actually favored over self-association of β-casein. Likewise, for 4:1 mixtures of αs1-casein and κ-casein, again close to the ratio at which they occur in milk, the large κ-casein micelles are disrupted by the presence of αs1-casein. Hence, it appears that interactions between caseins are highly co-operative and favorable. Interactions between κ-casein and αs1-casein are reported to be stronger than those between αs1-casein and β-casein. The latter are believed to be virtually entirely hydrophobic, and thus highly temperature-dependent. Based on association studies, Farrell et al. (2003a,b, 2013) indicated that mixed casein particles exist in sodium caseinate and Huppertz et al. (2017) proposed that these particles form the basis of casein micelle structure through cross-linking by calcium phosphate nanoclusters. The framework of these particles is formed by two dimers of αs1-casein, which are linked together by either an αs2-casein or κ-casein molecule. This association was found to be highly favorable from an energy minimization perspective. Two dimers of β-casein can then associate with this framework, completing what could be considered to be the basic casein particle (Kumosinski et al., 1994a,b; Farrell et  al., 2003a,b, 2013), which was recently termed a primary casein particle (PCP; Huppertz et al., 2017). Particles containing κ-casein can contain more protein molecules, due to noncovalent and covalent interactions in the N-terminal region of κ-casein, resulting in the inclusion of additional κ-casein.

3.3.3 Amyloid-like casein structures The ability of two of the caseins, that is, αs2-casein and κ-casein, to form large fibrillary structures under reducing conditions has been observed (Farrell et al., 2003a,b; Thorn et al., 2005, 2008; Ecroyd et al., 2008, 2010; Leonil et al., 2008). Based on staining by Congo Red and Thioflavin T (ThT), it has been proposed that these are ­amyloid-like structures. Given the role of amyloids in a number of degenerative diseases, for example, Alzheimer's Disease, it is natural to consider that the formation of amyloid(-like) structures by caseins is highly undesirable. The inclusion of casein-based sequences in corpora amylacea (calcium stones) isolated from the mammary gland has been reported (Niewold et  al., 1999), and Lencki (2007) suggested that these structures could also be present in casein micelles and caseinate. The presence of κ-casein and αs2-casein in milk, despite their ability to form amyloid-like structures, suggests an important role for these proteins, that is, linking of the framework of PCPs and the stabilization of casein micelles. It is critical to note that, in milk, the other caseins, αs1- and β-casein, actually have the ability to prevent amyloid formation by αs2- and κ-casein (Thorn et al., 2005, 2008; Ecroyd et al., 2008). In all cases, it seems that an approximate fourfold excess of αs1- or β-casein is sufficient to prevent amyloid formation, which is quite in line with the ratios at which these proteins occur in milk.

3.4 Casein-mineral interactions Key to the formation of casein micelles and the functional properties of casein micelles and many casein-based ingredients are casein-mineral interactions. These primarily relate to interactions of negatively charged side groups of amino acids with

The caseins: Structure, stability, and functionality65

cations, that is, the carboxylate group of Glu and Asp and the phosphate group of SerP. The most important ions in relation to caseins are calcium and magnesium. The low solubility of calcium and magnesium phosphates ensures that they readily interact with the SerP residues of caseins when present in solution. Interactions of Ca and Mg with Glu and Asp residues may also occur. Binding of Ca and Mg to SerP, Glu, and Asp residues reduces the net negative charge on the caseins, which can result in the loss of solubility as it facilitates interactions between caseins, and this is observed for αs1-, αs2-, and β-caseins, which are denoted as the calcium-sensitive caseins. κ-Casein, on the other hand, is not susceptible to Ca-induced loss of solubility and can actually stabilize the other caseins against Ca-induced loss of solubility (Farrell et al., 1988; Holt, 1992; de Kruif & Holt, 2003). Ca binding by caseins is strongly dependent on environmental conditions. With decreasing pH and increasing ionic strength, Ca binding by caseins decreases. In milk, virtually all SerP residues associate with either Ca or Mg, and 31P-NMR shows the presence of little or no free SerP residues (Thomsen et al., 1995). Dephosphorylation of casein, which can be achieved enzymatically, but also through heat treatment or exposure to alkaline conditions, reduces the Ca binding capacity of caseins markedly and proportionally to the degree of dephosphorylation (Yamuachi et al., 1967; Bingham et al., 1972; Aoki et al., 1985). In addition to binding Ca and Mg ions, caseins also possess the ability to prevent the precipitation of calcium phosphate (van Kemenade and De Bruyn, 1989). Detailed studies highlighted that the centers of phosphorylation, that is, at least 3 SerP residues in close proximity, which are present in αs1-, αs2-, and β-caseins that have the ability to adsorb onto the surface of calcium phosphate structures and prevent further growth (Holt et al., 1998; de Kruif & Holt, 2003). This mode of action is similar to how, as outlined in Section 3.5, caseins are believed to stabilize nanoclusters of amorphous calcium phosphate in casein micelles. Using phosphopeptides isolated from caseins, calcium phosphate nanoclusters with size, composition, and other properties closely resembling those found in casein micelles can be prepared. When mineral composition and SerP content resemble those of milk, calcium phosphate nanoclusters with a core mass of 61 kDa and a radius of ~2.5 nm can be prepared using the β-casein phosphopeptide f1–25 (Holt et al., 1998). In addition to providing valuable insights into casein micelle structures and nature's solution to avoiding pathological calcification of the mammary gland, calcium phosphate nanoclusters prepared using commercially available sources of casein phosphopeptides also offer opportunities for calcium fortification of products. As such, calcium phosphate particles stabilized by casein phosphopeptides are applied, for example, in dental care products (Reynolds, 2009).

3.5 Casein micelles One of the unique aspects of caseins is the form in which they exist in milk, that is, as casein micelles. Casein micelles may be described as sterically stabilized association colloids. Protein represents ~95% of the dry matter of casein micelles, with the remainder being minerals collectively referred to as micellar calcium phosphate (MCP,

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Proteins in Food Processing

also sometimes referred to as Colloidal Calcium Phosphate, CCP). MCP consists primarily of calcium and phosphate, with lesser amounts of magnesium, citrate, and other minerals. In addition to casein and MCP, casein micelles contain considerable amounts of water; typical hydration values for casein micelles are 3.0–3.5 g water/g dry matter (McMahon & Brown, 1984; Holt, 1992; Rollema, 1992; Holt & Horne, 1996; de Kruif & Holt, 2003; Dalgleish, 2011). A micrograph of a casein micelle is shown in Fig. 3.9. Electron micrographs of casein micelles suggest a near-spherical shape of the particles, with a radius in the range 50–150 nm (McMahon & McManus, 1998). Such a size range is also supported by light-scattering measurements on bulked milk (de Kruif & Huppertz, 2012; Bijl et al., 2014). Casein micelles in the milk from individual cows show a narrow size distribution, which does not change during the course of lactation or between lactations (De Kruif & Huppertz, 2012). However, considerable variation in casein micelle size is observed between milk of different cows, and mixing these leads to a considerably wider size distribution in bulked milk (de Kruif & Huppertz, 2012). Within bulked milk samples, fractionation of casein micelles into different size classes has indicated a higher proportion of κ-casein in fractions containing smaller casein micelles (Creamer et al., 1973; McGann et al., 1980; Dalgleish et al., 1989). Such findings have been important in establishing the hypothesis that κ-casein is found on the micellar surface (Holt & Horne, 1996; Dalgleish, 1998). However, when correlating casein composition of individual-cow milk samples to casein micelle size, no correlation between the proportion of κ-casein as a function of total casein and casein micelle size was observed, nor were correlations observed between micelle size and proportions of other caseins (de Kruif & Huppertz, 2012; Bijl et al., 2014). This apparent discrepancy

100 nm

Fig. 3.9  Scanning electron micrograph of a casein micelle.

The caseins: Structure, stability, and functionality67

can be explained when considering that not all casein is in the micellar phase, which is often separated from the serum phase by (ultra)centrifugation. Some of the casein is found in the nonmicellar phase, and positive correlations may thus be expected between casein micelle size and the proportion of nonmicellar κ-casein. In individual milk samples, correlations have also been found between genetic variations and posttranslational modification of casein and micelle size; smaller casein micelles were observed in milk containing κ-casein B rather than A and in milk with higher degrees of glycosylation of κ-casein (Bijl et al., 2014). The size and mass of the casein micelles mean that a micelle consists of several tens of thousands of casein molecules, as well as millions of calcium and inorganic phosphate ions, and hundreds of millions of water molecules. The distribution of mass within a casein micelle has been a topic of much study and debate for decades and will undoubtedly continue to be so in the future. Small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) measurements have been crucial in elucidating the structural properties of the MCP phase in the casein micelles. SANS and SAXS spectra show inflection points at Q ~0.35 nm−1, which persists when contrast matching of caseins by varying ratios of H2O and D2O in SANS measurements was carried out (Holt et  al., 2003; de Kruif et al., 2012; de Kruif, 2014). Therefore, the inflection point in the scattering spectra was attributed to the presence of calcium phosphate nanoclusters, which are spaced at distances of ~18.6 nm and have a radius of ~2.4 nm. A micelle with a radius of 55 nm is thus expected to contain almost 300 of such nanoclusters, whereas a micelle with a radius of 100 nm is expected to contain ~800 nanoclusters (Holt et al., 2003). The calcium phosphate nanoclusters in casein micelles are expected to be similar in size and composition to those which can be formed using the β-casein phosphopeptide f1–25 (Holt et al., 1998). For these nanoclusters, a core of amorphous calcium phosphate is stabilized by the adsorption of caseins or casein-derived phosphopeptides on their surface by centers of phosphorylation, that is, at least 3 SerP residues in close proximity. All caseins except κ-casein contain such a center of phosphorylation, with αs1- and αs2-casein containing multiple phosphorylation centers. Whereas the distribution of calcium phosphate nanoclusters throughout the casein micelles is quite homogeneous, the distribution of water in casein micelles is far less homogeneous. In particular, the surface of the casein micelles is highly hydrated, containing ~30% of the water but less than 10% of the protein; thus, the core of the micelle is less hydrated (Huppertz et al., 2017). SAXS and SANS measurements suggest that moisture distribution in the core of the micelle is not homogeneous either, which, in effect, means that the protein distribution is not homogeneous, that is, the core of casein micelles contains areas of high-protein density and low hydration and areas of low protein density and high hydration (Bouchoux et al., 2010; de Kruif et al., 2012). Such inhomogeneity in the substructure of casein micelles has been portrayed in different manners and at different length scales in different models for the substructure of the casein micelles that have been proposed. Submicellar models proposed by Schmidt (1982) and Walstra (1990) suggested the presence of spherical casein submicelles linked by calcium phosphate particles; inhomogeneity could occur within the submicelles, as well as between submicelles.

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Nanocluster models as proposed by Holt (1992), de Kruif and Holt (2003), and de Kruif et al. (2012) also assume inhomogeneity at these length scales. In these models, calcium phosphate nanoclusters are surrounded by caseins, and it is the casein-covered nanoclusters that are believed to associate to form casein micelles, with growth being terminated by the association of κ-casein on the micelle surface. de Kruif et al. (2012) suggested that areas of low protein density and high hydration were found around the nanoclusters, where most polar residues reside, whereas areas where casein interactions occur, that is, between casein-covered nanoclusters, are of low hydration and high-protein density. Inhomogeneity at different length scales was also proposed by Dalgleish (2011) and Bouchoux et al. (2010). The former proposed, based on electron micrographs and the ability of various proteins and enzymes and other constituents to dissociate into and out of casein micelles, that casein micelles contain so-called water channels. Bouchoux et al. (2010) suggested a casein micelle substructure containing hard incompressible regions of high-protein density and low hydration and soft compressible regions containing little or no protein and water, which can be removed under sufficient pressure. Bouchoux et al. (2010) reported that removal of water from the “soft” regions could be achieved by application of sufficient osmotic pressure; recent studies by Huppertz et al. (2017) also showed that ultracentrifugation at 400,000 × g for 72 h can achieve this. These authors proposed that the incompressible regions proposed by Bouchoux et al. (2010) are so-called PCPs, which are nonspherical and formed through the self-association of caseins as described by Farrell et al. (2013). The presence of incompressible regions in casein micelles is also suggested by the fact that the SAXS spectra of casein micelles in dried MPC powder resemble those of casein micelles in solution, that is, even after removal of (virtually) all water by spray-drying, the substructure of the casein micelles is retained and does not collapse (Mata et al., 2011). Huppertz et al. (2017) proposed that casein micelles consist of a network of PCPs linked by calcium phosphate nanoclusters, with 6 PCPs being associated with every nanocluster. Within this network, there are areas of entrapped water. This model of the structure of the casein micelle encompasses crucial elements of most aforementioned models, that is, (1) linking of casein particles by calcium phosphate as depicted in submicelle models, (2) the presence of MCP as nanoclusters which have centers of phosphorylation of caseins adsorbed onto the surface, as described in nanocluster models, (3) the presence of open areas (channels) through which proteins, enzymes, and other molecules can diffuse freely, and (4) the presence of incompressible and compressible regions. Deviations from submicelle models are the fact that submicelles are spherical particles, whereas PCPs are nonspherical, and the number of calcium phosphate nanoclusters interact with each casein particle (Huppetz et al., 2017). Deviations from previously described nanocluster models are also found in the considerably larger inhomogeneity in protein distribution, as well as the mode of formation of casein micelles in the mammary gland, whereby nanocluster models typically assume the formation of calcium phosphate nanoclusters to be the first step in casein micelle assembly. However, given the aforementioned self-­assembly behavior of caseins, the formation of PCPs appears to be the more likely first step in micelle formation, as also proposed in many previous models of casein micelles, reviewed by Farrell et al. (2006).

The caseins: Structure, stability, and functionality69

While, as outlined above, many different models have been proposed for the substructure of the casein micelle, with different structural features, there is general agreement between all models on the structural features of the surface of the casein micelles. The surface of the casein micelle is considered to be highly enriched in κ-­ casein, with the hydrophilic C-terminus of the protein protruding into the serum phase (Holt & Horne, 1996; Dalgleish, 1998). This brush of hydrophilic κ-casein tails, which have a length of ~10 nm, provides the casein micelle with stability against aggregation, as described further in Section 3.6.1. With respect to the localization of κ-casein on the micelle surface, it is important to consider that these are not in monomeric form but are in the form of disulphide-bonded dimers, tetramers, and potentially larger structures. However, the propensity of reduced carboxymethylated κ-casein to self-associate (Vreeman et al., 1977; Vreeman et al., 1981) and the fact that the addition of a reducing agent to milk does not increase the level of nonmicellar κ-casein (Nguyen et  al., 2012) indicates that considerable noncovalent interactions exist between κ-casein molecules. Furthermore, it is interesting to consider the estimates by Rollema et al. (1988) that only ~50% of κ-casein contributes to the κ-casein mobility observed by 1H-NMR measurements. This could be related to intermolecular interactions in the C-terminus of κ-casein. This, in turn, means that κ-casein distribution on the surface is uneven, with patches of high κ-­ casein density and patches depleted of κ-casein.

3.6 Stability of casein micelles 3.6.1 Colloidal stability From a colloidal perspective, casein micelles have been described as sterically stabilized colloids. Steric stabilization is provided primarily by the brush of κ-casein on the micelle surface, with the C-terminal region of the protein protruding into the aqueous phase and the N-terminus associating with the core of the casein micelles (Holt & Horne, 1996; Dalgleish, 1998, 2011; de Kruif, 1999). The solvency of the C-terminal part of κ-casein is governed by its high net negative charge, as a result of the presence of a high number of Glu and Asp residues, and the low level of positively charged residues. The glycosylation of Thr-residues, with sialic acid as part of the carbohydrate moieties, provides additional solvency to the κ-casein brush and hence colloidal stability to the micelles. As for other sterically stabilized colloids, colloidal stability of casein micelles depends on the density and the length of the κ-casein brush. These factors are affected by a number of processes commonly applied during dairy processing, that is, heat treatment, treatment with proteolytic enzymes, and acidification (Holt & Horne, 1996; Dalgleish, 1998, 2011; de Kruif, 1999). Acidification is the basis of conversion of milk into yogurt and other acid-­ coagulated dairy products, as well as the production of acid casein. During acidification, the negatively charged residues become progressively protonated, as a result of which solvency of the κ-casein brush is progressively lost and collapse of the brush occurs (de Kruif & Zhulina, 1996; de Kruif, 1999). Following collapse of the brush,

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casein micelles can aggregate, resulting in flocculation and, if the volume fraction of the particles is high enough, the formation of a self-supporting gel. In unheated milk, this typically occurs at pH ~4.8–5.0 but, in heated milk, which is used in the preparation of most acid-coagulated dairy products, this occurs at higher pH, that is, ~5.2–5.5. In this case, however, it is the loss of net negative charge on the denatured whey proteins, predominantly β-lactoglobulin, which induces the aggregation and subsequent flocculation and gelation of casein micelles (Holt & Horne, 1996; Dalgleish, 1998, 2011; de Kruif, 1999; de Kruif & Zhulina, 1996). Collapse of the κ-casein brush on the surface of the casein micelles can also be induced by the addition of solvents; the most studied phenomenon of this type is the ethanol stability of milk. When a sufficient amount of ethanol is added to milk, collapse of the κ-casein brush occurs as a result of a reduction in solvent quality and flocculation of casein micelles occurs. The ethanol stability of milk is dependent on many factors, most notably pH; ethanol stability increases with increasing pH and decreases strongly on acidification of milk (Horne, 2016). In addition to brush collapse, aggregation of casein micelles can be induced by (partial) removal of the κ-casein brush. The primary example is the enzymatic coagulation of milk, which is the first step in the manufacture of most cheese varieties. During this process, rennet, containing chymosin or other proteolytic enzymes with comparable specificity, hydrolyzes κ-casein. In the case of chymosin, this occurs at the Phe105–Met106 bond of κ-casein, but other proteases can cut κ-casein at different positions. Following hydrolysis, the C-terminal segment of κ-casein, the CMP, is released into the aqueous phase (Holt & Horne, 1996; de Kruif, 1999; Corredig & Salvatore, 2016). The N-terminal para-κ-casein remains associated with the micelles but provides insufficient residual stability, and aggregation of the para-casein micelles occurs, which can lead to flocculation and gelation. For unconcentrated milk, ~70% of κ-casein must be hydrolyzed before coagulation commences, but, for more concentrated milk, coagulation commences at a lower degree of κ-casein hydrolysis (Holt & Horne, 1996; de Kruif, 1999; Corredig & Salvatore, 2016). Rennet-induced coagulation of casein is affected by a number of factors, for example, pH, temperature, addition of minerals, and pretreatment (Holt & Horne, 1996; de Kruif, 1999; Corredig & Salvatore, 2016). The primary stage of rennet coagulation, that is, the enzymatic hydrolysis of κ-casein, is affected primarily through enzyme activity. Hence, reducing milk pH and increasing temperature increases the rate of enzymatic hydrolysis of κ-casein, with optima being observed around pH 5.5 and 45°C, respectively (Visser et  al., 1980; van Hooydonk et  al., 1984). Addition of CaCl2 or other minerals does not affect κ-casein hydrolysis, except through changes in milk pH (Sandra et al., 2012). Likewise, the association of denatured whey protein with casein micelles, for example, as a result of pre-heating of milk, does not affect the primary stage of rennet coagulation (Vasbinder et al., 2003; Kethireddipalli et al., 2010). The secondary stage of rennet coagulation, that is, the aggregation of para-casein micelles, also occurs more rapidly at higher temperature and lower pH. Also, the addition of CaCl2 to milk enhances the rate of aggregation of para-casein micelles, and indeed CaCl2 is typically added to cheese milk at a concentration of several mM (de Kruif, 1999; Corredig & Salvatore, 2016). The molecular basis for the effect of CaCl2

The caseins: Structure, stability, and functionality71

on aggregation of para-casein micelles has, perhaps surprisingly, thus far not been elucidated. Considering that para-κ-casein has a net positive charge and only few negatively charged residues, interaction of Ca with other caseins should perhaps be considered in this respect. The association of denatured whey proteins with casein micelles reduces the rate of aggregation of para-casein micelles considerably, which can be attributed to the fact that, while steric stabilization by the C-terminus of κ-­casein is reduced, considerable steric repulsion remains as a result of the association of denatured whey proteins with para-κ-casein (Vasbinder et al., 2003). Another form of enzymatic coagulation of casein micelles can occur during the storage of long shelf-life milk products, for example, UHT milk. Unlike the aforementioned application of enzymatic coagulation in cheese making, enzymatic coagulation of long shelf-life milk products is highly undesirable and is a common determinant of the shelf-life of the products. Enzymatic coagulation of UHT milk and related products can arise from the action of either bacterial proteases or the indigenous milk proteinase, plasmin (Harwalkar, 1992; Datta & Deeth, 2001; Nieuwenhuijse & van Boekel, 2003; Chavan et al., 2011; Deeth & Lewis, 2016). The main sources of bacterial proteases in milk are psychotropic spoilage bacteria, for example, Pseudomonas species, which can be secreted before heat treatment of milk. Whereas the bacteria are heat-labile and are inactivated by common thermal treatments applied to milk, the proteases are extremely heat-stable and are not inactivated by UHT treatment. As a result, the proteases can remain active in UHT-treated milk and hydrolyze κ-casein during storage, leading to coagulation not unlike rennet-induced coagulation of heated milk, only on a much longer time-scale. The indigenous milk proteinase, plasmin, is also not fully inactivated by UHT treatments applied to milk products and can cause the destabilization of casein micelles. In this case, however, it is not κ-casein that is hydrolyzed, as plasmin is unable to hydrolyze κ-casein; however, plasmin can hydrolyze the other caseins and therefore release κ-casein and the anchor points by which it is attached to the casein micelle. These κ-casein-depleted micelles subsequently can become prone to aggregation (Harwalkar, 1992; Datta & Deeth, 2001; Nieuwenhuijse & van Boekel, 2003; Chavan et al., 2011; Deeth & Lewis, 2016). In addition, depletion of κ-casein from casein micelles can occur as a result of heat treatment. Milk heated at a temperature >70°C typically has a considerably higher level of κ-casein in the serum phase than unheated milk, and the extent of heat-induced dissociation of κ-casein typically increases with increasing temperature and duration of heat treatment. In addition, the heat-induced dissociation of κ-casein is strongly pH-dependent. It typically does not occur at pH values lower than the natural pH of the milk, but occurs at pH values above the natural pH of milk and at a rate that increases with increasing pH (Singh & Fox, 1985, 1986, 1987a,b,c; Anema & Klostermeyer, 1997; Anema et al., 1993, 2004). At a given pH, heat-induced dissociation of κ-casein is also more extensive in concentrated milk than in unconcentrated milk (Anema & Klostermeyer, 1997; Anema et al., 1993, 2004). κ-Casein-depleted casein micelles are prone to coagulation, which may occur either rapidly during heat treatment, if the pH is sufficiently low and/or Ca2+ activity is sufficiently high (Huppertz, 2016). In addition, κ-casein-depleted casein micelles may aggregate during storage of heated milk, leading to either the formation of casein-rich κ-casein-depleted sediments or even gelation.

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3.6.2 Intramicellar stability In addition to colloidal stability, intramicellar stability, that is, the structural integrity of the casein micelle, should be considered. As outlined above, both casein-casein and casein-mineral interactions are important for maintaining the stability of the casein micelles. Casein-casein interactions are, also as outlined above, governed by, amongst others, hydrophobic interactions, hydrogen bonding, and electrostatic interactions. In addition, some covalent interactions also occur naturally, in the form of intra- and intermolecular disulphide bonds. However, as outlined above, the addition of reducing agents does not result in the dissociation of caseins from the micelle or disruption of casein micelle structure. Only the interactions of β-casein with other caseins is primarily hydrophobic (Farrell et al., 2013), as a result of which some β-casein dissociates from the casein micelles when hydrophobic interactions are reduced by cooling milk. Typically, up to 30% of β-casein dissociates from the casein micelles on cooling, whereas the remainder remains part of the casein micelles and, when milk is subsequently warmed to 30– 40°C, all dissociated β-casein reassociates with the micelles (Rose, 1968; Downey & Murphy, 1970; Creamer et al., 1977). The fraction of β-casein that readily dissociated from, and reassociates with, the casein micelles on cooling and warming, respectively, is β-casein that is associated with other caseins but not with nanoclusters of calcium phosphate. Even though a proportion of the β-casein can dissociate from the casein micelle, this does not appear to result in micellar disruption. Association of other caseins is not primarily hydrophobic and little dissociation occurs on cooling. However, dissociation can be achieved by the addition of chaotropic agents or surfactants. Chaotropic agents can disrupt the hydrogen bonding network between water molecules, which can lead to the dissociation of proteins. Guanidine hydrochloride and urea are two examples of chaotropic agents, with the latter most studied in relation to casein interactions. Addition of urea to milk at a concentration >3.5 M results in extensive reductions in the turbidity of milk and casein micelle suspension and large increases in nonsedimentable casein, both indicative of casein micelle disruption (Morr, 1967; McGann & Fox, 1974; Holt, 1998; Smiddy et al., 2006; Huppertz et al., 2007). Urea-induced disruption of casein micelles can be prevented by intermicellar covalent cross-linking using enzymes such as transglutaminase (Smiddy et al., 2006; Huppertz et al., 2007). Surfactants such as sodium dodecyl sulfate (SDS) can also result in micellar disruption (Lefebvre-Cases et  al., 1998; Smiddy et  al., 2006). Disruption of casein interactions can also be achieved by reducing intermolecular electrostatic interactions and enhancing electrostatic repulsion. Increasing the pH of milk enhances the net negative charge on caseins through deprotonation of the side groups of His, Lys, and Arg residues, as well as the phosphate moieties of SerP residues. Increasing pH to a value above ~9 reduces turbidity and increases the level of nonmicellar casein, indicative of micellar disruption (van Dijk, 1992; Vaia et al., 2006). Another way of disrupting casein micelles through modification of intermolecular electrostatic interactions is through enzymatic deamidation of casein. Using the enzyme protein glutaminase (PG), which converts accessible Gln residues into Glu

The caseins: Structure, stability, and functionality73

residues, Miwa et al. (2010) showed extensive disruption of casein micelles and increases in nonsedimentable casein, as well as nonsedimentable Ca, but not increases in 10 kDa-permeable Ca. This suggest that, while casein micelles were disrupted, calcium phosphate nanoclusters remained intact (Miwa et al., 2010). Casein micelle disruption as a result of treatment with PG is thus due to the disruption of casein-casein interactions as a result of an increase in net negative charge on the caseins. In addition to the disruption of casein micelles through protein dissociation, casein micelles can be disrupted through the solubilization of calcium phosphate nanoclusters, which can be achieved in various ways. For instance, a reduction in pH increases the solubility of calcium phosphate (Lucey & Horne, 2009) and thus result in the solubilization of MCP (Marchin et al., 2007). The addition of calcium-chelating agents, for example, citrate salts, polyphosphates, and EDTA, leads to the solubilization of MCP. Citrates and polyphosphates are used extensively in the preparation of processed cheese and cheese analogs and are often called melting salts in this specific application (Nakajima et  al., 1975). Disruption of casein micelles by addition of calcium-­ chelating agents can be reduced or prevented by enzymatic cross-linking of micellar caseins using transglutaminase or other cross-linking enzymes (Smiddy et al., 2006; Huppertz et al., 2007; Lam et al., 2017). Treatment with cation exchange resins can also be used to bind micellar calcium and disrupt casein micelles (Xu et al., 2016). Decreasing temperature also increases the solubility of calcium phosphate, but cooling milk to refrigeration temperature does not induce this to such an extent that notable disruption of casein micelles is observed, with the exception of the aforementioned cold dissociation of β-casein; however, this is related to the weakening of hydrophobic interactions and not to changes in calcium phosphate solubility. In addition to temperature, pressure also affects solubility, and an increase in pressure above the ambient increases the solubility of calcium phosphate. As a result, solubilization of MCP is observed when milk is subjected to high-pressure treatment (Hubbard et al., 2002; Huppertz & de Kruif, 2007) and, at ~400 MPa, all calcium phosphate in milk is solubilized. As a result, casein micelle structure is disrupted, as can be noted from reduced turbidity and light-scattering intensity of milk at high-­pressure; for milk, the maximum disruption of casein micelles under pressure coincides with the solubilization of calcium phosphate, which is also observed at ~400 MPa (Huppertz et al., 2006; Orlien et al., 2006). On releasing pressure, the solubility of calcium phosphate decreases again, leading to association with caseins (Huppertz et al., 2006). As a result, caseins reassociates into larger particles, accompanied by increases in turbidity. However, the original turbidity and micelle size are re-attained only if pressure is released slowly, that is, over 30 min or more (Merel-Rausch et al., 2007). If pressure is released quickly, that is, within 5 min, considerably smaller particles and lower turbidity are typically found (Huppertz et al., 2004; Anema et al., 2005). The requirement for a sufficiently long time for decompression to reform casein micelles of size close to those in original milk draws similarities to the formation of artificial casein micelles, where minerals are added to casein solutions. Artificial casein micelles with properties similar to those of casein micelles in milk can be produced only if mineral addition is sufficiently slow (Schmidt et al., 1974; Schmidt, 1979; Huppertz et al., 2017). This suggests that the (re)formation of calcium phosphate nanoclusters is

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a rather slow process that requires careful control. In some cases, however, the smaller particles found in milk subjected to more rapid decompression after high-pressure treatment can be beneficial. For instance, they have enhanced rennet coagulation and acid coagulation properties, in both cases showing more rapid coagulation and the formation of stronger gels (Lopez-Fandino et al., 1996; Needs et al., 2000; Zobrist et al., 2005). Rennet-induced coagulation of high-pressure-treated milk can even take place when considerable amounts of heat- or pressure-denatured whey protein are associated with the casein micelles, whereas this strongly inhibits rennet-induced coagulation in milk not subjected to high-pressure treatment (Huppertz et al., 2005). In concentrated milk systems, the disruption of casein micelles under pressure can be utilized to achieve the Ca-induced formation of casein networks on decompression; Huppertz et al. (2011) used this in the manufacture of ice cream, where the treatment of ice cream mix with high-pressure could be used to increase mix viscosity, increase melt stability of the ice cream, and improve texture and mouthfeel of the product.

3.7 Casein-based ingredients 3.7.1 Caseins and caseinates Caseins and caseinates are used as ingredients in a wide variety of food and nonfood products. Acid casein and rennet casein have been major commodity products for decades, while caseinates of the cations sodium, potassium, calcium, and magnesium are also widely produced, particularly sodium caseinate. Acid casein is produced from milk by acidification, which is achieved mostly by the addition of mineral acids, such as hydrochloric acid or sulphuric acid, although microbial acidification using lactic acid bacteria is also common. Acidification is typically carried out until pH ~4.2–4.6 is reached. As a result, two things happen; the casein micelles coagulate and the MCP is solubilized. After the required pH is reached, the curd is cooked to enhance whey removal; after removal of the acid whey, containing the lactose, whey proteins, and minerals, the acid casein curd is washed with water to achieve further removal of whey protein, lactose, and minerals and increase the purity of the casein. Acid casein curd is subsequently dried and milled to produce acid casein powder. Acid casein powder is insoluble in water and typically requires neutralization to make it soluble (Mulvihill & Ennis, 2003; Carr & Golding, 2016). Alternatively, washed acid casein curd may be used for the production of caseinates. To produce caseinates, the wet acid casein curd is milled and subsequently neutralized, to pH ~7.0, with the appropriate alkali, for example, sodium, potassium, calcium, or magnesium hydroxide, followed by spray-drying or roller-drying. Caseinates typically have high solubility in water (Mulvihill & Ennis, 2003; Carr & Golding, 2016). Suspensions of sodium or potassium caseinate contain particles which have a radius of ~20 nm (HadjSadok et al., 2008; Huppertz et al., 2017). Caseinate suspensions are rather viscous, which is in part due to the hydration of casein particles, but primarily to the nonspherical shape of the particles. Due to the small size of the particles, sodium and potassium caseinate solutions have low turbidity and are somewhat translucent.

The caseins: Structure, stability, and functionality75

Solutions of magnesium caseinate, and particularly calcium caseinate, are more turbid and less translucent, indicative of the presence of larger particles, as is confirmed by particle size analysis (Moughal et al., 2000). The larger particles in calcium caseinate probably arise as a result of casein aggregation at high temperature in the presence of calcium during processing, as careful neutralization of acid casein with calcium hydroxide does not yield turbid suspensions. Calcium caseinate and magnesium caseinate solutions typically have lower viscosity than sodium and potassium caseinate, which can be attributed to primarily the association of Ca or Mg ions with SerP residues, which reduces hydration of the caseinate particles. The production of rennet casein is also based on coagulation of casein and removal of whey. Subsequently, as for acid casein, the casein curd is cooked and washed to remove whey protein, lactose, and soluble salts, and the washed rennet casein curd is subsequently dried (Mulvihill & Ennis, 2003; Carr & Golding, 2016). Compared with acid casein and caseinates, rennet casein has a higher mineral content, due to the fact that rennet casein production does not involve acidification, and MCP is thus retained in the product. Due to the fact that rennet casein contains aggregated para-casein micelles, it is insoluble in water. Solubilization is typically achieved by the addition of a calcium-chelating agent, for example, citrate or phosphate salts, as in the preparation of processed cheese and cheese analogs, which is one of the main applications of rennet casein (Ennis et al., 1998; Mizuno & Lucey, 2005).

3.7.2 Milk protein and micellar casein concentrates and isolates Milk protein concentrate (MPC), milk protein isolate (MPI), and micellar casein isolate (MCI) are a class of casein-dominant ingredients in which, unlike caseins and caseinates, casein micelles are retained in a (near-)native form. To achieve this, mild separation technologies such as membrane filtration are applied. For MPC and MPI, ultrafiltration (UF) is used to concentrate caseins and whey proteins and remove lactose and soluble salts. Typical molecular weight cut-offs for UF membranes used in the preparation of MPC and MPI are 5–20 kDa. Using concentration by UF and further washing by diafiltration (DF), virtually all lactose and soluble salts can be removed, yielding products with up to 90% protein in dry matter, the remainder being MCP and ions as counter-ions for charged amino acid residues. For MCI production, microfiltration (MF) rather than UF is used, which, in addition to lactose and soluble salts, also removes whey proteins and thus concentrates only casein micelles. Typical pore sizes for MF membranes used in MCI production are 0.05–0.2 μm and, as for MPC/MPI production, most soluble material can be removed using this process when washing steps with DF are included. After UF/MF followed by DF, retentates may be evaporated to increase dry matter content, followed by spray-drying (Carr & Golding, 2016). The composition of MCI is similar to that of calcium caseinate; it exhibits good rennet coagulation properties and is particularly well suited for increasing the protein content of cheesemilk, thereby improving the quality of cheese and increasing the capacity of a cheese plant (Kelly et al., 2000). Garem et al. (2000) described the manufacture of a milk powder with improved cheesemaking properties, using a process involving removal of whey proteins by a combination of microfiltration and ultrafiltration.

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Whereas MCI typically has a high-protein level, that is, >80% protein in dry matter, MPCs are produced with a protein content ranging from ~50% to ~90% in dry matter. With increasing protein content in the product, lactose content decreases, whereas ash content remains relatively constant. The latter can be attributed to the fact that, while the soluble minerals are removed, the micellar minerals are concentrated during UF/ DF. While the casein micelles remain relatively unchanged during UF/MF and DF, the serum phase changes considerably, most notably in terms of an increase in pH and a decrease in ionic strength. At a given pH, Ca2+ activity is also considerably higher in high-protein MPCs, due to the reduced ionic strength (Crowley et al., 2014). This higher Ca2+ activity also results in reduced heat stability of MPCs (Crowley et  al., 2014, 2015). The lower heat stability is important for applications of these ingredients in sterilized products, but also for stability during drying of MPCs, which can lead to insolubility of the powdered products. Insolubility of MPC/MCI occurs primarily in high-protein products, and increases with storage time and with storage temperature; a higher heat load during drying also contributes to insolubility (Anema et al., 2006; Havea, 2006; Gazi & Huppertz, 2015). Analysis of the insoluble fraction in MPCs indicates that it is primarily the micellar casein that becomes insoluble, whereas the whey proteins and other constituents do not lose solubility (Anema et al., 2006; Gazi & Huppertz, 2015). Development of insolubility can be prevented by the addition of calcium-chelating agents, treatment with cation exchange resins, as well as by performing membrane filtration at low pH, which reduces the MCP content and increases nonmicellar casein content (Carr & Golding, 2016). A number of new methods, for example, cryoprecipitation and ethanol precipitation, for the preparation of casein products with interesting properties have been developed but have not been applied industrially. There have also been significant recent developments in the purification of individual caseins, in particular β-casein, the temperature-dependent dissociation and micellization properties of which can be exploited in a range of purification strategies (Atamer et al., 2017).

3.7.3 Casein hydrolysates Being relatively open-structured rheomorphic proteins, the caseins are excellent substrates for hydrolysis by a wide range of proteolytic enzymes, and indeed such breakdown is critical to the development of flavor and texture of cheese. In addition, the production of hydrolysates of casein for specific functional or ingredient-related applications has been studied widely, and commercial products based on such hydrolysis have been available for decades. In particular, focus in the area of hydrolysis of casein has concerned the production of biologically functional peptides, as the bovine caseins contain a range of peptide sequences which have specific biological activities when released by enzymatic hydrolysis, including the following: ●

●

●

●

phosphopeptides caseinomacropeptide (CMP) caseinomorphines immunomodulating peptides

The caseins: Structure, stability, and functionality77 ●

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blood platelet-modifying (antithrombic) peptides (e.g., casoplatelin) anginotensin converting enzyme (ACE) inhibitors, sometimes referred to as casokinins bactericidal peptides

Casein-derived bioactive peptides have been the subject of considerable research for several years and the very extensive literature has been reviewed by Fox and Flynn (1992), Gobetti et al. (2002), Fitzgerald and Meisel (2003), Urista et al. (2011), Hernandez-Ldesma et al. (2014), and Nongomiera and Fitzgerald (2016). As an example of the bioactive peptides, β-casomorphins, which are derived from the sequence residues 60–70 of β-casein, may inhibit gastrointestinal motility and the emptying rate of the stomach by direct interaction with opioid receptors. The antimicrobial properties of dairy-derived peptides was reviewed by Akalin (2014). As mentioned in Section 3.2.4, CMP (κ-CN f106–169) results from hydrolysis of the Phe105–Met106 bond of κ-casein on renneting; this peptide diffuses into the whey. Relatively high levels of CMP are present in whey (~4% of total casein, 15%–20% of protein in cheese whey, 180 × 103 tonnes per annum are available globally from whey), and can be quite easily recovered therefrom. CMP has several interesting biological properties, as it: ●

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has no aromatic amino acids and hence is suitable for individuals suffering from phenylketonuria (although it lacks several essential amino acids); inhibits viral and bacterial adhesion; promotes the growth of bifidobacteria; suppresses gastric secretions; modulates immune system responses; inhibits the binding of bacterial toxins (e.g., cholera and E. coli toxins).

Peptides derived from CMP by proteolysis may have antithrombotic properties or may act as growth promoters for L. lactis subsp. lactis. A further area of interest in the generation of hydrolysates of casein has been in the reduction of the risk of allergenicity on consumption of dairy-based products, and casein (and whey protein) hydrolysates are common constituents of hypoallergenic infant formulae (Monaci et al., 2006; Hays & Wood, 2005). Currently, few milk-derived biologically active peptides are produced commercially although many of them have been purified partially by UF and hence are amenable to large-scale production. In recent years, approaches informed by bioinformatics have been applied to intelligently design more targeted approaches to the generation of specific sequences within hydrolysates (Nongomiera & Fitzgerald, 2016). In addition, a key area of research has concerned the protection of casein-derived peptides, for example, through encapsulation, to improve their targeted delivery within the body, increase their bioavailability, and overcome frequent challenges with bitterness of casein hydrolysates (Mohan et al., 2015). In general, the biological activity of many casein-derived peptides in vivo remains to be demonstrated. Perhaps the peptides most likely to be commercially viable in the short-term are the caseinophospeptides, which contain clusters of serine phosphate residues. It is claimed that these peptides promote the absorption of metals (Ca, Fe, and Zn),

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through chelation and acting as passive transport carriers for the metals across the distal small intestine (Naqvi et al., 2016; Sun et al., 2016). Caseinophosphopeptides are currently used in some dietary and pharmaceutical supplements, for example in treatment of dental caries. A further area of interest in relation to casein hydrolysates involves their ­techno-functional properties, such as improved solubility or emulsifying properties compared with intact caseins (Van der Ven, 2001, Van Der Ven et al., 2002a,b; Banach et  al., 2013). Hydrolysis of caseins (or sodium caseinate) results in products with much greater solubility across a range of pH values, particularly around the isoelectric point (Rajarathman et al., 2016), increasing the range of potential applications in acidic beverages, while the more rapid mobility and possible arrangements of peptides versus intact protein can result in improved functional emulsifying and foaming properties (Walsh et al., 2008; Luo et al., 2014). In terms of gelation characteristics, hydrolysis reduces the ability of caseins to form rennet or acid gels, as would be expected, and their presence affects the microstructure and rheological properties of such gels (Hidalgo et al., 2015).

3.7.4 Applications of caseins in dairy and nondairy products Functional milk proteins have been of major significance to the dairy and general food industry for many years. Owing to the ease with which casein can be produced by isoelectric precipitation or rennet-induced coagulation, it has been produced commercially since the early 20th century. Initially, casein was used only for industrial applications, for example, glues, plastics, and paper glazing, and was essentially a by-product of minor economic importance. Pioneering work in New Zealand and Australia in the 1960s, however, up-graded casein for use as a food ingredient; consequently, it became a much more valuable product and is now one of the principal functional food proteins. The production of functional food-grade casein in the 1960s coincided with the development of processed food products that require functional proteins. The most important applications of casein products are in cheese analogs, especially pizza cheese, as an emulsifier in coffee whiteners, synthetic whipping creams, and cream liqueurs, and in fabricated meats, some cereal products, and various dietetic foods. In addition, there has been some recent interest in the production of high-protein liquid concentrates which can undergo temperature-dependent gelation, such as high solid suspensions of micellar casein which can be refrigerated or frozen as gels and which liquefy on warming to room temperature (Amelia and Barbano, 2013), or can be stored frozen in a stable state (Lu et al., 2015). Such preparations can also have applications in cheese making (Lu et al., 2016). A future area of significant application of casein-based ingredients may relate to their ability to act as molecular chaperones, which may have applications in the treatment and understanding of diseases and disorders which involve the formation of amyloid fibrils, such as Alzheimer's and Parkinson's disease (Thorn et al., 2009). Randaheera et al. (2016) reviewed the potential applications of casein-based ingredients for the delivery of sensitive food ingredients, including bioactive compounds, in forms including encapsulated structures, hydrogels, emulsions, and edible films and

The caseins: Structure, stability, and functionality79

coatings. There may also be interest in using individual caseins in biomedical applications; for example, Shapira et al. (2010) reported on the use of β-casein micelles as vehicles for the delivery of therapeutic drugs.

3.8 Conclusions and future perspectives The proteins of milk have been the subject of research for > 100 years, especially since 1950. It is not surprising, then, that the milk proteins are probably the best characterized food protein system; today, all the principal milk proteins and many of the minor ones have been well characterized at the molecular level, while the understanding of the structure of the casein micelle is, arguably, reaching a consensus model. Further progress on the chemistry of milk proteins will depend on developments in protein chemistry generally. Developments which are likely to have a significant impact on the technological aspects of milk proteins are: ●

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improved fractionation techniques; application of molecular biology techniques to modify proteins through point mutations; modification of proteins by chemical (which was not discussed here), physical, or enzymatic methods; application of highly sensitive proteomic tools to characterize the microheterogeneity of caseins and hydrolysates thereof; more extensive interspecies comparison of various aspects of milk proteins—the milk proteins of very few of the ~4500 mammalian species have been studied to date. It is highly probable that very interesting new milk proteins remain to be discovered.

It is thus very likely that useful studies on the chemistry and technology of caseins and products that include these proteins will continue for many years to come.

References Adzhubei, A.A., Sternberg, M.J.E., 1993. Left-handed polyproline II helices commonly occur in globular proteins. J. Mol. Biol. 229, 472–493. Akalin, A.S., 2014. Dairy-derived antmiocrobial peptides: action mechanisms, pharmaceutical uses and production proposals. Trends Food Sci. Technol. 36, 79–95. Amelia, I., Barbano, D.M., 2013. Production of an 18% protein liquid micellar casein concentrate with a long refrigerated shelf life. J. Dairy Sci. 96, 3340–3349. Andrews, A.L., Atkinson, D., Evans, M.T.A., Finer, E.G., Green, J.P., Phillips, M.C., Robertson, R.N., 1979. The conformation and aggregation of bovine β-casein A. I. Molecular aspects of thermal aggregation. Biopolymers 18, 1105–1121. Anema, S.G., Klostermeyer, H., 1997. Heat-induced, pH-dependent dissociation of casein micelles on heating reconstituted skim milk at temperatures below 100°C. J. Agric. Food Chem. 45, 1108–1115. Anema, S.G., Singh, H., Creamer, L.K., 1993. The relative importance of protein and mineral concentrations on the dissociation of κ-casein from the micelles in heated reconstituted milk. In: Protein Fat Globule Modifications by Heat Treatment, Homogenization and Other

80

Proteins in Food Processing

Technological Means for High Quality Dairy Products. International Dairy Federation, Brussels, pp. 227–235 (Special Issue 9303). Anema, S.G., Lowe, E.K., Lee, S.K., 2004. Effect of pH at heating on the acid-induced aggregation of casein micelles in reconstituted skim milk. LWT Food Sci. Technol. 37, 779–787. Anema, S.G., Lowe, E.K., Stockmann, R., 2005. Particle size changes and casein solubilisation in high-pressure-treated skim milk. Food Hydrocoll. 19, 257–267. Anema, S.G., Pinder, D.N., Hunter, R.J., Hemar, Y., 2006. Effects of storage temperature on the solubility of milk protein concentrate (MPC85). Food Hydrocoll. 20, 386–393. Aoki, T., Toyooka, K., Kako, Y., 1985. Role of phosphate groups in the calcium sensitivity of αs2-casein. J. Dairy Sci. 68, 1624–1629. Arima, S., Niki, R., Takase, K., 1979. Structure of β-casein. J. Dairy Res. 46, 281–282. Atamer, Z., Post, A.E., Schubert, T., Holder, A., Boom, R.M., Hinrichs, J., 2017. Bovine β-casein: Isolation, properties and functionality. A review. Int. Dairy J. 66, 115–125. Banach, J.C., Lin, Z., Lamsal, B.P., 2013. Enzymatic modification of milk protein concentrate and characterization of resulting functional properties. LWT Food Sci. Technol. 54, 397–403. Bijl, E., de Vries, R., van Valenberg, H., Huppertz, T., Van Hooijdonk, T., 2014. Factors influencing casein micelle size in milk of individual cows: genetic variants and glycosylation of κ-casein. Int. Dairy J. 34, 135–141. Bingham, E.W., Farrell, H.M., Carroll, R.J., 1972. Properties of dephosphorylated αs1-casein. Precipitation by calcium ions and micelle formation. Biochemistry 11, 2450–2454. Bonsing, J., Ring, J.M., Stewart, A.F., Mackinlay, A.G., 1988. Complete nucleotide sequence of the bovine beta-casein gene. Aust. J. Biol. Sci. 41, 527–537. Bouchoux, A., Gesan-Guiziou, G., Pérez, J., Cabane, B., 2010. How to squeeze a sponge: casein micelles under osmotic stress, a SAXS study. Biophys. J. 99, 3754–3762. Brignon, G., Ribadeau-Dumas, B., Mercier, J.-C., 1976. Premiers elements de structure primaire des caseines αs2 bovines. FEBS Lett. 71, 111–116. Buchheim, W., Schmidt, D.G., 1979. On the size of monomers and polymers of β-casein. J. Dairy Res. 46, 277–280. Byler, D.M., Susi, H., 1986. Examination of the secondary structure of proteins by deconvoluted FTIR spectra. Biopolymers 25, 469–487. Byler, D.M., Farrell, H.M., Susi, H., 1988. Raman spectroscopic study of casein structure. J. Dairy Sci. 71, 2622–2629. Caessens, P.W.J.R., De Jongh, H.H.J., Noide, W., Gruppen, H., 1999. The absorption-induced secondary structure of β-casein and of distinct parts of its sequence. Biochim. Biophys. Acta 1430, 73–83. Carr, A., Golding, M., 2016. Functional milk proteins production and utilization: casein-based ingredients. In: McSweeney, P.L.H., O'Mahony, J.A. (Eds.), Advanced Dairy Chemistry, fourth ed. Proteins. Springer, New York, pp. 35–66. Chavan, R.S., Chavan, S.R., Khedkar, C.D., Jana, A.H., 2011. UHT milk processing and effect of plasmin activity on shelf life: a review. Compr. Rev. Food Sci. Food Saf. 10, 251–268. Coolbear, K.P., Elgar, D.F., Ayers, J.S., 1996. Profiling of genetic variants of bovine κ-casein macropeptide by electrophoretic and chromatographic techniques. Int. Dairy J. 6, 1055–1068. Corredig, M., Salvatore, E., 2016. Enzymatic coagulation of milk. In: McSweeney, P.L.H., O'Mahony, J.A. (Eds.), Advanced Dairy Chemistry. Proteins: Applied Aspects, vol. 1B. Springer, New York, pp. 287–307. Creamer, L.K., Wheelock, J.V., Samuel, D., 1973. The distribution of glyco-κ-casein and ­carbohydrate-free κ-casein between large and small bovine casein micelles, and its implication in micelle structure. Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 317, 202–206.

The caseins: Structure, stability, and functionality81

Creamer, L.K., Berry, G.P., Mills, O.E., 1977. A study of the dissociation of b -casein from the bovine casein micelle at low temperature. N. Z. J. Dairy Sci. Technol. 12, 58–66. Creamer, L.K., Richardson, T., Parry, D.A.D., 1981. Secondary structure of bovine αs1- and β-casein structure. Arch. Biochem. Biophys. 211, 689–696. Creamer, L.K., Plowman, J.E., Liddell, M.J., Smith, M.H., Hill, J.P., 1998. Micelle stability: κ-casein structure and function. J. Dairy Sci. 81, 3004–3012. Crowley, S.V., Megemont, M., Gazi, I., Kelly, A.L., Huppertz, T., O'Mahony, J.A., 2014. Heat stability of reconstituted milk protein concentrate powders. Int. Dairy J. 37, 104–110. Crowley, S.V., Boudin, M., Chen, B., Gazi, I., Huppertz, T., Kelly, A.L., O'Mahony, J.A., 2015. Stability of milk protein concentrate suspensions to in-container sterilisation heating conditions. Int. Dairy J. 50, 45–49. Dalgleish, D.G., 1998. Casein micelles as colloids: surface structures and stabilities. J. Dairy Sci. 81, 3013–3018. Dalgleish, D.G., 2011. On the structural models of bovine casein micelles—review and possible improvements. Soft Matter 7, 2265–2272. Dalgleish, D.G., Horne, D.S., Law, A.J.R., 1989. Size-related differences in bovine casein micelles. Biochim. Biophys. Acta Gen. Subj. 991, 383–387. Datta, N., Deeth, H.C., 2001. Age gelation of UHT milk—a review. Food Bioprod. Process. 79, 197–210. De Kruif, C.G., 1999. Casein micelle interactions. Int. Dairy J. 9, 183–188. De Kruif, C.G., 2014. The structure of casein micelles: a review of small-angle scattering data. J. Appl. Crystallogr. 47, 1479–1489. De Kruif, C.G., Grinberg, V.Y., 2002. Micellisation of β-casein. Colloids Surf. A 210, 183–190. De Kruif, C.G., Holt, C., 2003. Casein micelle structure, functions and interactions. In: Fox, P.F., McSweeney, P.L.H. (Eds.), Advanced Dairy Chemistry, third ed. Proteins, vol. 1. Kluwer Academic/Plenum Publishers, New York, pp. 233–276. De Kruif, C.G., Huppertz, T., 2012. Casein micelles: size distribution in milks from individual cows. J. Agric. Food Chem. 60, 4649–4655. De Kruif, C.G., May, R.P., 1991. κ-Casein micelles: structure, interaction and gelling studied by small-angle neutron scattering. Eur. J. Biochem. 200, 431–436. De Kruif, C.G., Zhulina, E.B., 1996. κ-casein as a polyelectrolyte brush on the surface of casein micelles. Colloids Surf. A 117, 151–159. De Kruif, C.G., Tuinier, R., Holt, C., Timmins, P.A., Rollema, H.S., 2002. Physicochemical study of κ- and β-casein dispersion and the effect of cross-linking by transglutaminase. Langmuir 18, 4885–4891. De Kruif, C.G., Huppertz, T., Urban, V.S., Petukhov, A.V., 2012. Casein micelles and their internal structure. Adv. Colloid Interf. Sci. 171, 36–52. Deeth, H., Lewis, M., 2016. Protein stability in sterilised milk and milk products. In: McSweeney, P.L.H., O'Mahony, J.A. (Eds.), Advanced Dairy Chemistry. Proteins: Applied Aspects, vol. 1B. Springer, New York, pp. 247–286. Dong, C., Ng-Kwai-Hang, K.F., 1998. Characterization of a nonelectrophoretic genetic variant of β-casein by peptide mapping and mass spectroscopic analysis. Int. Dairy J. 8, 967–972. Downey, W.K., Murphy, R.F., 1970. The temperature-dependant dissociation of β-casein from bovine casein micelles and complexes. J. Dairy Res. 37, 361–375. Ecroyd, H., Koudelka, T., Thorn, D.C., Williams, D.M., Devlin, G., Hoffman, P., Carver, J.A., 2008. Dissociation from the oligomeric state is the rate-limiting step in fibril formation by κ-casein. J. Biol. Chem. 283, 9012–9022. Ecroyd, H., Thorn, D.C., Lui, Y., Carver, J.A., 2010. The dissociated form of κ-casein is the precursor to its amyloid fibril formation. Biochem. J. 429, 251–260.

82

Proteins in Food Processing

Eigel, W.N., Butler, J.E., Ernstrom, C.A., Farrell, H.M., Harwalkar, V.R., Jenness, R., Whitney, R.M., 1984. Nomenclature of proteins of cow's milk: fifth revision. J. Dairy Sci. 67, 1599–1631. Ennis, M.P., O'Sullivan, M.M., Mulvihill, D.M., 1998. The hydration behaviour of rennet caseins in calcium chelating salt solution as determined using a rheological approach. Food Hydrocoll. 12, 451–457. Erhardt, G., 1996. Detection of a new κ -casein variant in the milk of Pinzgauer cattle. Anim. Genet. 27, 105–107. Evans, M.T.A., Phillips, M.C., 1979. The conformation and aggregation of bovine beta-casein A. II. Thermodynamics of thermal association and the effects of changes in polar and apolar interactions on micellization. Biopolymers 18, 1123–1140. Fang, Z.H., Visker, M.H.P.W., Miranda, G., Delacroix-Buchet, A., Bovenhuis, H., Martin, P., 2016. The relationships among bovine αS-casein phosphorylation isoforms suggest different phosphorylation pathways. J. Dairy Sci. 99, 8168–8177. Farrell Jr., H.M., Kumosinski, T.F., Malin, E.L., Brown, E.M., 1988. The caseins of milk as calcium-binding proteins. Biochemistry 23, 5912–5923. Farrell, H.M., Kumosinski, T.F., Cooke, P.H., King, G., Hoagland, P.D., Wickham, E.D., Dower, H.J., Groves, M.L., 1996. Particle size of purified κ-casein: metal effect and correspondence with predicted three-dimensional molecular models. J. Protein Chem. 15, 435–445. Farrell, H.M., Wickham, E.D., Groves, M.L., 1998. Environmental influences on purified κ-­ casein: disulphide interactions. J. Dairy Sci. 81, 2974–2984. Farrell, H.M., Wickham, E.D., Unruh, J.J., Qi, P.X., Hoagland, P.D., 2001. Secondary structure studies of bovine caseins: temperature dependence of β-casein structure as analyzed by circular dichroism and FTIR spectroscopy and correlation with micellization. Food Hydrocoll. 15, 341–354. Farrell, H.M., Qi, P.X., Brown, E.M., Cooke, P.H., Tunick, M.H., Wickham, E.D., Unruh, J.J., 2002. Molten globule structures in milk proteins: implications for potential new s­ tructure-function relationships. J. Dairy Sci. 85, 459–471. Farrell, H.M., Cooke, P.H., Wickham, E.D., Piotrowski, E.G., Hoagland, P.D., 2003a. Environmental influences of bovine κ-casein: reduction and conversion to fibrillar (amyloid) structures. J. Protein Chem. 22, 259–273. Farrell Jr., H.M., Brown, E.M., Hoagland, P.D., Malin, E.L., 2003b. Higher order structures of the caseins: a paradox? In: Fox, P.F., McSweeney, P.L.H. (Eds.), Advanced Dairy Chemistry. Proteins, vol. 1. Springer, New York, pp. 203–231. Farrell, H.M., Malin, E.L., Brown, E.M., Qi, P.X., 2006. Casein micelle structure: what can be learned from milk synthesis and structural biology? Curr. Opin. Colloid Interface Sci. 11, 135–147. Farrell, H.M., Malin, E.L., Brown, E.M., Mora-Gutierrez, A., 2009. Review of the chemistry of αs2-casein and the generation of a homolgous molecular model to explain its properties. J. Dairy Sci. 92, 1338–1353. Farrell Jr., H.M., Brown, E.M., Malin, E.L., 2013. Higher order structures of the caseins: a paradox? Advanced Dairy Chemistry. Springer, New York. pp. 161–184. Fitzgerald, R.J., Meisel, H., 2003. Milk protein hydrolysates and bioactive peptides. In: Fox, P.F., McSweeney, P.L.H. (Eds.), Advanced Dairy Chemistry, third ed. Proteins, Vol. 1B. Kluwer Academic/Plenum Publishers, New York. Fox, P.F., 1982. Developments in Dairy Chemistry. Proteins, vol. 1. Applied Science Publishers, London. Fox, P.F., 1989. Developments in Dairy Chemistry. Functional Proteins, vol. 4. Elsevier Applied Science Publishers, London.

The caseins: Structure, stability, and functionality83

Fox, P.F., Brodkorb, A., 2008. The casein micelle: historical aspects, current concepts and significance. Int. Dairy J. 18, 677–684. Fox, P.F., Flynn, A., 1992. Biological properties of milk proteins. Adv. Dairy Chem. 1, 255–284. Fox, P.F., Uniacke-Lowe, T., McSweeney, P.L.H., O'Mahony, J.A., 2015. Dairy Chemistry and Biochemistry. Springer, New York. Gagnard, S., Zuev, Y., Gaudin, J.-C., Fedotov, V., Choiset, Y., Axelos, M.A.V., Chobert, J.-M., Haertle, T., 2007. Modifications of the charges at the N-terminus of bovine β-casein: consequences on its structure and its micellisation. Food Hydrocoll. 21, 180–190. Garem, A., Schuck, P., Maubois, J.L., 2000. Cheesemaking properties of a new dairy-based powder made by a combination of microfiltration and ultrafiltration. Le Lait 80, 25–32. Garnier, J., 1966. Conformation of β-casein in solution. Analysis of a thermal transition between 5 and 40°C. J. Mol. Biol. 19, 586–590. Garnier, J., Osguthorpe, D.J., Robinson, B., 1978. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120, 97–120. Gazi, I., Huppertz, T., 2015. Influence of protein content and storage conditions on the solubility of caseins and whey proteins in milk protein concentrates. Int. Dairy J. 46, 22–30. Gobetti, M., Stepaniak, L., De Angelis, M., Corsetti, A., Di Cagno, R., 2002. Latent bioactive peptides in milk proteins: proteolytic activation and significance in dairy processing. Crit. Rev. Food Sci. Nutr. 42, 223–239. Graham, E.R.B., Malcolm, G.N., McKenzie, H.A., 1984. On the isolation and conformation of bovine β-casein A1. Int. J. Biol. Macromol. 6, 155–161. Griffin, M.C.A., Price, J.C., Martin, S.R., 1986. Effect of alcohols on the structure of caseins: circular dichroism studies of κ-casein A. Int. J. Biol. Macromol. 8, 367–371. Grosclaude, F., Mercier, J.-C., Ribadeau-Dumas, B., 1969. Sur la localisation, dans la séquence COOH-terminale de la caseine alpha-S1 bovine, de la substitution GLU-GLY différenciant les variants génétiques B et C. CR Acad. Sci. D: Sci Nat. 268, 3133–3136. Grosclaude, F., Mahe, M.-F., Mercier, J.-C., Ribadeua-Dumas, B., 1970. Localisation, dans la partie NH2-terminale de la caséine αs1 bovine, d'une délétion de 13 acides aminés différenciant le variant a des variants B et C. FEBS Lett. 11, 109–112. Grosclaude, F., Mahe, M.-F., Mercier, J.-C., Ribadeau-Dumas, B., 1972. Caracterisation des variants genetiques des caseines αs1 et β bovines. Eur. J. Biochem. 26, 328–337. Grosclaude, F., Mahe, M.-F., Mercier, J.-C., 1974a. Comparison du polymorphisme genetique des lactoproteines du zebu et des bovines. Ann. Genet. Sel. Anim. 6, 305–329. Grosclaude, F., Mahe, M.-F., Voglino, G.-F., 1974b. Le variant βE et le code de phosphorylation des caseins bovines. FEBS Lett. 45, 3–5. Grosclaude, F., Mahe, M.-F., Mercier, J.-C., Bonnemaire, J., Teissier, J.H., 1976. Polymorphisme des lactoproteines de bovines Nepalais. Polymorphisme des caseine αs2-mineurs. Ann. Genet. Sel. Evol. 8, 461–479. Grosclaude, F., Joudrier, P., Mahe, M.-F., 1978. Polymorphisme de la caseine αS2 bovine: etroite liaison du locus αS2-Cn avec les loci αs1-Cn, β-Cn et κ-Cn; mise en evidence d'une deletion dans le variant αS2-Cn D. Ann. Genet. Sel. Anim. 10, 313–327. Groves, M.L., Wickham, E.D., Farrell, H.M., 1998. Environmental effects on disulphide bonding patterns of bovine κ-casein. J. Protein Chem. 17, 73–84. HadjSadok, A., Pitkowski, A., Nicolai, T., Benyahia, L., Moulai-Mostefa, N., 2008. Characterisation of sodium caseinate as a function of ionic strength, pH and temperature using static and dynamic light scattering. Food Hydrocoll. 22, 1460–1466. Hammarsten, O., 1883. Zur Frage, ob das Caseïn ein einheitlicher Stoff sei. Z. Phys. Chem. 7, 227–273.

84

Proteins in Food Processing

Han, S.K., Shin, Y.C., Byun, H.D., 2000. Biochemical, molecular and physiological characterization of a new β-casein variant detected in Korean cattle. Anim. Genet. 31, 49–51. Harwalkar, V.R., 1992. Age gelation of sterilized milks. In: Fox, P.F. (Ed.), Advanced Dairy Chemistry, second ed. Proteins, vol. 1. Elsevier Applied Science, London, pp. 691–734. Havea, P., 2006. Protein interactions in milk protein concentrate powders. Int. Dairy J. 16, 415–422. Hays, T., Wood, R.A., 2005. A systematic review of the role of hydrolyzed infant formulas in allergy prevention. Arch. Pediatr. Adolesc. Med. 159, 810–816. Hernandez-Ldesma, B., Garcia-Nebot, M.J., Fernandez-Tome, S., Amigo, L., Recio, I., 2014. Dairy protein hydrolysates: peptides for health benefits. Int. Dairy J. 38, 82–100. Herskovits, T.T., 1966. On the conformation of casein. ORD properties. Biochemistry 5, 1018–1026. Hidalgo, M.E., Correa, M.P.F., Canales, M.M., Daroit, D.J., Brandelli, A., Risso, P., 2015. Biological and physicochemical properties of bovine sodium caseinate hydrolysates obtained by a bacterial protease preparation. Food Hydrocol. 53, 510–520. Ho, C., Waugh, D.F., 1965. Interactions of bovine αs-casein with small ions. J. Am. Chem. Soc. 87, 110–117. Hoagland, P.D., Unruh, J.J., Wickham, E.D., Farrell, H.M., 2001. Secondary structure of bovine αs2-casein: theoretical and experimental approaches. J. Dairy Sci. 84, 1944–1949. Holland, J.W., Deeth, H.C., Alewood, P.F., 2004. Proteomic analysis of κ-casein microheterogeniety. Proteomics 4, 743–752. Holland, J.W., Deeth, H.C., Alewood, P.F., 2005. Analysis of O-glycosylation site occupancy in bovine κ-casein glycoforms separated by two-dimensional gel electrophoresis. Proteomics 5, 990–1002. Holland, J.W., Deeth, H.C., Alewood, P.F., 2006. Resolution and characterization of multiple isoforms of bovine κ-casein by 2-DE followed by reversible cysteine-tagging enrichment strategy. Proteomics 6, 3087–3095. Holt, C., 1992. Structure and stability of bovine casein micelles. Adv. Protein Chem. 43, 63–151. Holt, C., 1998. Casein micelle substructure and calcium phosphate interactions studied by Sephacryl column chromatography. J. Dairy Sci. 81, 2994–3003. Holt, C., Horne, D.S., 1996. The hairy casein micelle: evolution of the concept and its implications for dairy technology. Neth. Milk Dairy J. 50, 85–127. Holt, C., Timmins, P.A., Errington, N., Leaver, J., 1998. A core‐shell model of calcium phosphate nanoclusters stabilized by β‐casein phosphopeptides, derived from sedimentation equilibrium and small‐angle X‐ray and neutron‐scattering measurements. Eur. J. Biochem. 252, 73–78. Holt, C., De Kruif, C.G., Tuinier, R., Timmins, P.A., 2003. Substructure of bovine casein micelles by small-angle X-ray and neutron scattering. Colloids Surf. A Physicochem. Eng. Asp. 213, 275–284. Horne, D.S., 2016. Ethanol stability and milk composition. In: McSweeney, P.L.H., O'Mahony, J.A. (Eds.), Advanced Dairy Chemistry. Proteins: Applied Aspects, vol. 1B. Springer, New York, pp. 225–246. Hubbard, C.D., Caswell, D., Lüdemann, H.D., Arnold, M., 2002. Characterisation of pressure‐ treated skimmed milk powder dispersions: application of NMR spectroscopy. J. Sci. Food Agric. 82, 1107–1114. Huppertz, T., 2016. Heat stability of milk. In: McSweeney, P.L.H., O'Mahony, J.A. (Eds.), Advanced Dairy Chemistry. Proteins: Applied Aspects, vol. 1B. Springer, New York, pp. 179–216.

The caseins: Structure, stability, and functionality85

Huppertz, T., de Kruif, C.G., 2007. High pressure-induced solubilisation of micellar calcium phosphate from cross-linked casein micelles. Colloids Surf. A Physicochem. Eng. Asp. 295, 264–268. Huppertz, T., Fox, P.F., Kelly, A.L., 2004. High pressure treatment of bovine milk: effects on casein micelles and whey proteins. J. Dairy Res. 71, 97–106. Huppertz, T., Hinz, K., Zobrist, M.R., Uniacke, T., Kelly, A.L., Fox, P.F., 2005. Effects of high pressure treatment on the rennet coagulation and cheese-making properties of heated milk. Innovative Food Sci. Emerg. Technol. 6, 279–285. Huppertz, T., Kelly, A.L., De Kruif, C.G., 2006. Disruption and reassociation of casein micelles under high pressure. J. Dairy Res. 73, 294–298. Huppertz, T., Smiddy, M.A., de Kruif, C.G., 2007. Biocompatible micro-gel particles from cross-linked casein micelles. Biomacromolecules 8, 1300–1305. Huppertz, T., Smiddy, M.A., Goff, H.D., Kelly, A.L., 2011. Effects of high pressure treatment of mix on ice cream manufacture. Int. Dairy J. 21, 718–726. Huppertz, T., Gazi, I., Luyten, H., Nieuwenhuijse, H., Alting, A., Schokker, E., 2017. Hydration of casein micelles and caseinates: implications for casein micelle structure. Int. Dairy J. 74, 1–11. Jann, O., Ceriotti, G., Caroli, A., Erhardt, G., 2002. A new variant in exon VII of the bovine β-casein gene (CSN2) and its distribution among European cattle breeds. J. Anim. Breed. Genet. 119, 65–68. Kajiwara, K., Niki, R., Urakawa, H., Hiragi, Y., Donkai, N., Nagura, M., 1988. Micellar structure of β-casein observed by small-angle X-ray scattering. Biochim. Biophys. Acta 955, 128–134. Kethireddipalli, P., Hill, A.R., Dalgleish, D.G., 2010. Protein interactions in heat-treated milk and effect on rennet coagulation. Int. Dairy J. 20, 838–843. Kumosinski, T.F., Brown, E.M., Farrell, H.M., 1991. Three-dimensional molecular modeling of bovine caseins: κ-casein. J. Dairy Sci. 74, 2879–2887. Kumosinski, T.F., Brown, E.M., Farrell, H.M., 1993. Three-dimensional molecular modeling of bovine caseins: a refined, energy-minimized κ-casein structure. J. Dairy Sci. 76, 2507–2520. Kumosinski, T.F., King, G., Farrell Jr., H.M., 1994a. An energy-minimized casein submicelle working model. J. Protein Chem. 13, 681–700. Kumosinski, T.F., King, G., Farrell Jr., H.M., 1994b. Comparison of the three-dimensional molecular models of bovine submicellar caseins with small-angle X-ray scattering. Influence of protein hydration. J. Protein Chem. 13, 701–714. Lam, E., Holt, C., Edwards, P., McKinnon, I., Otter, D., Li, N., Hemar, Y., 2017. The effect of transglutaminase treatment on the physico-chemical properties of skim milk with added ethylenediaminetetraacetic acid. Food Hydrocoll. 69, 329–340. Leclerc, E., Calmettes, P., 1997a. Interactions in micellar solutions of β-casein. Physica B 234– 236, 207–209. Leclerc, E., Calmettes, P., 1997b. Interactions in micellar solutions of β-casein. Phys. Rev. Lett. 78, 150–153. Leclerc, E., Calmettes, P., 1998. Structure of β-casein micelles. Physica B 241–243, 1141–1143. Lefebvre-Cases, E., Gastaldi, E., de la Fuente, B.T., 1998. Influence of chemical agents on interactions in dairy products: effect of SDS on casein micelles. Colloids Surf. B 11, 281–285. Lencki, R.W., 2007. Evidence for fibril-like structure in bovine casein micelles. J. Dairy Sci. 90, 75–89.

86

Proteins in Food Processing

Leonil, J., Henry, G., Jouanneau, D., Delage, M.-M., Forge, V., Putaux, J.-L., 2008. Kinetics of fibril formation of bovine κ-casein indicate a conformational rearrangement as a critical step in the process. J. Mol. Biol. 381, 1267–1280. Lopez-Fandino, R., Carrascosa, A.V., Olano, A., 1996. The effects of high pressure on whey protein denaturation and cheese-making properties of raw milk. J. Dairy Sci. 79, 929–936. Lu, Y., McMahon, D.J., Metzger, L.E., Kommeneni, A., Vollmer, A.H., 2015. Solubilization of rehydrated highly concentrated micellar casein for use in liquid food applications. J. Dairy Sci. 98, 5917–5930. Lu, Y., McMahon, D.J., Vollmer, A.H., 2016. Investigating cold gelation properties of highly concentrated micellar casein and cream for use in cheese making. J. Dairy Sci. 99, 5132–5143. Lucey, J.A., Horne, D.S., 2009. Milk salts: technological significance. Advanced Dairy Chemistry. Springer, New York, pp. 351–389. Luo, Y.C., Pan, K., Zhong, Q.X., 2014. Physical, chemical and biochemical properties of casein hydrolyzed by three proteases: partial characterization. Food Chem. 155, 146–154. Mahe, M.-F., Grosclaude, F., 1982. Polymorphisme de la caseine αS2 des bovines: characterization du variant C du yak (Bos grunniens). Ann. Genet. Sel. Anim. 14, 401–416. Mahe, M.-F., Miranda, G., Queral, R., Bado, A., Souvenir-Zafidrajaona, P., Grosclaude, F., 1999. Genetic polymorphism of milk proteins in African Bos taurus and Bos indicus populations characterization of variants αS1-Cn H and κ-Cn J. Genet. Sel. Evol. 31, 239–253. Malin, E.L., Brown, E.M., Wickham, E.D., Farrell, H.M., 2005. Contributions of terminal peptides to associative behavior of αs1-casein. J. Dairy Sci. 88, 2318–2328. Manson, W., Carolan, T., Annan, W.D., 1977. Bovine αs0-casein; a phosphorylated homologue of αs1-casein. Eur. J. Biochem. 78, 411–417. Marchin, S., Putaux, J.L., Pignon, F., Léonil, J., 2007. Effects of the environmental factors on the casein micelle structure studied by cryo transmission electron microscopy and small-angle x-ray scattering/ultrasmall-angle x-ray scattering. J. Chem. Phys. 126, 01B617. Mariani, P., Zanzucchi, G., Masoni, M., Serventi, P., 1995. Percentage distribution of the caseins and the κ-casein fractions, separated by RP-HPLC, in Italian Brown cows with different genotype at the κ-CN locus. Scienza E Tecnica Lattiero Casearia 46, 20–20. Mata, J.P., Udabage, P., Gilbert, E.P., 2011. Structure of casein micelles in milk protein concentrate powders via small angle X-ray scattering. Soft Matter 7, 3837–3843. McGann, T.C.A., Fox, P.F., 1974. Physicochemical properties of casein micelles reformed from urea-treated milk. J. Dairy Res. 41, 45–53. McGann, T.C., Donnelly, W.J., Kearney, R.D., Buchheim, W., 1980. Composition and size distribution of bovine casein micelles. Biochim. Biophys. Acta Gen. Subj. 630, 261–270. McKenzie, H.A., 1970. Milk Proteins: Chemistry and Molecular Biology. vol. 1. Academic Press, New York. McKenzie, H.A., 1971. Milk Proteins: Chemistry and Molecular Biology. vol. II. Academic Press, New York. McMahon, D.J., Brown, R.J., 1984. Composition, structure, and integrity of casein micelles: a review 1. J. Dairy Sci. 67, 499–512. McMahon, D.J., McManus, W.R., 1998. Rethinking casein micelle structure using electron microscopy 1. J. Dairy Sci. 81, 2985–2993. McSweeney, P.L.H., Fox, P.F., 2013. Advanced Dairy Chemistry. In: fourth ed.. Proteins: Basic Aspects. vol. 1A. Springer, New York. Mercier, J.-C., 1981. Phosphorylation of the caseins, present evidence for an amino acid triplet code posttranslationally recognized by specific kinases. Biochimie 63, 1–17.

The caseins: Structure, stability, and functionality87

Mercier, J.-C., Brignon, G., Ribadeau-Dumas, B., 1973. Structure primarie de la caseine κ-­ bovine B. Sequence complete. Eur. J. Biochem. 35, 222–235. Merel-Rausch, E., Kulozik, U., Hinrichs, J., 2007. Influence of pressure release rate and protein concentration on the formation of pressure-induced casein structures. J. Dairy Res. 74, 283–289. Mikheeva, L.M., Grinberg, N.V., Grinberg, V.Y., Khokhlov, A.R., De Kruif, C.G., 2003. Thermodynamics of micellization of bovine β-casein by high-sensitivity differential scanning calorimetry. Langmuir 19, 2913–2921. Minkiewicz, P., Slangen, C.J., Lagerwerf, F.M., Haverkamp, J., Rollema, H.S., Visser, S., 1996. Reversed-phase high-performance liquid chromatographic separation of bovine κ-casein macropeptide and characterization of isolated fractions. J. Chromatogr. A 743, 123–135. Miranda, G., Anglade, P., Mahe, M.-F., Erhardt, G., 1993. Biochemical characterization of the bovine genetic kappa-casein C and E variants. Anim. Genet. 4, 27–31. Miwa, N., Yokoyama, K., Wakabayashi, H., Nio, N., 2010. Effect of deamidation by ­protein-glutaminase on physicochemical and functional properties of skim milk. Int. Dairy J. 20, 393–399. Mizuno, R., Lucey, J.A., 2005. Effects of emulsifying salts on the turbidity and calcium-­ phosphate–protein interactions in casein micelles. J. Dairy Sci. 88, 3070–3078. Mohan, A., Rajendran, S.R.C.K., He, Q.S., Bazinet, L., Udenigwe, C.C., 2015. Encapsulation of food protein hydrolysates and peptides: a review. RSC Adv. 97, 79270–79278. Molle, D., Leonil, J., 1995. Heterogeneity of the bovine κ-casein caseino-macropeptide, resolved by liquid chromatography on-line with electrospray ionization mass spectrometry. J. Chromatogr. A 708, 223–230. Monaci, L., Tregoat, V., van Hengel, A.J., Anklam, E., 2006. Milk allergens, their characteristics and their detection in food: a review. Eur. Food Res. Technol. 223, 149–179. Morr, C.V., 1967. Effect of oxalate and urea upon ultracentrifugation properties of raw and heated skim milk casein micelles. J. Dairy Sci. 50, 1744–1751. Moughal, K.I., Munro, P.A., Singh, H., 2000. Suspension stability and size distribution of particles in reconstituted, commercial calcium caseinates. Int. Dairy J. 10, 683–690. Mulvihill, D.M., Ennis, M.P., 2003. Functional milk proteins: production and utilization. In: Fox, P.F., McSweeney, P.L.H. (Eds.), Advanced Dairy Chemistry. Proteins, vol. 1. Springer, New York, pp. 1175–1228. Nakajima, I., Kawaniski, G., Furuichi, E., 1975. Reaction of melting salts upon casein micelles and their effects on calcium, phosphorus and bound water. Agric. Biol. Chem. 39, 979–987. Naqvi, M.A., Irani, K.A., Katanishooshtari, M., Rousseau, D., 2016. Disorder in milk proteins: formation, structure, function, isolation and applications of casein phosphopeptides. Curr. Protein Pept. Sci. 17, 368–379. Needs, E.C., Stenning, R.A., Gill, A.L., Ferregut, V., Rich, G.T., 2000. High-pressure treatment of milk: effects on casein micelle structure and on enzymic coagulation. J. Dairy Res. 67, 31–42. Nguyen, N.H., Wong, M., Anema, S.G., Havea, P., Guyomarc'h, F., 2012. Effects of adding low levels of a disulfide reducing agent on the disulfide interactions of β-lactoglobulin and κ-casein in skim milk. J. Agric. Food Chem. 60, 2337–2342. Nieuwenhuijse, J.A., van Boekel, M.A.J.S., 2003. Protein stability in sterilised milk and milk products. In: Fox, P.F., McSweeney, P.L.H. (Eds.), Advanced Dairy Chemistry, third ed. Proteins, vol. 1. Kluwer Academic/Plenum, New York, pp. 947–974. Niewold, T.A., Murphy, C.L., Hulskamp-Koch, C.A., Tooten, P.C., Gruys, E., 1999. Casein related amyloid, characterization of a new and unique amyloid protein isolated from bovine corpora amylacea. Amyloid 6, 244–249.

88

Proteins in Food Processing

Niki, V.R., Takase, K., Arima, S., 1977. Über die gestalt and gröβe des temperaturabhängigen assoziats von β-casein. Milchwissenschaft 32, 577–582. Noelken, M., Reibstein, M., 1968. The conformation of β-casein B. Arch. Biochem. Biophys. 123, 397. Nongomiera, A.B., Fitzgerald, R.J., 2016. Strategies for the discovery, identification and validation of milk protein-derived bioactive peptides. Trends Food Sci. Technol. 50, 26–43. O'Connell, J.E., Grinberg, V.Y., De Kruif, C.G., 2003. Association behavior of β-casein. J. Colloid Interface Sci. 258, 33–39. Ono, T., Yada, R., Yunati, K., Nakai, S., 1987. Comparison of the comformations of κ-casein, para-κ-casein and glycomacropeptide. Biochim. Biophys. Acta 911, 318–325. Orlien, V., Knudsen, J.C., Colon, M., Skibsted, L.H., 2006. Dynamics of casein micelles in skim milk during and after high pressure treatment. Food Chem. 98, 513–521. Osborne, T.B., Wakeman, A.J., 1918. Some new constituents of milk third paper. A new protein, soluble in alcohol. J. Biol. Chem. 33, 243–251. Parry, R.M., Carroll, R.J., 1969. Location of κ-casein in milk micelles. Biochim. Biophys. Acta 194, 138–150. Payens, T.A.J., Heremans, K., 1969. Effect of pressure on the temperature-dependent association of β-casein. Biopolymers 8, 335–345. Payens, T.A.J., Schmidt, P.G., 1965. The thermodynamic parameters of the association of αs1-casein C. Biochim. Biophys. Acta 109, 214–222. Payens, T.A.J., Schmidt, D.G., 1966. Boundary spreading of rapidly polymerizing αs1-casein B and C during sedimentation. Arch. Biochem. Biophys. 115, 136–145. Payens, T.A.J., Van Markwijk, B.W., 1963. Some features of the association of β-casein. Biochim. Biophys. Acta 71, 517–530. Payens, T.A.J., Brinkhuis, J.A., Van Markwijk, B.W., 1969. Self-association in non-ideal systems. Combined light scattering and sedimentation measurements in β-casein solutions. Biochim. Biophys. Acta 175, 434–437. Pepper, L., Farrell, H.M., 1982. Interactions leading to formation of casein submicelles. J. Dairy Sci. 65, 2259–2266. Pisano, A., Packer, N.H., Redmond, J.W., Williams, K.L., Gooley, A.A., 1994. Glycobiology 4, 837–844. Prinzenberg, E.-M., Hiendleder, S., Ikonen, T., Erhardt, G., 1996. Molecular genetic characterization of new bovine κ-casein alleles CSN3-F and CSN3-G and genotyping by PCR.RFLP. Anim. Genet. 27, 347–349. Prinzenberg, E.-M., Krause, I., Erhardt, G., 1999. SCCP analysis of the bovine CSN3 locus discriminates six alleles corresponding to known protein variants (A, B, C, E, F, G) and three new DNA polymorphism (H, I, A1). Anim. Biotechnol. 10, 49–62. Qi, P.X., Wickham, E.D., Farrell, H.M., 2004. Thermal and alkaline denaturation of bovine β-casein. Protein J. 23, 389–402. Qi, P.X., Wickham, E.D., Piotrowski, E.G., Fagerquist, C.K., Farrell, H.M., 2005. Implication of C-terminal deletion on the structure and stability of bovine β-casein. Protein J. 24, 431–444. Raap, J., Kerling, K.E.T., Vreeman, H.J., Visser, S., 1983. Peptide substrates for chymosin (rennin): conformational studies of κ-casein and some κ-casein-related oligopeptides by circular dichroism and secondary structure prediction. Arch. Biochem. Biophys 221, 117–124. Rasmussen, L.K., Hojrup, P., Petersen, T.E., 1992. Localization of two interchain disulfide bridges in dimers of bovine αS2-casein. Eur. J. Biochem. 203, 381–386.

The caseins: Structure, stability, and functionality89

Rasmussen, L.K., Hojrup, P., Petersen, T.E., 1994. Disulphide arrangement in bovine caseins: localization of intrachain disulphide bridges in monomers of κ- and αs2-casein from bovine milk. J. Dairy Res. 61, 485–493. Ribadeau-Dumas, B., Grosclaude, F., Mercier, J.-C., 1970. Localization in the peptide chain of bovine beta casein of the His-Gln substitution differentiating the A2 and A3 genetic variants. CR Acad. Sci. D: Sci. Nat. 270, 2369–2372. Rajarathman, E., Nongomeira, A.B., O'Sullivan, D., Flynn, C., FitzGerald, R.J., 2016. Impact of enzyme preparation and degree of hydrolysis on peptide profile and nitrogen solubility of sodium caseinate hydrolysates. Int. J. Food Sci. Technol. 51, 2123–2131. Randaheera, C.S., Liyanaarachchi, W.S., Chandrapala, J., Dissaayakje, M., Vasiljevic, T., 2016. Utilizing unique properties of caseins and the casein micelle for delivery of sensitive food ingredients and bioactives. Trends Food Sci. Technol. 57, 178–187. Reynolds, E.C., 2009. Casein phosphopeptide-amorphous calcium phosphate: the scientific evidence. Adv. Dent. Res. 21, 25–29. Rollema, H.S., 1992. Casein association and micelle formation. In: Fox, P.F. (Ed.), Advanced Dairy Chemistry, vol. 1, pp. 111–140. Rollema, H.S., Brinkhuis, J.A., Vreeman, H.J., 1988. 1H-NMR studies of bovine kappa-casein and casein micelles. Neth. Milk Dairy J. 42, 233–248. Rose, D., 1968. Relation between micellar and serum casein in bovine milk. J. Dairy Sci. 51, 1897–1902. Saito, T., Itoh, T., 1992. Variations and distributions of O-glycosidically linked sugar chains in bovine κ-casein. J. Dairy Sci. 75, 1768–1774. Sandra, S., Ho, M., Alexander, M., Corredig, M., 2012. Effect of soluble calcium on the renneting properties of the casein micelles as measured by rheology and diffusing wave spectroscopy. J. Dairy Sci. 95, 75–82. Sawyer, L., Holt, C., 1993. The secondary structure of milk proteins and their biological function. J. Dairy Sci. 76, 3062–3078. Schmidt, D.G., 1970a. Differences between the association of the genetic variants B, C and D of αs1-casein. Biochim. Biophys. Acta 221, 140–142. Schmidt, D.G., 1970b. The association of αs1-casein B at pH 6.6. Biochim. Biophys. Acta 207, 130–138. Schmidt, D.G., 1979. Properties of artificial casein micelles. J. Dairy Res. 46, 351–355. Schmidt, D.G., 1982. Association of caseins and casein micelle structure. In: Fox, P.F. (Ed.), Developments in Dairy Chemistry 1. Elsevier Applied Science, London, pp. 61–86. Schmidt, D.G., Payens, T.A.J., 1972. The evaluation of positive and negative contributions to the second virial coefficient of some milk proteins. J. Colloid Interface Sci. 39, 655–662. Schmidt, D.G., Van Markwijk, B.W., 1968. Further studies on the associating subunit of αs1-­ casein. Biochim. Biophys. Acta 154, 613–614. Schmidt, D.G., Van Der Spek, C.A., Buchheim, W., Hinz, A., 1974. On the formation of artificial casein micelles. Milchwissenschaft 29, 455–459. Senocq, D., Molle, D., Pochet, S., Leonil, J., Dupont, D., Levieux, D., 2002. A new bovine β-casein genetic variant characterized by a Met93 – Leu93 substitution in the sequence A2. Lait 82, 171–180. Shapira, A., Assaraf, Y.G., Epstein, D., Livney, Y.D., 2010. Beta-casein nanoparticles as an oral delivery system for chemtherapeutic drugs: impact of drug structure and properties on co-assembly. Pharmaceutical Res. 27, 2175–2186. Singh, H., Fox, P.F., 1985. Heat stability of milk: pH-dependent dissociation of micellar κ-casein on heating milk at ultra-high temperatures. J. Dairy Res. 52, 529–538.

90

Proteins in Food Processing

Singh, H., Fox, P.F., 1986. Heat stability of milk: further studies on the pH-dependent dissociation of micellar κ-casein. J. Dairy Res. 53, 237–248. Singh, H., Fox, P.F., 1987a. Heat stability of milk: influence of modifying sulphydryl-disulphide interactions on the heat coagulation time–pH profile. J. Dairy Res. 54, 347–359. Singh, H., Fox, P.F., 1987b. Heat stability of milk: role of β-lactoglobulin in the pH-dependent dissociation of micellar κ-casein. J. Dairy Res. 54, 509–521. Singh, H., Fox, P.F., 1987c. Heat stability of milk: influence of colloidal and soluble salts and protein modification on the pH-dependent dissociation of micellar κ-casein. J. Dairy Res. 54, 523–534. Smiddy, M.A., Martin, J.E., Kelly, A.L., De Kruif, C.G., Huppertz, T., 2006. Stability of casein micelles cross-linked by transglutaminase. J. Dairy Sci. 89, 1906–1914. Smyth, E., Syme, C.D., Blanch, E.W., Hecht, L., Vasak, M., Barron, L.D., 2001. Solution structure of native proteins with irregular folds from Raman optical activity. Biopolymers 58, 138–151. Snoeren, T.H.M., Van Markwijk, B., Van Montfort, R., 1980. Some physicochemical properties of bovine αs2-casein. Biochim. Biophys. Acta 622, 266–276. Sulimova, G.E., Sokolova, S.S., Semikozova, O.P., Nguet, L.M., Berberov, E.M., 1992. Analysis of DNA polymorphisms of clustered genes in cattle: casein genes and genes of the major histocompatibility complex (BOLA). Tsitol. Genet. 26, 18–26. Sun, N., Wu, H.T., Du, M., Tang, Y., Lui, H.W., Hu, Y.H., Zhu, B.W., 2016. Food protein-­ derived calcium chelating peptides: a review. Trends Food Sci. Technol. 58, 140–148. Swaisgood, H.E., 1992. Chemistry of the caseins. In: Fox, P.F. (Ed.), Advanced Dairy Chemistry, second ed. Proteins, vol. 1. Elsevier Applied Science, New York, pp. 63–110. Swaisgood, H.E., Timasheff, S.N., 1968. Association of αs1-casein C in the alkaline pH range. Arch. Biochem. Biophys. 125, 344–361. Swaisgood, H.E., Brunner, J.R., Lillevik, H.A., 1964. Physical parameters of κ-casein from cow's milk. Biochemistry 3, 1616–1623. Syme, C.D., Blanch, E.W., Holt, C., Jakes, R., Goedert, M., Hecht, L., Barron, L.D., 2002. A Raman optical activity study of rheomorphism in casein, synucleins and tau. New insight into the structure and behavior of natively unfolded proteins. Eur. J. Biochem. 269, 148–156. Tai, M.-S., Kegeles, G., 1984. A micelle model for the sedimentation behavior of bovine β-­ casein. Biophys. Chem. 20, 81–87. Takase, K., Niki, R., Arima, S., 1980. A sedimentation equilibrium study of the temperature-­ dependent association of bovine β-casein. Biochim. Biophys. Acta 622, 1–8. Talbo, G.H., Suckau, D., Malkoski, M., Reynolds, E.C., 2001. MALDI-PSD-MS analysis of the phosphorylation sites of caseinomacropeptide. Peptides 22, 1093–1098. Talbot, B., Waugh, D.F., 1970. Micelle-forming characteristics of monomeric and covalent polymeric κ-caseins. Biochemistry 9, 2807–2813. Tauzin, J., Miclo, L., Roth, S., Molle, D., Gaillard, J.-L., 2003. Tryptic hydrolysis of bovine αs2-casein: identification and release kinetics of peptides. Int. Dairy J. 13, 15–27. Thompson, A., Boland, M., Singh, H. (Eds.), 2014. Milk Proteins: From Expression to Food. Academic Press, Amsterdam, The Netherlands. Thomsen, J.K., Jakobsen, H.J., Nielsen, N.C., Petersen, T.E., Rasmussen, L.K., 1995. Solid‐ state magic‐angle spinning 31P‐NMR studies of native casein micelles. Eur. J. Biochem. 230, 454–459. Thorn, D.C., Meehan, S., Sunde, M., Rekas, A., Gras, S.L., MacPhee, C.E., Dobson, C.M., Wilson, M.R., Carver, J.A., 2005. Amyloid fibril formation by bovine milk κ-casein and its inhibition by the molecular chaperones αs- and β-casein. Biochemistry 44, 17027–17036.

The caseins: Structure, stability, and functionality91

Thorn, D.C., Ecroyd, H., Sunde, M., Poon, S., Carver, J.A., 2008. Amyloid fibril formation by bovine milk αs2-casein occurs under physiological conditions yet is prevented by its natural counterpart, αs1-casein. Biochemistry 47, 3926–3936. Thorn, D.C., Ecroyd, H., Carver, J.A., 2009. The two-faced nature of milk casein proteins: amyloid fibril formation and chaperone-like activity. Aust. J. Dairy Technol. 64, 34–40. Thurn, A., Burchard, W., Niki, R., 1987. Structure of casein micelles. I. Small angle neutron scattering and light scattering from β-casein and κ-casein. Colloid Polym. Sci. 265, 653–666. Trieu-Cuot, P., Gripon, J.-C., 1981. Electrofocusing and two-dimensional electrophoresis of bovine caseins. J. Dairy Res. 48, 303–310. Urista, C.M., Fernandez, R.A., Rodriguez, F.R., Cenca, A.A., Jurado, A.T., 2011. Review: production and functionality of active peptides from milk. Food Sci. Technol. Int. 17, 293–317. Vaia, B., Smiddy, M.A., Kelly, A.L., Huppertz, T., 2006. Solvent-mediated disruption of bovine casein micelles at alkaline pH. J. Agric. Food Chem. 54, 8288–8293. Van der Ven, C., Gruppen, H., de Bont, D.B.A., Voragen, A.G.J., 2001. Reversed phase and size exclusion chromatography of milk protein hydrolysates: relation between elution from reversed phase column and apparent molecular weight distribution. Int. Dairy J. 11, 83–92. Van der Ven, C., Muresen, S., Gruppen, H., de Bont, D.B.A., Merck, K.B., Voragen, A.G.J., 2002a. FTIR spectra of whey and casein hydrolysates in relation to their functional properties. J. Agric. Food Chem. 50, 6943–6950. Van der Ven, C., Gruppen, H., de Bont, D.B.A., Voragen, A.G.J., 2002b. Correlations between biochemical characteristics and foam-forming and -stabilizing abilities of whey and casein hydrolysates. J. Agric. Food Chem. 50, 2938–2946. Van Dijk, H.J.M., 1992. The properties of casein micelles. VI: Behaviour above pH 9, and implications for the micelle model. Nederlands melk en Zuiveltijdschrift 46, 101–113. Van Hooydonk, A.C.M., Olieman, C., Hagedoorn, H.G., 1984. Kinetics of the chymosin-­ catalysed proteolysis of κ-casein in milk. Neth. Milk Dairy J. 38, 207–222. Van Kemenade, M.J.J.M., De Bruyn, P.L., 1989. The influence of casein on the kinetics of hydroxyapatite precipitation. J. Colloid Interface Sci. 129, 1–14. Vasbinder, A.J., Rollema, H.S., de Kruif, C.G., 2003. Impaired rennetability of heated milk; study of enzymatic hydrolysis and gelation kinetics. J. Dairy Sci. 86, 1548–1555. Visser, S., van Rooijen, P.J., Slangen, C.J., 1980. Peptide substrates for chymosin (rennin). Isolation and substrate behaviour of two tryptic fragments of bovine κ-casein. Eur. J. Biochem. 108, 415–421. Visser, S., Slangen, C.J., Lagerwerf, F.M., Van Dongen, W.S., Haverkamp, J., 1995. Identification of a new variant of bovine beta-casein using reversed-phase high-performance liquid-­ chromatography and mass-spectrometric analysis. J. Chromatogr. A 711, 141–150. Vreeman, H.J., 1979. The association of bovine SH-κ-casein at pH 7.0. J. Dairy Res. 46, 271–276. Vreeman, H.J., Both, P., Brinkhuis, J.A., Van der Spek, C., 1977. Purification and some physicochemical properties of bovine κ-casein. Biochim. Biophys. Acta 491, 93–103. Vreeman, H.J., Brinkhuis, J.A., Van der Spek, C., 1981. Some association properties of bovine SH-κ-casein. Biophys. Chem. 14, 185–193. Vreeman, H.J., Visser, S., Slangen, C.J., Van Riel, J.A.M., 1986. Characterization of bovine κ-casein fraction and the kinetics of chymosin-induced macropeptide release from ­carbohydrate-free and carbohydrate-containing fractions determined by high-performance gel-permeation chromatography. Biochem. J. 240, 87–97. Walsh, D.J., Russell, K., Fitzgerald, R.J., 2008. Stabillisation of sodium caseinate hydrolysate foams. Food Res. Int. 41, 43–52. Walstra, P., 1990. On the stability of casein micelles. J. Dairy Sci. 73, 1965–1979.

92

Proteins in Food Processing

Walstra, P., Jenness, R., 1984. Dairy Chemistry and Physics. John Wiley & Sons, New York. Xu, Y., Liu, D., Yang, H., Zhang, J., Liu, X., Regenstein, J.M., Zhou, P., 2016. Effect of calcium sequestration by ion-exchange treatment on the dissociation of casein micelles in model milk protein concentrates. Food Hydrocoll. 60, 59–66. Yamuachi, K., Takemoto, S., Tsugo, T., 1967. Calcium-binding property of dephosphorylated casein. Agric. Biol. Chem. 31, 54–63. Zobrist, M.R., Huppertz, T., Uniacke, T., Fox, P.F., Kelly, A.L., 2005. High-pressure-induced changes in the rennet coagulation properties of bovine milk. Int. Dairy J. 15, 655–662.