15 Genetic Polymorphism of Milk Proteins 491 being taken as a reference in the UniProt data- hydrophobic proteins as found in MFGM. base (accession number P09462). However, Recent advances in the field of proteomics, although the two predicted sequences differ including development of one-dimensional gel for four amino acid residues, they were not electrophoresis approach coupled to tandem attributed to any of the two known variants mass spectrometry (GeLC-MS-MS), led to the observed so far, named A and B (Schmidt and identification of hundreds of proteins associated Ebner, 1972). to the MFGM in several species, including humans (Hettinga et al., 2011), cattle (Reinhardt 15.5 Genetic Polymorphism in and Lippolis, 2006) and sheep (Pisanu et al., Milk-Fat Globule Membrane 2011). Thus, the number of MFGM proteins, as Proteins well as biological functions associated with MFGM proteins (including fat metabolism, cell The secretion process of milk lipids initiates in trafficking or signalling, or immune-related the endoplasmic reticulum with the budding of functions), is still growing. lipid droplets. These lipid droplets then migrate to the apical pole of the mammary epithelial 15.5.1 Genetic Polymorphisms cell (MEC), where they are progressively envel- Associated with MFGM Protein- oped by the plasma membrane and released as Encoding Genes fat globules in the milk (Mather and Keenan, 1998). Hence, most of MFGM-associated pro- Genetic polymorphisms within MFGM protein- teins belong to the ER and plasma membrane encoding genes reported to date are summarised compartments. In addition, cytoplasmic inclu- in Table 15.16. As expected, since MFGM pro- sions, known as “crescents”, are often trapped teins are involved in lipid secretion processes, between the outer membrane and the lipid core most of reported genetic polymorphisms are during the secretion process (Wooding et al., associated with dairy traits related to quantitative 1970). These crescents contain soluble, cyto- (milk-fat yield) or qualitative (fatty acid compo- plasmic proteins which can be also identified in sition) aspects of fat in milk. MFGM isolates. 15.5.1.1 MUC-1 Biochemical approaches (SDS-PAGE fol- Mucins are large proteins containing more than lowed by diverse staining procedures) or molec- 50% carbohydrates by weight which are present ular biology techniques have been initially at the interface between epithelia and their employed to characterise MFGM proteins. extracellular environment. They play an essen- Roughly, MFGM material can be resolved by tial role in forming protective mucous barriers SDS-PAGE into eight protein bands correspond- on epithelial surfaces. MUC-1 is undoubtedly ing to MUC-1 (mucin-1), fatty acid synthase the best characterised milk mucin, and, histori- (FAS), xanthine oxidoreductase (XOR), MUC cally, MUC-1 polymorphism is probably the 15 (PASIII), CD36, butyrophilin (BTN1A1), earliest genetic polymorphism reported to date milk-fat globule EGF factor 8 (MFG-E8) or lac- for MFGM proteins. tadherin (LDH), and adipophilin (ADRP, adi- pose differentiation-related protein). Major Bovine MUC-1 is a highly glycosylated pro- MFGM proteins have been reviewed extensively tein of 580 amino acid residues comprising a sig- (Mather, 2000). Historically, two-dimensional nal peptide of 22 residues and encoded by a rather gel electrophoresis coupled to mass spectrome- compact gene (ca. 4 kb), known to contain a try methods was used to identify MFGM- highly polymorphic variable number of tandem associated proteins (Fortunato et al., 2003; Fong repeats (VNTR) domain. General features of the et al., 2007; Barello et al., 2008). However, protein are a large extracellular region with highly these methods are not suited to analyse large conserved tandem repeats of 20 amino acids
Table 15.16 Genetic polymorphisms identified within milk-fat globule membrane protein-encoding genes and associated dairy traits 492 P. Martin et al. MFGM protein- Associated dairy trait References encoding gene Species MUC-1 Bos taurus, Ovis aries, Capra hircus, Bos Milk protein and fat percentages (weak) Patton and Patton (1990), Hens et al. (1995), Jiang et al. (2004), grunniens (yak) Sacchi et al. (2004), Rasero et al. (2007) and Sando et al. FASN (2009) BTN1A1 Bos taurus MFGE-8 Milk fatty acid composition Roy et al. (2006), Morris et al. (2007) and Schennink et al. ABCG2 Bos taurus, Bubalus bubalis (water buffalo), (2009) FABP Ovis aries, Capra hircus Capra hircus Milk yield, milk fat yield, total solids Bhattacharya et al. (2007) and Qu et al. (2010) Bos taurus, Bos indicus (zebu), Bubalus Milk fat yield, total solids Qu et al. (2010) bubalis Milk yield, fat and protein percentages Cohen-Zinder et al. (2005), Ron et al. (2006), Tantia et al. Ovis aries (2006), Olsen et al. (2007) and Yue et al. (2011) Fat contents Calvo et al. (2004) MUC-1 mucin-1; FASN fatty acid synthase; BTN butyrophilin; MFG-E8 milk fat globule EGF factor 8, lactadherin; ABCG2 ATP-binding cassette subfamily G, member 2; FABP fatty acid-binding protein, heart-type
15 Genetic Polymorphism of Milk Proteins 493 (VNTR domain), a membrane-proximal SEA Ruminants can be a useful model to study the (sperm protein, enterokinase, and agrin) module, mechanisms by which the variation in the repeat which is a 120 amino-acid domain frequently number and the extracellular domain size can associated with heavily O-glycosylated proteins, modulate the effectiveness of MUC-1 as a cell- a transmembrane region and a short (70 amino surface shield (Rasero et al., 2007). Remarkably, acids) cytoplasmic tail (Pallesen et al., 2001). the polymorphic nature of the gene has been lost in mice and other rodents (Spicer et al., 1991). Because each codominant inherited allele may contain a variable number tandem repeat (VNTR) MUC-1 is an extensively glycosylated mucin encoding the 20-amino-acid motif, different sizes that may act as a decoy receptor for numerous of MUC-1 are observed by SDS-PAGE analysis. pathogens invading epithelial tissues, including Heterozygous individuals display two bands for the mammary gland. Accordingly, numerous MUC-1 protein in SDS-PAGE whereas a single health-related (mostly, anti-infectious) benefits band is observed for homozygous individuals. have been proposed for MUC-1 in milk (Schroten, Genetic polymorphism of MUC-1 has been dem- 1998; Patton, 1999). Two studies investigated onstrated for several dairy species, including potential associations between the MUC-1 bovine (Patton and Patton, 1990), caprine (Sacchi genetic polymorphism and important dairy traits, et al., 2004; Cebo et al., 2010) and ovine (Rasero including milk fat or protein percentages or et al., 2007) species. It has been demonstrated somatic cell counts in milk, an indirect measure that the number of tandem repeats vary between of mastitis susceptibility in dairy cattle. Both species as well between breeds. Indeed, up to five studies concluded no association between the alleles were reported in a Holstein population of MUC-1 genotype and somatic cell counts in milk. dairy cows whereas a reduced number of MUC-1 In addition, only weak associations between the alleles was found in Ayrshire, Jersey, Italian number of tandem repeats in MUC-1 and milk Friesian and Piedmontese or Brown Swiss cattle protein and fat percentages were found in Holstein (Huott et al., 1995; Sacchi et al., 1995; Rasero dairy cows (Hens et al., 1995; Sando et al., et al., 2002). The existence of 15 different alleles, 2009). showing a repetitive region ranging in size between 1,500 and 3,000 bp, has been reported in 15.5.1.2 Fatty Acid Synthase the goat MUC-1 gene (Sacchi et al., 2004) Fatty Acid Synthase (FASN) is a complex whereas only four alleles, showing a 1,500 bp homodimeric enzyme which catalyses de novo repetitive region, were reported in sheep (Rasero biosynthesis of long-chain fatty acids from et al., 2007). Accordingly, in SDS-PAGE, MUC-1 acetyl-coenzyme A and malonyl-coenzyme A. from sheep milk appears smaller in size than its FASN is therefore a strong candidate gene for fat caprine counterpart (Cebo and Martin, 2012). content in milk. In addition, several studies reported quantitative trait loci on Bos taurus The number of tandem repeats truly represents chromosome 19 (BTA19) where FASN maps a matter of interspecies differences. In humans, it (Roy et al., 2001; Boichard et al., 2003). varies from 21 to 125 with 41 and 85 repeats being the most frequently encountered motif in Two SNP were analysed in bovine FASN with the Northern European population. As a conse- regard to their associations with milk-fat content: quence, apparent molecular masses in SDS- they were located in exon 1 (g.763G > C) and in PAGE for human MUC-1 range from 240 to exon 34 (g.16009A > G) of the bovine FASN 450 kDa whereas those for bovine MUC-1 are genomic sequence (GenBank # AF285607). The considerably lower (Gendler et al., 1990; Pallesen g.763G > C substitution alters a putative Sp1 tran- et al., 2001). Analysis of the polymorphism in six scription factor-binding site in the untranslated Italian breeds showed that the sheep repetitive exon 1, whereas the g.16009A > G substitution in region seemed to be less variable and smaller in exon 34 generates a non-conservative substitu- size than the repetitive region of the goat. tion of a threonine with an alanine residue within the domain having enoyl and ketoacyl reductase
494 P. Martin et al. activities of FAS. Roy et al. (2006) reported with their frequencies in two ovine breeds significant frequency differences in Holstein (Karakul and Muzzafarnagari), Holstein- cows with high and low breeding values for milk- Friesian × Hariana cross-bred cattle and Murrah fat content. Additional SNP in bovine FASN buffaloes (Bhattacharya et al., 2004, 2007). significantly associated with C14:0, C18:2cis9,12 More recently, a novel SNP was reported in and C18:1cis9 fatty acid levels in milk were exon 5 of caprine BTN1A1 (Qu et al., 2010). A reported recently (Morris et al., 2007; Schennink single-nucleotide change (C to T) resulted in a et al., 2009). missense mutation at position 377 of BTN1A1 (leucine to phenylalanine substitution). This 15.5.1.3 Butyrophilin SNP was significantly associated with milk Butyrophilin (BTN1A1) is one of the most yield, as well as with milk fat and total solids abundant proteins in the MFGM (Mather, 2000). percentage in Xinong Saanen dairy goat milk Butyrophilin belongs to the B7/butyrophilin- (Qu et al., 2010). Taken together, these studies like proteins, a subset of the immunoglobulin provide substantial evidence for the existence of superfamily. The main features of butyrophilin genetic polymorphism in BTN1A1 gene in asso- are an extracellular part containing two Ig-like ciation with important dairy traits. domains, a short transmembrane region and a long carboxy-terminal cytoplasmic domain 15.5.1.4 MFGE8 called B30.2 domain. Interestingly, BTN1A1 Milk-fat globule-epidermal growth factor gene which is positioned within the cluster of (MFGE8) or lactadherin (LDH) is a major pro- BTN genes (seven genes including the two sub- tein of the MFGM (Mather, 2000). The main fea- families, BTN2 and BTN3, arranged in pairs) is ture of bovine LDH is the presence of two located close to the leukocyte antigen class I epidermal growth factor (EGF)-like domains in genes on human chromosome 6, thus linking the N-terminal region of the protein with an argi- butyrophilin to proteins involved in the immune nine-glycine-aspartic acid (RGD) sequence in the response (Rhodes et al., 2001). second EGF-like domain and of two C terminal regions of about 150 amino acids called F5/8 type Butyrophilin plays a key role in the regulated C or C1/C2-like domains also present in coagula- secretion of milk lipids (Ogg et al., 2006). The tion factors V and VIII (Hvarregaard et al., 1996). prevailing model for milk lipid droplet formation The C-terminal domain of the second F5/8 repeat in the MEC favours that adipophilin (ADRP or has been shown to be responsible for membrane Perlipin2/PLIN2), at the surface of lipid droplets, binding through a phosphatidylserine-binding mediates interactions with butyrophilin at the motif (Shi and Gilbert, 2003; Shi et al., 2004). cytoplasmic face of the MEC apical membrane. The RGD sequence is a cell-adhesion motif able These interactions, stabilised by XOR, initiate to bind to avb3/5 integrins therefore mediating cell oligomerisation of butyrophilin leading to the adhesion as well as cell transduction mechanisms final budding of lipid droplets and the release of (Taylor et al., 1997). fat globules in the luminal compartment (Heid and Keenan, 2005; MacManaman et al., 2007). Multiple forms of LDH have been reported in However, this model is challenged by several the mammary gland and other tissues (Giuffrida studies suggesting that butyrophilin homophilic et al., 1998; Häggqvist et al., 1999: Oshima et al., interactions solely orchestrate fat globule extru- 1999). We have recently shown that LDH from sion from MEC (Robenek et al., 2006a, b). goat milk appears as a single polypeptide chain contrary to its bovine counterpart which appears Previous studies have reported genetic vari- as two glycosylated polypeptides (Cebo et al., ability within exon 8 of the butyrophilin gene in 2010). Furthermore, four polypeptides were sheep (EMBL# AY491475), cattle (EMBL# identified among MFGM proteins from mares’ Z93323) and buffalo (EMBL# AY491471). Two milk, and LDH is expressed as two major protein alleles (A, B) and three genotypes (AA, AB, variants in the camel (Cebo et al., 2012; Cebo BB) were reported for butyrophilin together
15 Genetic Polymorphism of Milk Proteins 495 and Martin, 2012). Thus, although the occurrence caused the aberrant inclusion of a cryptic exon in of splicing variants has been reported for LDH, the human transcript. This point mutation gener- the molecular diversity of LDH across species is ates a premature termination codon resulting in a mostly explained by differential glycosylation of C-terminal truncated form of MFGE8 protein a single polypeptide backbone. leading to the development of the disease in SLE patients (Yamaguchi et al., 2010). LDH is involved in a wide range of biological functions, including apoptosis, mammary gland 15.5.1.5 ABCG2 involution after lactation and subsequent lacta- ABCG2 (also known as BRCRP, breast cancer tion (Hanayama and Nagata, 2005; Raymond resistance protein) belongs to the ATP-binding et al., 2009). It is therefore expected that genetic cassette (ABC) family of transmembrane trans- polymorphism occurring within MFGE8 gene porters. ABCG2 is strongly expressed during late may affect lactation biology or milk composition pregnancy and lactation, and a role for secretion in cattle. of riboflavin (Vitamin B2) into milk has been demonstrated for this transporter (van Herwaarden The LDH gene (MFGE8) has been sequenced et al., 2007). In addition, ABCG2 has been recently in the goat species (GenBank number identified in bovine MFGM (Reinhardt and GQ344829). Goat MFGE8 consists of nine exons Lippolis, 2006) as well as in the MFGM from covering a 3,844 bp genomic sequence. A recent goat and camel milk (Cebo et al., unpublished study (Qu et al., 2010) investigated variations in data). Another member of the ABC family, the MFGE8 and its associations with growth namely, ABCG1, has been recently identified in traits and milk performance in the goat (Capra both basal and apical regions of mammary cells hircus taxon, Guanzhong dairy breed). and in milk-fat globules (Mani et al., 2011). Interestingly, the authors identified four loci in Because ABCG1 is involved in the active trans- the goat MFGE8 which were highly polymor- port of sterols across the membrane, and may phic. They were in the 5¢-untranslated region (5¢ thus control cholesterol levels in milk, such a role UTR), the fourth intron, the seventh exon and the for ABCG2 may not be excluded. seventh intron. The g.14892T > C mutation resulted in a synonymous mutation, AAT Segregating QTL for milk, fat and protein (Asp) > AAC (Asp), located in the seventh exon were reported on Bos taurus chromosome 6 of the gene. Research for codon preference in the (BTA6), and a 420 kb region near ABCG2 was goat LDH gene indicated that the preferred codon determined (Olsen et al., 2005). A missense for Asp was AAC, the AAT sequence being the mutation was finally identified within exon 14 of less preferred codon in this gene. This mutation, bovine ABCG2. The single-nucleotide change which changes from a low preference codon to a (A to C), induces a nonconservative tyrosine to much higher preference one, may affect the serine (Y581S) substitution. ABCG2A (allele A expression level of the protein by modifying the of ABCG2 capable of encoding a tyrosine resi- mRNA stability. due), which was the most frequent allele in the Israeli Holstein population, was associated with Genetic polymorphism in the MFGE8 gene an increased fat and protein concentration in has been shown recently to be associated with milk (Cohen-Zinder et al., 2005). Interestingly, systemic lupus erythematosus (SLE) disease in analysis of the corresponding ABCG2 sequence humans. SNP were identified in codons 3 (argin- in Indian breeds of cattle (Bos indicus) and buf- ine to serine substitution) and 76 (leucine to meth- falo (Bubalus bubalis) revealed fixed ABCG2A ionine substitution) of human MFGE8. The alleles, thus suggesting that ABCG2A is the MFGE8 genotypic combination with arginine and ancestral allele and that the Y581S substitution methionine at positions 3 and 76 of the resulting (ABCG2C allele) occurred after the separation of protein, respectively, was identified as the most B. indicus and Bos taurus lineages over 200,000 predisposing genotype to SLE in a case–control years ago (Ron et al., 2006; Tantia et al., 2006). study (Hu et al., 2009). In addition, an A to G mutation located in intron 6 of human MFGE8
496 P. Martin et al. More recently, two novel SNP (45599A > C and 1995). Four levels of as1-casein synthesis have been 45610A > G) were reported in bovine ABCG2. described, ranging between 3.5 and 0 g/L for strong These SNP, located within the seventh intron of alleles (A, B and C) and null alleles (O), respec- ABCG2, were associated with milk yield and tively (Martin et al., 2002). Moreover, in null ani- somatic cell scores (Yue et al., 2011). mals for as1-casein, secretory pathways are severely affected. In the absence of as1-casein, an accumula- 15.5.1.6 FABP3 tion of immature proteins (mainly caseins) is Fatty acid-binding proteins (FABP) constitute a observed, leading to a dramatic distension of the family of small intracellular proteins (~15 kDa) rough ER (Chanat et al., 1999). Since the first steps which are involved in fatty acid transport from of milk-fat synthesis occur in the ER, this process the plasma membrane to the sites of b-oxidation might be affected by the general dysfunction of and triacylglycerol or phospholipid synthesis. secretion pathways observed in as1-casein null To date, nine genes have been identified in goats. Accordingly, it was recently demonstrated mammals, and FABP were initially named that both milk-fat globule size and zeta potential according to their tissue of origin (Storch and are related to the as1-casein genotype. At mid-lac- Thumser, 2010). tation, goats displaying strong genotypes for as1- casein (A/A goats) produced larger fat globules The MFGM form of FABP has been shown to than goats with a null genotype at the CSN1S1 be heart-type FABP (FABP3) in the bovine locus (O/O goats). Moreover, dramatic differences (Mather, 2000) or, more recently, in the ovine were found with regard to MFGM composition (Pisanu et al., 2011) species. Because of its (including both MFGM proteins and polar lipids) role in fatty acids transport and its expression in in the milk from goats with extreme genotype at the the lactating mammary gland, FABP3 is a func- CSN1S1 locus (Cebo et al., in press). Although the tional candidate gene for milk traits. Two SNP polar lipids composition of the MFGM have (one in exon 2 and the other one in intron 3) have been shown to be modified by diet (Lopez et al., been reported in ovine FABP3 (Calvo et al., 2008), breed (Graves et al., 2007) or even lactation 2002). Both SNP were tested with regard to asso- stage (Bitman and Wood, 1990), this is to our ciations with milk yield, protein and fat contents. knowledge the first report of the impact of genetic Results strongly suggest a role for FABP3 geno- polymorphism of milk proteins on the MFGM type on milk-fat content (Calvo et al., 2004). composition. 15.5.2 Genetic Polymorphisms 15.6 Linkage Between Casein Associated with Milk Proteins Genes: The Haplotype Notion Encoding Genes Which Affect MFGM Composition One of the important features of milk proteins genetics, first highlighted by Grosclaude et al. Besides the growing number of studies describ- (1965), is the tight linkage of the genes encoding ing genetic variability in MFGM protein-encod- the four caseins. This situation has been confirmed inggenes,itappearsthatthegeneticpolymorphism 25 years later (Ferretti et al., 1990; Threadgill and previously reported for non-MFGM milk pro- Womack, 1990). Casein genes are organised as a teins may affect both milk-fat structure (i.e. bio- cluster spanning a ca. 250 kb DNA fragment physical properties of milk-fat globules) together located on chromosome 6 in cattle and goats. The with MFGM composition. haplotype notion, coined in human histocompati- bility studies, has been introduced to designate a Indeed, the extensive genetic polymorphism combination of alleles of closely linked genes described in the goat at the CSN1S1 locus (encod- (Ng-Kwai-Hang and Grosclaude, 1992). The non- ing the as1-casein protein in milk) is associated independent inheritance of casein alleles in popu- with strong differences in milk protein and milk-fat content (Grosclaude et al., 1994; Barbieri et al.,
15 Genetic Polymorphism of Milk Proteins 497 lations is an example of “linkage disequilibrium” milk-fat or milk protein content, and the haplo- (non-random association of alleles). The strongest type concept has been refined. A high-resolution linkage occurs between alleles at CSN1S1 and SNP map of the bovine 2009 region was recently CSN2 loci which are convergently transcribed and constructed by Nilsen et al. (2009) to study asso- between 10 and 20 kb apart, across species. Even ciations with milk traits in Norwegian Red cattle though recombination may occur at a very low (NRC). Consistent with the structure of the casein frequency between so close loci, the casein cluster cluster they suggested to separate the casein clus- is considered as a unit of inheritance: a haplotype. ter, into two haplotype blocks, one consisting of This haplotype notion is useful to improve the CSN1S1, CSN2 and CSN1S2 and the other con- estimation of the relationship between casein vari- sisting of CSN3. Associations both with protein ants and milk production traits. Considering the and milk yield were found highly significant haplotype instead of single alleles at the four within the CSN1S1-CSN2-CSN1S2 haplotype casein loci was first suggested by Grosclaude block. In contrast, no significant association was et al. (1979) in cattle and further developed in the found within the CSN3 block. The authors pointed same species (Ikonen et al., 2001; Boettcher et al., towards CSN2 and CSN1S2 as the most likely 2004; Caroli and Erhardt, 2004). genes harbouring the underlying causative DNA variation. The most significant results involved 15.6.1 Relationship Between Casein the CSN2_67 SNP, a transversion C > A in codon Variants and Milk Production 67 that results in Pro to His exchange, with C Traits being consistently associated with greater protein and milk yield. More recently, the same team Practical applications emerging from the study of (Sodeland et al., 2011), using high coverage re- genetic polymorphism of milk proteins have been sequencing, enabled molecular characterisation extensively reviewed by Ng-Kwai-Hang and of a long-range haplotype encompassing genes Grosclaude (2003) in the third edition of CSN2 and CSN1S2 from a Swedish Holstein- Advanced Dairy Chemistry-1: Proteins. Using Friesian bull introduced into the NRC population. milk protein genes as genetic markers for increas- Haplotype analysis of a large number of descen- ing milk production, improving production- dants from this bull indicated that the haplotype related traits and altering milk composition which was not markedly disrupted by recombina- remain a matter of interest. However, a new tech- tion in this region was associated both with nology called genomic selection (Meuwissen increased milk protein content (SNPs possibly et al., (2001) is revolutionising dairy cattle breed- affecting transcription and/or translation of ing. Genomic selection refers to selection deci- CSN1S2) and increased susceptibility to mastitis sions based on genomic breeding values (GEBV) (polymorphisms close to a cluster of genes encod- which are calculated as the sum of the effects of ing CXC chemokines). dense genetic markers (SNPs), or haplotypes of these markers, across the entire genome, thereby In a recent investigation of the Dutch Holstein- potentially capturing all the quantitative trait loci Friesian population (Heck et al., 2009), it has (QTL) that contribute to variation in a trait (Hayes been shown that genetic variants and casein hap- et al., 2009). This approach which is a variant of lotypes have a major impact on the protein com- marker-assisted selection has become feasible, position of milk and explain a considerable part thanks to the large number of SNPs discovered of the genetic variation in milk protein composi- by genome sequencing and new methods to tion. It was concluded from this study that selec- efficiently genotype large number of SNP tion for both the B allele at the LGB locus (Goddard and Hayes, 2009). (LGB*B) and the CSN2*A2-CSN3*B haplotype will result in cows that produce milk more suit- However, there are still many reports that able for cheese production. A linkage study per- relate genetic polymorphism of milk proteins to formed to screen the whole bovine genome identified ten chromosomal regions (QTL) affect-
498 P. Martin et al. ing milk protein composition (casein, whey pro- centage and fat amount were significant, as were tein and specific protein content). Regions on Bos the effects of CSN3 haplotypes on fat percentage taurus autosomes (BTA) 6, 11 and 14 showed the and protein percentage. A deletion in exon 12 of largest effect on milk protein composition, and CSN1S1, so far reported only in the Norwegian some QTL could partially be explained by poly- goat population and at a high frequency (0.73), morphisms in milk protein genes (Schopen et al., explained the effects of CSN1S1 haplotypes on 2009). Using a whole-genome association study, fat amount, but not protein percentage. three genomic regions associated with major Investigation of linkage disequilibrium between effects on milk protein composition or protein all possible pairs of SNPs revealed higher levels percentage were confirmed on BTA6, BTA11, of linkage disequilibrium for SNP pairs within and BTA14 in addition to several regions with casein loci than for SNP pairs between casein smaller effects involved in the regulation of milk loci, likely reflecting low levels of intragenic protein composition (Schopen et al., 2011). recombination. Further, they provide evidences for a site of preferential recombination between Taken together, these results strongly sug- CSN2 and CSN1S2. Casein haplotypes were gest that there are opportunities to increase the found to have large effects on production traits, casein index which is a desirable breeding goal and the possibility to use haplotypes associated for cheesemaking properties and cheese pro- with the increase in fat and protein percentages, duction and that it would be advisable to in haplotype-assisted selection, would have combine information on polymorphism of potential. In a second study, in which both geno- known genes (casein cluster) with genomic type and phenotype information on milk-produc- selection (anonymous markers). ing goats were recorded, casein SNP dominance and additive effects were investigated (Dagnachew Several association studies have analysed the et al., 2011). Unlike in the previous study, the effects of the polymorphism of casein genes on deletion occurring in exon 12 appeared to be dairy performance and milk quality in different significantly associated with protein and fat con- goat breeds (Grosclaude et al., 1994; Grosclaude tents and with milk taste. Similar results have and Martin, 1997; Ådnøy et al., 2003; Hayes been reported previously by Grosclaude et al. et al., 2006; Chilliard et al., 2006; Chiatti et al., (1994) and Grosclaude and Martin (1997) who 2007). They revealed that polymorphisms at the underlined the unexpected effect of defective CSN1S1 locus have significant effects on casein alleles at the CSN1S1 locus (alleles E and F) on content, total protein content, fat content and fat content, correlated with a dysfunction in technological properties of milk. It has also been secretory mechanisms triggered by the accumu- reported that k-casein (CSN3) variants have a lation of caseins in the endoplasmic reticulum of significant influence on milk production traits the MEC (Chanat et al., 1999). They also reported (Angulo et al., 1994; Chiatti et al., 2007; Caravaca that the polymorphism at the CSN1S1 locus in et al., 2009). French dairy flocks (Alpine and Saanen breeds) has significant effect on the diameter and calcium Among these studies, one of the most geneti- content of casein micelles which were both lower cally detailed was performed on Norwegian goats in AA milks. Moreover, in traditional cheese- by Hayes et al. (2006) who identified and used 39 making of Pélardon des Cévennes-type cheeses, polymorphisms (SNPs) within the casein genes the goat flavour tended to be less pronounced in to assess the effect of these haplotypes on milk AA milks. The “goaty” flavour in cheese was production traits. Most of these SNPs are located partly due to lipolysis occurring in milk before in the promoter regions of the genes (particularly clotting. On the other hand, they did not observe for CSN3). The numbers of unique haplotypes any association between genetic variants of as1- found in a large population of Norwegian bucks casein and milk yield. in each locus were 10, 6, 4 and 8 for CSN1S1, CSN2, CSN1S2 and CSN3, respectively. The effects of the CSN1S1 haplotypes on protein per-
15 Genetic Polymorphism of Milk Proteins 499 15.6.2 Genetic Variants of Milk Proteins cattle breeds, including Portuguese breeds and for Population and Phylogeny the Austrian breed. Ibeagha-Awemu et al. (2007) Studies demonstrated, using alleles CSN1S2*B and CSN3*H as markers, that zebu-specific attributes Haplotypes at the casein cluster as well as genetic may be more widely distributed in European cat- variants of milk proteins were also intensively tle breeds than expected from previous data. used for characterising breeds and as markers for population and phylogeny studies, establishing Sacchi et al. (2005) investigated the genetic geographical diversity (see review by Ng-Kwai- structure of the casein gene cluster in five Italian Hang and Grosclaude, 2003). Jann et al. (2004) goat breeds to evaluate the haplotype variability investigated the diversity of the casein locus in within and among populations. Goats from the context of the origin and phylogeny of taurine Vallesana, Roccaverano, Jonica, Garganica and cattle, including variants which had not been yet Maltese breeds were genotyped at the four casein the subject of phylogeny studies. The 19 alleles at loci (CSN1S1, CSN2, CSN1S2 and CSN3) using the 5-linked loci (including one SNP in the pro- genomic techniques and milk protein analysis. moter region of CSN1S1) were combined in 83 Allele and haplotype distributions indicated con- haplotypes. Genotyping of 30 cattle breeds from siderable differences across breeds. CSN2 four continents revealed that casein haplotype appeared to be monomorphic for the A allele. frequencies are geographically distributed and The haplotype CSN1S1*F-CSN1S2*F-CSN3*D defined mainly by frequencies of alleles at the occurred in all breeds and was the most common CSN1S1 and CSN3 loci. The genetic diversity haplotype in the Southern breeds. A high fre- within Bos taurus breeds in Europe was found to quency of CSN1S1*0-CSN1S2*C-CSN3*A hap- decrease significantly from the south to the north lotype was found in the Vallesana population and from the east to the west. Such geographic (0.162). Principal component analysis clearly patterns of cattle genetic variation at the casein separated the Northern and Southern breeds. The gene cluster may be a result of the domestication authors suggest that the variability of the caprine process of modern cattle as well as geographi- casein loci and variety of resulting haplotypes cally differentiated natural or artificial selection. should be exploited in the future using specific The comparison of African Bos taurus and Bos breeding programmes aiming to preserve biodi- indicus breeds allowed the identification of sev- versity and to select goat genetic lines for specific eral B. indicus-specific haplotypes not found in protein production. Interestingly, a Maltese goat pure taurine breeds. The occurrence of such hap- heterozygous for CSN2*0 was homozygous for lotypes in southern European breeds also sug- the F allele at CSN1S1 locus. This indicates the gests that an introgression of indicine genes into occurrence of a CSN1S1*F-CSN2*0 haplotype, taurine breeds could have contributed to the dis- which is associated with a very low casein con- tribution of the genetic variation observed. Such tent even if linked to a strong CSN1S2 allele. a hypothesis is substantiated by several studies performed on Portuguese cattle breeds (Beja- A similar investigation was carried out by Pereira et al., 2002), on the Original Pinzgauer Caroli et al. (2006) with local Lombardy breeds red and white cattle breed, from the Pinzgau and confirmed that the casein-haplotype structure region of the federal state of Salzburg in Austria is highly different among breeds. Combining (Caroli et al., 2010) and on 26 breeds from ten haplotype with the molecular knowledge of each countries spanning three continents (Ibeagha- locus, it was postulated that the ancestral haplo- Awemu et al., 2007). It was found in the first two type might be CSN1S1*B-CSN2*A-CSN1S2*A- studies that the CA2 haplotype (abbreviation of CSN3*B. A casein cluster evolutionary model CSN1S1*C-CSN2*A2) which is the most fre- considering the whole casein haplotype was pro- quent haplotype in zebu breeds (Mahé et al., posed, and strong evidence of recombination 1999) also predominates in several European events, not only among but also within casein genes, was found. This is consistent with the interallelic recombination hypothesis put forward
500 P. Martin et al. to account for the occurrence of the M as1-casein 15.7 Milk Proteins Polymorphism variant in the goat species and the postulated and Human Nutrition existence of two ancestral allelic lineages at the CSN1S1 locus (Bevilacqua et al., 2002). Our nutritional perception of milk has grown sub- stantially from a time when it was seen purely as In addition to assess the phylogenetic relation- an excellent source of protein and calcium. Milk ships, genetic polymorphisms of milk proteins proteins provide the suckling neonate with a source have also been used to investigate the genetic of amino acids, highly bioavailable calcium, but diversity within and between populations to also potentially health-promoting bioactive pep- establish geographical diversity. Tadlaoui Ouafi tides (i.e. antimicrobial, antihypertensive, immune et al. (2002) have shown that CSN1S1 alleles modulating). Thus, over the last decade, attention associated with a high expression level (mainly A has shifted from the contribution of milk proteins and B) are predominant in Moroccan goat breeds to human nutrition in terms of essential amino (74% and 94% in Draa and Noire-Rahalli, respec- acids (summarized in Table 15.17) to more specific tively), whereas allele E, which is rather frequent human health issues, not only restricted to the in European goat breeds, is rare (2–3%). neonate, such as the presence of bioactive peptides encrypted in milk proteins and allergy to milk pro- To characterise the diversity within the four teins. Biological activities of peptides released casein genes in two geographically distant goat from milk protein digestion are directly impacted populations, the Sicilian Girgentana breed and by amino acid substitutions or internal deletions the Norwegian goat breed, goats were haplotyped, arising from gene mutations. The existence of based on 22 SNPs and one deletion (Finocchiaro defective alleles associated with a reduced content et al., 2008). The SNP haplotype frequencies for of different caseins is also of interest for the pro- the four casein genes were calculated, and despite duction of hypoallergenic milk with a low casein the large geographical distance and phenotypic content. Since PTMs such as phosphorylation divergence between these two breeds, a propor- seem to reduce the allergenicity of cow caseins in tion of casein loci haplotypes were found to be children with selective allergy to goat and sheep identical between both Norwegian and Girgentana milk (Cases et al., 2011), mutations impacting the goats. The level of linkage disequilibrium between phosphorylation level of caseins might be a selec- the casein genes was less in the Girgentana popu- tion goal. lation than in the Norwegian population. 15.7.1 Bioactive Peptides Milk protein polymorphism was also investi- gated in nine Indian goat breeds/genetic groups Milk proteins are essentially the only source of from varied agro-climatic zones to analyse the amino acids for the newborn mammal and a com- genetic structure of the casein cluster and milk mon source of proteins for adults, but their larger protein diversity at six milk protein loci (Rout physiological significance has started to be et al., 2010). Frequencies of the A allele at the acknowledged only by the end of the twentieth CSN1S1 locus varied from 0.45 to 0.77, and a century. Fiat and Jolles (1989) reviewed the struc- total of 16 casein haplotypes were observed in tural and physiological aspects of caseins and the seven breeds. The distribution of casein haplo- presence of potentially bioactive peptides, but the types was specific to breed and geographical amount of information concerning the biological regions. The average number of alleles was low- effect of these compounds in vivo was still scarce est in Ganjam (1.66 ± 0.81) and highest in Sirohi at the time. At present, milk proteins are regarded goats (2.50 ± 1.05). Expected heterozygosity at as a major source of bioactive peptides, and the six different loci (caseins a-lactalbumin and number of biologically active sequences as well as b-lactoglobulin) demonstrated genetic diversity and breed fragmentation. There was about 17% variability due to differences between breeds; neighbour-joining tree built using Nei’s distance, indicating a strong subdivision.
15 Genetic Polymorphism of Milk Proteins 501 Table 15.17 Supply of essential amino acids (g/100 mL) from bovine, caprine and ovine milka and percentage of Recommended adult Daily dietary Allowances (RDA) fulfilled (g/day) Tryptophan Cow milk % RDA Goat milk % RDA Sheep milk % RDA RDA (g/day) Threonine Supply 8 Supply 8 Supply 16 0.50 Isoleucine 0.04 16 0.04 16 0.08 28 1.00 Leucine 0.16 14.2 0.16 15.8 0.28 24.2 1.40 Lysine 0.2 14.6 0.22 14.6 0.34 27.2 2.20 Methionine 0.32 16.2 0.32 18.8 0.6 32.4 1.60 Cystine 0.26 3.6 0.3 3.6 0.52 2.20 Phenylalanine 0.08 – 0.08 – 0.16 7.2 – Tyrosine 0.04 7.2 0.04 7.2 0.04 – 2.20 Valine 0.16 – 0.16 – 0.28 12.8 – 0.14 13.8 0.18 15 0.28 – 1.60 0.22 0.24 0.44 27.6 Adapted from Haenlein (2001) aAverage composition of milk: cow, 12.01% total solids, 3.29% protein; goat: 12.97% total solids, 3.56% protein; sheep: 19.30% total solids, 5.98% protein the spectrum of activities is constantly increasing. from type-I diabetes mellitus, to atherosclerosis Bioactive peptides are specific protein fragments and coronary-heart diseases, and later to central that have a positive effect on physiological func- nervous system disorders, such as autism. A tions or conditions and might ultimately influence recent report by the European Food Safety health (Kitts and Weiler, 2003). They are said to be Authority (EFSA, 2009) extensively revises the encrypted (latent) within the primary structure or possible implication of BCM7 and other b-caso- parent proteins and need to be released upon pro- morphins in the development of human infirmity teolysis to exert a physiological effect. Their activ- and concluded that “a cause-effect relationship ity is based on their inherent amino acid sequence. between the oral intake of BCM7 or related pep- The size of active peptides may vary from 2 to 20 tides and aetiology or course of any suggested amino acid residues, and many peptides are known non-communicable diseases cannot be estab- to have multifunctional properties (Meisel and lished”. In this respect, caution should be taken FitzGerald, 2003). The origins and biological when claiming the health effects of milk bioac- effects of such peptides have been reviewed by tive peptides, be it beneficial or detrimental. several authors (Shah, 2000; Clare and Swaisgood, Nevertheless, the possible presence of bioactive 2000; Hayes et al., 2007; Korhonen, 2009). peptides encrypted in different milk protein vari- ants was definitely acknowledged and widened Despite the importance of the amino acid the horizons of this research field. sequence of such peptides, little or no attention was paid to the genetic polymorphisms of milk Two interesting examples on this subject were proteins until the outbreak in the late 1990s of the reviewed by Caroli et al. (2009). In the first one, “A2 milk case”, as critically reviewed by Truswell Weimann et al. (2009) compared AA sequences (2005). Briefly, the single amino acid substitution of several bovine k-CN variants and predicted P67H in bovine b-CN A1, compared to A2, the sequence of four different antihypertensive allows the cleavage by pepsin, leucine aminopep- peptides (ASP, variant B and C; VSP, variant F1; tidase and elastase, releasing the opioid peptide AHHP, variant C; ACHP, variant G2). Such pep- known as b-casomorphin-7 (BCM7), correspond- tides were synthesised and their ACE inhibitory ing to the sequence 60–66 of bovine b-CN (Tyr- potency was demonstrated in vitro. In the second Pro-Phe-Pro-Gly-Pro-Ile). In turn, the possible one, Tulipano et al. (2010) investigated the presence of BCM7 in dairy products was alleg- effects of four chemically synthesised peptides, edly associated with an array of diseases, ranging encrypted in bovine caseins (b-and as2-casein)
502 P. Martin et al. and corresponding to genetic variants showing could be a way to theoretically reduce the immu- one amino acid change in their sequence, on nogenicity of milk proteins. Preliminary works osteoblast mineralisation in vitro. Results sug- by Chessa et al. (2008) in silico, suggested that gested that distinct peptides in protein hydro- genetic variability could impact the structure of lysates may differentially affect calcium IgE-binding epitopes structure of milk proteins. deposition in the extracellular matrix and that Moreover, it can be argued that mutations affect- the genetic variation within the considered ing the phosphorylation of caseins could affect sequences may profoundly impact their biolog- the immunogenicity of proteins, as observed in ical activities. More recently, Norberg et al. certain internally deleted variants or when the (2011) assayed the antimicrobial potency of phosphorylation consensus sequence is modified. synthetic homologues of two previously This hypothesis was tested by Bernard et al. reported bovine as1-casein peptides (caseicin A, (2000), who measured in vitro the specific IgE IKHQGLPQE; caseicin B, VLNENLLR) against response to naturally occurring common variants many Gram-negative pathogens and Staphy- of b- and as2-casein, both in the native and lococcus aureus. The effect of single amino dephosphorylated forms, and a purified as2-casein acid substitutions was compared to the original variant D, lacking one major phosphorylation. sequences, and it appeared that the importance Results showed that the IgE response to caseins of specific residues within the caseicin peptides was significantly reduced by modifying or elimi- is dependent on the strain being targeted. nating the major phosphorylation site. Taken together, these findings suggest that An even more interesting strategy, however, milk protein polymorphisms and amino acid sub- is offered by the quantitative polymorphisms stitutions naturally occurring not only in the cat- described in a previous section of this chapter, tle but also in the caprine and ovine species, namely, by the presence of “null” alleles, as greatly increase the possibility of discovering observed in the goat species. In particular, new sequences of biologically active peptides. selected dairy animals can be considered as nat- ural knockout for major milk allergens, as1-, b- 15.7.2 Milk Allergy and as2-casein. Only little attention has been paid, to date, to the possibility of breeding dairy As reviewed by Crittenden and Bennett (2005), animals to target milk protein allergy patients. in spite of the substantial differences in the types Bevilacqua et al. (2001) compared the effect of and sequence of proteins that are present in rumi- diets containing either cow milk proteins or nant and human milk, in most people the immune goat milk proteins with high and low as1-casein system is able to recognise these proteins as content, on a guinea pig model, and analysed harmless and tolerate them. In some individuals, the sensitisation of animals against BLG. however, the immune system becomes sensitised Interestingly, it was observed that animals fed to the milk proteins and mounts a damaging the goat milk proteins diet low in as1-casein had inflammatory response, for reasons that are not significantly low anti-goat BLG IgG1 antibodies. still completely understood. In a recent work, The authors suggested that the high genetic Wal (2004) stated that most of milk proteins may polymorphism of goat milk proteins could be regarded at as potential allergens, as several account for different responses in terms of epitopes, both conformational and linear, have allergy to cow and goat milk. Marletta et al. been described so far. (2004) tested the in vitro allergenicity of milk from goats: homozygous N/N at the CSN1S2 Under this viewpoint, amino acid substitutions locus (N being any allele except O), homozy- could occur in the areas of highly conserved milk gous O/O or heterozygous N/O goats. The protein epitopes described so far, and this could absence or the reduction of as2-CN in goat milk limit their allergenicity. Thus, the selection of decreased but did not erase the allergenic animals on specific qualitative polymorphisms potency of the casein fraction. More recently,
15 Genetic Polymorphism of Milk Proteins 503 Ballabio et al. (2011) assessed the immunoge- tion on loci showing a major effect has to be nicity of milk from goats with different CSN1S1 included. genotypes, both in vitro (SDS-PAGE and immu- noblotting with sera from milk allergic patients) Variation in milk protein composition is due and in vivo (skin prick tests). A lower reaction primarily to mechanisms other than protein was observed with milk samples from goats sequence polymorphisms. Mechanisms, such as homozygous O1/O1 and O1/F at the CSN1S1 transcriptional and translational regulation of locus. Moreover, skin prick tests, carried out on milk protein-encoding genes, contribute to milk six allergic children, were negative to O1/O1 composition variation. Non-coding regions of goat milk. The authors concluded that milk the genome, particularly those with putative from animals carrying CSN1S1 “weak” or “null” regulatory function, have been investigated alleles should be tested in the preparation of intensively, thanks to an easier access to NGS modified formulas for selected groups of allergic technologies, and will be still further explored for patients. mutations (SNP, indels) in the future. During very recent years, the shift from coding to non-coding 15.8 Concluding Remarks sequences is probably the most striking evolution recorded as far as milk protein polymorphism Advances made over the past decade in areas analysis is concerned. Expression takes more such as molecular biology, genomics and pro- and more importance. Mutations altering protein teomics mainly due to major technological prog- structure and expression, and therefore milk ress have greatly accelerated the process of composition and properties, have been found in acquiring knowledge. In particular, the primary coding sequences, promoters, 3¢- and 5¢-untrans- structure of most milk proteins and the genomic lated regions and in intragenic as well as in inter- organisation of the relevant genes have been elu- genic regions. Novel mechanisms of regulation cidated providing access to a better understand- such as those found at the RNA level controlling ing of the mechanisms regulating gene traductibility and stability have been described expression. This information has also opened (e.g. miRNA regulation of human lactoferrin many avenues of research and permits the devel- expression). opment of more powerful selection tools. However, most traits of economic importance in Interestingly, quite the same events have been livestock are either quantitative or complex. found in different species: insertion of repetitive Despite considerable efforts, there have been elements (LINE) in the last non-coding exon of only rare successes in identifying the causal CSN1S1 in goat and cattle and mutations impact- polymorphisms responsible for variation in these ing the splicing process more or less deeply (exon traits. Genomic tools, such as SNP chips, have skipping, cryptic splice site usage). Differential been used to identify genes involved in the splicing, affecting particularly genes whose struc- expression of many traits, including dairy traits ture is highly fragmented, or the occurrence of and to select genetically desirable livestock. This nonsense mutations (premature stop codons) was has led to the discovery of the causal mutations found to be a factor of heterogeneity and struc- for several single-gene traits but not for complex tural diversity as well as quantitative, with some- traits. Genome-wide panels of SNPs have led to times unexpected dramatic consequences. a new method of selection called “genomic selection” in which dense SNP genotypes cover- These mutational events consequently affect ing the whole genome are used to predict the either the coding message, the mRNA stability or breeding value. Even though this approach is both, leading to truncated protein products in expected to double the rate of genetic improve- weak amount or even their absence (null alleles). ment per year in many livestock systems The occurrence of premature stop codons, due to (Goddard and Hayes, 2009), molecular informa- single-nucleotide deletion (CSN1S1 and CSN2 in goat) or to SNP, triggers cellular response (mRNA decay) ensuring improper mRNAs to be degraded. Alleles CSN1S1*G, in goat, and CSN1S1*A, in cattle, both give rise to a messenger lacking exon
504 P. Martin et al. Synthesis of a short β-casein (94 aa-long) Intron Splicing Enhancer Cryptic splice site Promoter region 1 2 34 5 6 78 9 Full lenght β-casein 2 34 6 7 89 (226 aa-long) 1 Lenasi et al., 2006 Skipping / exon 5 218 aa-long β-casein Miranda et al., 2004 Fig. 15.4 A schematic representation of the multiple splic- a cryptic splice site in exon 7 leading to the synthesis of a ing patterns of the horse b-casein encoding gene (CSN2). A short b-casein (94 aa long). It also induces a casual (sto- mutation in a splicing enhancer (yellow circle) within the chastic) skipping of exon 5 (a weak exon) responsible for first intron of the horse b-casein gene induces the usage of an internal deletion of eight amino acid residues 4 which is skipped consecutively to a SNP at Acknowledgements This article is dedicated to the position +1 or +6 in the 5¢ consensus splicing memory of François Grosclaude on his untimely death. donor sequence of intron 4, respectively, result- ing in the upstream exon skipping along the References course of primary transcripts processing, with reduced as1-casein expression. Ådnøy, T., Vegarud, G., Devold, T.G., Nordbø, R., Colbjørnsen, I., Brovold, M., Markovic, B., Roseth, A. Another situation exemplifying the impor- and Lien, S. (2003). Effects of the 0- and F- alleles of tance of the splicing process is provided in the alpha S1-casein in two farms of Northern Norway. equine species by a cis-element in intron 1 Proceedings of the International Workshop on Major (intronic splicing enhancer 1, ISE1) of the gene Genes and QTL in Sheep and Goat, INRA Toulouse encoding b-casein. This splicing enhancer France, communication no. 2–20, December 8–11, increases the inclusion of all weak exons in its pp. 1–5. mRNA and is responsible for a cryptic splice site usage (Fig. 15.4) leading to the loss of more than Alexander, L.J., Hayes, G., Pearse, M.J., Beattie, C.W., a half of the coding sequence (Lenasi et al., Stewart, A.F., Willis, I.M. and McKinlay, A.G. (1989). 2006). Thus, by producing different mRNA forms Complete sequence of the bovine beta-lactoglobulin from a single gene, such mechanisms contribute cDNA. Nucleic Acids Res. 17, 6739. to milk proteome diversity and complexity. Ali, S., McClenaghan, M., Simons, J.P. and Clark, A.J. Nowadays, tools are available and milk pro- (1990). Characterisation of the alleles encoding ovine tein polymorphisms are well documented, at least b-lactoglobulins A and B. Gene, 91, 201–207. for the dairy ruminant species. It remains to gain further insights into the variability of proteins Amigo, L., Recio, I. and Ramos, M. (2000). Genetic poly- from the MFGM as well as quantitatively minor morphism of ovine milk proteins: Its influence on whey proteins and to acquire knowledge on the technological properties of milk—A review. Int. Dairy consequences of described polymorphisms on J. 10, 135–149. biological functions of milk proteins before initi- ating selection for milk production differentiated Andersson, L. and Georges, M. (2004). Domestic-animal according to the targeted goals. genomics: deciphering the genetics of complex traits. Nat. Rev. Genet. 5(3), 202–212. Angiolillo, A., Yahyaoui, M.H., Sanchez, A., Pilla, F. and Folch, J.M. (2002). Characterization of a new genetic variant in the caprine k-casein gene. J. Dairy Sci. 85, 2679–2680. Angulo, C., Diaz Carrillo, E., Munoz, A., Alonso, A., Jimenez, I. and Serradilla, J.M. (1994). Effect of elec-
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Nutritional Quality of Milk Proteins 16 L. Pellegrino, F. Masotti, S. Cattaneo, J.A. Hogenboom, and I. de Noni 16.1 Introduction easily feasible. Few studies have demonstrated significant changes in milk protein composition Milk has been a subject of nutrition research for by balancing AAs in the dairy cows’ diet via many years because it represents a major source supplementation with different proteins or rumen- of nutrients, especially of protein, for populations protected individual AAs, especially lysine and worldwide. Generally, the protein content of methionine. cows’ milk varies among cattle populations, between breeds and between individuals, with Taking into account all of the aspects men- genetics accounting for most of the differences as tioned above, bovine milk contains about 32 g protein content is more highly heritable than protein/L, caseins (~80%) and whey proteins other components. Apart from genetic factors, (~20%) representing the two main fractions. Both natural variations in milk protein content are due protein groups have been recognised as funda- to the stage of lactation, milk production, the mental in maintaining health and well-being, cow’s age, number of lactations and mastitic especially for the newborn, and as important infections. Variations of milk protein content are components of a balanced diet. also related to the cow’s feeding system parame- ters, such as energy intake (positively), lipid In general, a lack of protein will have a nega- supplementation (negatively), protein levels and tive impact, especially when protein requirements their source and additional amino acid (AA) are higher (growth, pregnancy, feeding, ageing, supplementation (in general positively). Many illness), and, for this reason, it becomes indis- attempts have been made to increase protein yield pensable that the daily recommended protein and change the relative synthesis of the indi- intake be assured. Clearly, this would be least vidual proteins. These results would improve problematic for populations in industrialised both the technological and nutritional properties countries. There are new aspects, however, of of milk. However, dietary manipulation of the increasing concern for consumers when choosing amount and the composition of milk proteins is not among different sources of dietary protein, such as cost and the resources needed and the environ- L. Pellegrino (*) • F. Masotti • S. Cattaneo mental impact associated with their production. • J.A. Hogenboom • I. de Noni Dipartimento di Scienze per gli Alimenti, la Nutrizione e Multidisciplinary research programmes are l’Ambiente, Università degli Studi di Milano, increasingly dedicated to investigating policy Milan, Italy options for defining more sustainable diets through e-mail: [email protected] a reduction of environmental pressures related to protein production. Scoring food models based on the nutrient density and taking ecological, eco- nomic and cultural aspects into consideration are P.L.H. McSweeney and P.F. Fox (eds.), Advanced Dairy Chemistry: Volume 1A: Proteins: Basic Aspects, 515 4th Edition, DOI 10.1007/978-1-4614-4714-6_16, © Springer Science+Business Media New York 2013
516 L. Pellegrino et al. approaches being applied to studies of sustainable various functions other than maintaining body and affordable nutrition. Among these models, the protein mass has appeared in literature. Among Nutrient Rich Foods Index was used in conjunc- several metabolic actions, Millward et al. (2008) tion with a food price database to rank different reported the regulation of body composition, food groups (Drewnowski, 2010). Using this bone health, gastrointestinal function and bacte- approach, milk proved to have the lowest overall rial flora, glucose homeostasis, cell signalling nutrient-to-price ratio and to be the lowest-cost and satiety. Meanwhile, it is well known that source of calcium, whereas milk products, along protein deficiency depresses immune function with eggs and legumes, were the lowest-cost and defence against diseases. A deficiency of sources of protein. protein reduces the plasma availability of most AAs, especially glutamine, arginine, methionine, 16.2 The Nutritional Quality tryptophan and cysteine, whose roles in enhanc- of Dietary Proteins ing immune function have been well established (Li et al., 2007; Tan et al., 2009; Wu, 2009), The nutritional quality of a dietary protein is the whereas high-protein diets may increase the expression of its bioavailability and metabolic acidity of the urine leading to an increased loss utilisation. In other words, it represents the capac- of minerals, in particular magnesium and calcium ity of a food protein to achieve defined nutritional (Remer, 2001). and metabolic actions related to a specified requirement. The evaluation of protein quality Currently, the N balance method, which con- allows one to establish a ranking of food sources sists of measuring the difference between protein on the basis of their potential nutritive value and intake and loss, is the favoured approach for esti- to predict the contribution of a protein to satisfy mating protein requirements in humans. A detailed the nitrogen (N) and AA requirements for growth definition of protein requirements was provided by and maintenance of the human body. The classic the FAO/WHO Ad Hoc Expert Committee (1973), approach to protein quality evaluation involves which defines it as ‘the lowest level of dietary pro- the coverage of human requirements to be tein intake that will balance the losses of N from assessed for each food source separately. Although the body, and thus maintain the body protein mass, such a concept is useful in terms of protein in persons at energy balance with modest levels of classification or measuring the effect of processing physical activity, plus, in children or pregnant/lac- on the nutritional quality of single proteins, no tating women, the needs associated with the depo- prediction of diet quality can be made on this sition of tissues or the secretion of milk at rates basis. However, the evaluation of dietary protein consistent with good health’. The study of Rand quality should involve multiparametric studies and Young (1999), consisting of a meta-analysis of taking into account both the composition of the previously published data for the estimation of diet and the effect of the interaction of proteins protein requirements in healthy adults, is the basis with other components. At present, protein quality for the most recent international recommenda- is determined from growth measurements, protein tions. The FAO/WHO/UNU Expert Consultation and N digestibility data, N balance, N retention or (2007) proposed an estimated ‘average require- protein turnover at a whole-body level or in ment’ of 0.66 g protein/kg body weight/day to specific tissue protein pools (Tomé, 2010). achieve a zero N balance in healthy adults. Taking into account the interindividual variability, a ‘safe 16.2.1 Protein Requirements level of intake’ of 0.83 g protein/kg/day is expected to satisfy the requirements of most (97.5%) of the The understanding of proteins’ actions has healthy population. Such requirements must be improved through the years, and evidence of integrated with additional amounts of protein for proteins’ complex roles in the regulation of children and pregnant/lactating women. In fact, the dietary requirement is the amount of protein that must be supplied in the diet to satisfy the met- abolic demand and should, therefore, take into
16 Nutritional Quality of Milk Proteins 517 Fig. 16.1 Schematic representation of the metabolic demands for amino acids account such factors as biological value and digest- Nutritionally, DAAs can be synthesised in ade- ibility of the protein. The representation of the quate amounts by the body in any situation, metabolic demands in Fig. 16.1 explains why bal- whereas CIAAs are those that can normally be ance methods are extremely difficult to apply and synthesised in adequate amounts by the organism thus are imprecise. Milk proteins play a major role but that must be provided by the diet to meet opti- in ensuring that this intake is met, and by way of mal needs under particular conditions when their example, a survey by the French Agency for Food utilisation is more rapid than their synthesis. This Safety (AFSSA, 2007) indicated that milk and classification is of the utmost importance because milk products supplied 20.6% and 17.2% of the bioavailability level of IAAs in a protein dietary proteins for children (4–14 years) and source is the key factor in determining its nutri- adults, respectively. Despite the many unanswered tional value. The mixture of dispensable AAs and nutritional issues, milk proteins provide a wide CIAAs as supplied by adequate intake of food range of important functional and biological prop- proteins will assure that both the N and specific erties that are continuously being discovered and AA needs are met. for which scientific evidence is accumulating. Besides their fundamental function as build- 16.2.2 Amino Acid Requirements ing blocks for proteins, AAs also act as cell sig- nalling molecules and regulate numerous Although over 300 AAs exist in nature, only 20 important metabolic pathways necessary for of them, all in the l configuration, are used as maintenance, growth, reproduction and immu- structural units for building proteins. Nevertheless, nity. Furthermore, they are key precursors of hor- it has been demonstrated that nonprotein a-AAs mones and other substances of great biological (like ornithine, citrulline, homocysteine), as well importance, like polyamines, glutathione, tau- as non-a AAs (like taurine or b-alanine), play rine, nitric oxide and serotonin. Tyrosine, for important metabolic roles (Perta-Kajan et al., example, is the precursor for the synthesis of epi- 2007; Manna et al., 2009). nephrine, norepinephrine, dopamine and thyroid hormones, whereas glutamine and leucine Amino acids are classified as nutritionally increase insulin release from pancreatic b-cells indispensable (IAA), dispensable (DAA) or con- (Newsholme et al., 2005). An excess or deficiency ditionally indispensable (CIAA) for humans of even one single AA can disturb whole-body (Table 16.1). Indispensable AAs, all present in homeostasis, can provoke problems of growth milk proteins, are defined as either those AAs and development and may in some cases lead to whose carbon skeletons cannot be synthesised or death (Orlando et al., 2008; Willis et al., 2008). those that are inadequately synthesised by the body and therefore must be provided by the diet. Some AAs have metabolic roles proportional to dietary intake. For instance, there is a dose– response relationship between IAA concentration
518 L. Pellegrino et al. Table 16.1 Indispensable, dispensable and conditionally requirements for IAAs, together with the AA dispensable amino acids in the human diet content of some food sources, are shown in Fig. 16.2. Indispensable Dispensable Conditionally Histidine Alanine indispensable A detailed description of individual AA Leucine Aspartic acid Arginine requirements for humans at different ages is Isoleucine Asparagine Cysteine reported in the Report on Dietary Reference Lysine Glutamic acid Glutamine Intakes of the US National Academy of Sciences Methionine Serine Glycine (2005) and critically discussed in the review by Phenylalanine Proline Boutry et al. (2008). Threonine Tyrosine Tryptophan 16.3 Methods for Evaluating Valine the Nutritional Quality of Dietary Proteins in the blood and muscle protein synthesis, the lat- ter being maximally stimulated with a postexer- Assessing protein quality with respect to its cise consumption of IAAs at a dose of efficiency in supporting body protein metabolism approximately 10 g/day. In particular, leucine has should firstly take into consideration the capacity been demonstrated to be a key activator of muscle of the protein to provide a suitable source of N and protein synthesis (Phillips, 2011). IAAs. For this purpose, the nutritional quality of dietary proteins was classically evaluated with the The small intestine is a major site for exten- chemical score (CS) obtained by calculating the sive catabolism of IAAs in humans and animals content of each IAA of a food source as a percent- (Stoll et al., 1998), whereas branched-chain AAs age of the same AA in a reference protein, such as (BCAA) are primarily metabolised in skeletal egg, which is regarded as being well balanced in muscle, where they serve both as an important AA content in relation to human needs. Compared energy substrate and as precursors for the synthe- with egg, human milk shows a CS of 100% and sis of other AAs and proteins (Platell et al., 2000). cow milk of 95%, with methionine and cysteine BCAAs serve as an oxidative fuel source and being the limiting IAAs. Although this method is stimulate the synthesis of glutamine, an impor- fast and cost-effective in comparison to in vivo tant nutrient for rapidly dividing cells, especially measurements, its shortcoming is that protein in the gut and immune system. In the brain, digestibility and AA bioavailability are not taken BCAAs also act as amino group donors in the into consideration. Another limitation is that pro- synthesis of glutamate, the major excitatory neu- tein metabolism may be influenced by the excess rotransmitter of the mammalian nervous system of other AAs or by the presence of anti-nutritional and the most important AA neurotransmitter. At factors in the food. least one third of the amino groups of brain gluta- mate are derived from BCAAs, with leucine A significant improvement in the routine assess- donating no less than 25% (Yudkoff, 1997). ment of dietary protein quality took place with the introduction of the PDCAAS (FAO/WHO, 1991). The scientific community has reached some In this case, the CS is calculated for the first limit- consensus on IAAs requirements for men (FAO/ ing AA in the test protein with respect to that of a WHO/UNU, 2007), whereas in women, infants, reference (scoring) pattern. The reference pattern children and the elderly, IAAs requirements need is derived from the IAA requirements of 1–3-year- to be studied further. This aspect is of key rele- old children, to cover all ages from 1 year on vance because the indices used for evaluating the (Schaafsma, 2000). The value is then corrected for nutritional quality of dietary proteins are based protein digestibility, which is determined in rats on their IAA composition with reference to the over a 5-day period. A value >100%, measured in recommended requirements. The renewed series most animal proteins, means that the protein source of recommended (FAO/WHO/UNU, 2007)
16 Nutritional Quality of Milk Proteins 519 Fig. 16.2 Distribution of indispensible amino acids branched-chain AAs (leucine, isoleucine, valine); AAAs (IAAs) in food proteins and current dietary IAA require- aromatic AAs (phenylalanine, tyrosine); SAAs sulphur ments recommended by the FAO/WHO/UNU (2007) AAs (cysteine, methionine)) (Source: FAO/WHO/UNU, 2007; Tomé, 2010. BCAA provides the IAAs at a level higher than the by the FAO/WHO, whereas it has been recom- requirements. However, calculated scores >100% mended that the anti-nutritional factors affecting are truncated so that the capability of a high- protein digestibility, including those formed dur- quality protein to compensate proteins deficient in ing processing, should be investigated further. one or more IAA is underestimated. For instance, milk has a non-truncated PDCAAS = 120%, 16.3.1 Protein Digestibility casein = 123% and whey protein = 115% (Phillips, 2011), but no discrimination among these products A second important issue in nutritional quality eval- is possible if all values are truncated to 100%. As a uation of a protein relates to its bioavailability, or consequence, this approach underestimates the digestibility, or the capacity to provide metaboli- power of a high-quality protein to balance the IAA cally available N and AAs to tissues and organs. composition of other proteins and thus fails to be useful as a comparative tool. Protein digestibility can be evaluated by mea- suring the free AA (FAA) or specific soluble N Despite these limitations, the PDCAAS has fractions released from the test protein after been considered the most suitable regulatory in vitro digestion with one or more proteolytic method for evaluating the protein quality of foods enzymes under conditions mimicking the in vivo and infant formulae. Because this method is digestive process. Several procedures have been based on human AA requirements, it is inher- proposed (Mandalari et al., 2009), making it ently more appropriate than animal assays used difficult to compare results from different authors, for predicting the protein quality of foods (FAO/ whereas a standardised protocol is under study WHO, 1991). Since 1993, the Food and Drug within a project funded by the European Administration adopted the PDCAAS as a stan- Commission (Food and Agriculture COST dard method to calculate protein quality. The Action, 2011). Much of the interest in simulated validity of the PDCAAS method was endorsed
520 L. Pellegrino et al. digestion studies comes from the possibility of ratio (PER), net protein ratio (NPR), biological assessing the allergenic potential of either intact value (BV) and net protein utilisation (NPU) proteins or the released peptides. This aspect will (Pellett and Young, 1980). The PER in rats repre- be addressed in the following sections. sents the weight gain per gram of protein con- sumed, evaluated over a period of 28 days. The classical approach for calculating protein Although time-consuming, the PER has been digestibility is based on N balance determined used extensively for ranking dietary protein in vivo both in animal models (Gaudichon et al., sources and for calculating the US Recommended 1994) and in humans (Bos et al., 1999; Gaudichon Daily Allowance (USRDA) for labelling regula- et al., 1999; Mariotti et al., 2001). Basically, the tions. The PER has been criticised for not meet- difference between the amounts of ingested and ing the criteria of a valid routine test and because excreted N, expressed relative to ingested N, is modifications in the total food intake give rise to obtained through measurements repeated over an increase in its levels, thus reducing the capa- several days. The excreted N can be calculated bility to compare proteins. from the faecal N (‘apparent faecal digestibility’, including the N metabolism of the microorgan- The calculation of the NPR differs from that isms present in the large intestine) or from digesta of the PER in that a protein-free control group is sampled at the end of the small intestine (‘ileal considered. In fact, the NPR is obtained by add- digestibility’) (Darragh and Hodgkinson, 2000). ing the weight gain, per g of protein consumed, With respect to milk, Bos et al. (1999) recorded of rats fed the test protein and the weight loss of similar values of faecal and true ileal digestibility the animal group fed the protein-free diet. This (96.6% and 95.5%, respectively) in human sub- parameter, measured over a 2-week period, jects. In any case, ileal digesta also contain a although characterised by a high relationship to significant proportion of non-dietary AAs from protein quality, has the limitation of being a sin- mucus, cells, digestive enzymes and bile (endog- gle-dose method (Boutrif, 1991). Both the NPR enous N), which should be quantified in subjects and the PER values can be corrected to a 0–100 consuming a protein-free diet. In this regard, a scale, thereby providing the corresponding rela- more reliable technique, whose limitation is its tive RNPR and RPER indices. high cost, is based on the use of proteins labelled with stable isotopes (15N or 13C). Intrinsically The percentage of N /Nretained absorbed gives the labelled animal or plant proteins can be BV, which is an expression of the utilisation of obtained and adopted for studies on humans the absorbed dietary N, whereas the NPU index (Pennings et al., 2011). In this way, it is possible allows the evaluation of the ingested amount of N to calculate the ‘true faecal digestibility’ as that is retained (NPU = Nretained/Ningested). True [Ningested − (Nfaeces − Nfaecal endogenous)]/Ningested. digestibility is reflected in this parameter, being NPU = BV × digestibility. The NPU is assessed The true faecal digestibility of protein is con- from long-term balance measurements in sidered a good approximation of the bioavailabil- humans. ity of AAs of properly processed food products (FAO/WHO, 1991). As shown in Table 16.2, milk The whole-body protein balance method, and milk products (like other animal proteins) are whose classic parameter is the PDCAAS, has highly digestible by humans in comparison to been criticised because of the existence of a plant proteins. diurnal cycling of N retention and loss between the fasted and fed states, which leads alternately 16.3.2 Efficiency of Protein Utilisation to postprandial N accretion and post-absorption N loss phases (Bos et al., 2000). The acute utili- The results of the in vivo tests to evaluate the sation of N from a dietary protein may be evalu- efficiency of protein utilisation can be expressed ated by N deposition in the postprandial phase. through such parameters as the protein efficiency As the postprandial phase is critical for dietary protein utilisation, the measurement of the immediate retention of dietary N following meal
16 Nutritional Quality of Milk Proteins 521 Table 16.2 True faecal digestibility and PDCAAS values of milk, milk products and other dietary sources Protein source Digestibility (%) PDCAAS (%) Milk and milk products Milk 94a–95b,c 100b Casein 95b,c–99a 100b Whey protein 98b 100b Whey protein concentrate 100d 100d Whey protein hydrolysate 99d 100d Skim milk powder 93d 100d Others Egg 97b,c 100b Beef salami 99e 100f Tuna 94e 100f Soy 91b 99b Wheat 86b 54b Rice 88b 55b Sources: aSarwar (1997), bAFSSA (2007), cSarwar Gilani et al. (2005), dGilani and Sepehr (2003), eSarwar et al. (1989), and fSchaafsma (2005) ingestion (net postprandial protein utilisation, 16.4 Nutritional Quality of Proteins NPPU) represents a reliable approach for evalu- in Milk and Milk Products ating dietary protein efficiency (Tomé, 2010). Assessment of the NPPU of dietary proteins Cow milk is considered a source of dietary pro- may represent an appropriate approach for the tein with excellent nutritional value (Tomé, further validation of the PDCAAS scoring 2010). Such quality, ascribed to the supplementa- method because the latter parameter is known to tion of both N and IAAs necessary to meet human be influenced by protein turnover. The non-trun- requirements, is also attributable to the presence cated PDCAAS values reported by Tomé and in milk of a wide range of biologically active pro- Bos (2000) for milk, soy, pea and wheat (120, teins and peptides having specific nutritional and 99, 73 and 36, respectively) and the NPPU val- physiological properties (Bosze, 2008). However, ues reported by Fouillet et al. (2002) for the technological processes used in the manufacture same proteins (75, 71, 71 and 62, respectively) of milk products may impair this value. All of are in quite good agreement, confirming the these topics are addressed in the following capability of the NPPU method to discriminate sections. protein quality (Reeds et al., 2000). The mea- surement of short-term retention of dietary N 16.4.1 Amino Acid Composition (Bos et al., 1999) is made possible by the use of 15N-labelled proteins. The AA composition of milk proteins is reported in Table 16.3. The IAA composition of milk pro- Tomé (2010) reported that, in humans adapted teins is similar to that of other animal proteins, to a normal protein diet (1 g/kg/day), about such as egg, but with limited amounts of sulphur- 65–80% of dietary N is retained 12 h after a containing AAs (SAA). Despite this limitation, mixed meal. In particular, 30–50% is distributed the IAA pattern of milk abundantly covers the in the splanchnic tissues (i.e. intestine, liver, etc.) requirements defined by the FAO/WHO/UNU to and 20–30% in peripheral tissues (muscle, skin, satisfy AA needs in adults. The IAA content of etc.). The wide range of reported values is due to milk proteins, expressed as a percentage of the the different kinetics of dietary N distribution in daily IAA requirement, when protein is supplied the anabolic and catabolic pathways of different tissues after protein ingestion.
522 L. Pellegrino et al. Table 16.3 Amino acid composition (% of total AAs) of milk proteins Whey protein 10.5 Amino acid Casein 7.0 Aspartic acid/asparagine 7.1 4.8 Threonine 4.9 17.6 Serine 6.3 5.9 Glutamic acid/glutamine 1.8 Proline 22.4 4.9 Glycine 11.3 2.3 Alanine 1.7 Cysteine 2.7 5.7 Methionine 3.0 6.4 Valine 0.34 10.3 Isoleucine 2.8 2.9 Leucine 7.2 3.1 Tyrosine 6.1 2.4 Phenylalanine 9.2 8.7 Tryptophan 6.3 1.7 Lysine 5.0 2.3 Histidine 1.7 Arginine 8.2 3.1 4.1 at the mean requirement (i.e. 0.66 g/kg/day), Knowing the nutritional and physiological roles demonstrates that milk represents a well-balanced of the different AAs, it would be possible to food source. In particular, the Expert Consultation develop functional foods based on milk proteins of the FAO/WHO/UNU (2007) calculated that having AA compositions suitable to meet the lysine, SAAs and aromatic AAs (AAA) represent needs of people interested in controlling diabetes, more than 150% of the daily requirements and losing fat or building muscle protein (Etzel, tryptophan is the most relatively abundant IAA 2004). a-La, for example, has a high cysteine (417% of the required value). Furthermore, milk content, and b-lg is rich in glutamine, whereas can fully satisfy all IAA requirements when GMP has an extraordinarily high threonine con- ingested as the only protein source (Reeds et al., tent and completely lacks cysteine, histidine, 2000), and in a mixed diet, milk can be consid- arginine, tyrosine, tryptophan and phenylalanine. ered complementary to dietary sources lacking It has been demonstrated that GMP, when supple- threonine and especially lysine, such as grains. mented with IAAs, may represent a safe and highly acceptable primary protein source in the Milk proteins consist mainly of casein (~80%) nutritional management of phenylketonuria (Ney and whey protein (~20%), having different AA et al., 2009). compositions (Table 16.3). In particular, whey protein has higher amounts of SAAs (methionine Among the different protein sources, milk is and cysteine), lysine, threonine and tryptophan relatively abundant (21%) in BCAA, and leucine (Fig. 16.3). Moreover, whey proteins have a represents 10% of the total AAs and approxi- slightly higher true ileal digestibility for all AA. mately 50% of the BCAAs (Table 16.4). In fact, whey protein is a mixture of a-lactalbu- min (a-la), b-lactoglobulin (b-lg), immunoglob- As already mentioned, in addition to protein ulins (Ig), blood serum albumin (BSA) and synthesis, BCAAs play important roles in many perhaps other components, including glyco- metabolic functions. Specifically, leucine is a key macropeptide (GMP) (which is cleaved from activator in regulating the turnover of muscle k-casein by chymosin during cheese production), proteins (Rennie et al., 2006). Because of their each of which has a distinct AA composition. high content of leucine, compared with other pro- teins (Table 16.4), milk and whey proteins are
16 Nutritional Quality of Milk Proteins 523 Fig. 16.3 Distribution of indispensible amino acids recommended by the FAO/WHO/UNU (2007) (Source: (IAAs) in milk, milk protein fractions and glycomacro- FAO/WHO/UNU, 2007; Boutrif, 1991; USDEC, 2011; peptide (GMP) in comparison to human requirements Etzel, 2004) Table 16.4 Content of leucine and branched-chain casein and soy (Phillips, 2011). These findings amino acids (BCAA) (leucine, isoleucine and valine) in are apparently in contrast with those obtained by milk and other food sources whole-body measurements (Lacroix et al., 2006), but it is useful to recognise that only 25% of the Protein Leucine BCAA whole-body response is due to muscle protein. Whey isolate (g/100 g protein) Currently, hydrolysed whey protein and/or casein Milk 14 26 is largely used in the formulation of sports/recov- Egg 10 21 ery integrators. Soy isolate 8.5 20 Navy beans 8 18 The SAAs, methionine and cysteine, which Whole wheat 16 represent approximately 3% of milk proteins, are flour 7.6 15 important for the synthesis of proteins in the 7 immune system (Grimble, 2006). Sources: Layman and Baum (2004) (source: USDA Food Methionine is a methyl group donor that par- composition tables) and Young and Pellet (1990) ticipates in the methylation of DNA and proteins, the synthesis of spermidine and spermine and the receiving much attention for the nutrition of ath- regulation of gene expression (Wu et al., 2006). letes (Phillips, 2011) and also in clinical nutrition Furthermore, methionine is a substrate for the of the elderly to prevent sarcopaenia (Paddon- synthesis of choline and, thus, phosphatidylcho- Jones and Rasmussen, 2009). The main unan- line and acetylcholine, which are essential for swered questions in this regard are how the source nerve function and leucocyte metabolism (Kim of dietary protein and the degree of hydrolysis of et al., 2007a). Cysteine is the precursor of gluta- the ingested protein can affect increases in mus- thione (GSH), which scavenges free radicals (e.g. cle protein synthesis. Consumption of hydrolysed hydroxyl radical ) and other reactive oxygen spe- whey protein results in a rapid and pronounced cies (e.g. H2O2), and conjugates with various rise in aminoacidaemia and leucinaemia, com- electrophiles and xenobiotics for their pared with isonitrogenous quantities of both detoxification (Fang et al., 2002). According to
524 L. Pellegrino et al. Fratelli et al. (2005), the intracellular concentra- well-balanced AA composition with respect to tions of GSH play an important role in regulating human needs. The data in Table 16.6 suggest that cellular signalling pathways in response to immu- the type of protein can specifically influence the nological challenges. Furthermore, GSH concen- kinetics and the profile of AA delivery, which trations in antigen-processing cells modulate influences protein metabolism and N retention. immune responses, including antibody produc- tion (Peterson et al., 1998). Nowadays, a number The quality of selected milk protein fractions of whey products rich in BCAAs and SAAs are has been assessed using indices based on rat commercially available, including whey protein growth (Table 16.7). concentrates, whey protein isolate, reduced lac- tose whey, demineralised whey and hydrolysed The RNPR values were higher than those of whey, and many clinical trials have demonstrated RPER because the former index considers pro- the efficacy of these products in the treatment of tein used for both growth and maintenance. The several diseases, such as cancer, AIDS, hepatitis PDCAAS values of the different milk protein B, cardiovascular disease and osteoporosis fractions are identical, all of them being truncated (Marshall, 2000). Whey protein also acts as an to 100. Casein integrated with methionine antimicrobial agent and enhances athletic increases the scores for all of the indices. performance. Casein and whey protein fractions are digested Bovine milk contains a small amount of FAAs, differently. Casein shows a delayed gastric empty- which represent a ready source of substrate for ing due to its clotting in the stomach. In contrast, bacterial growth. Table 16.5 reports the FAA con- whey protein remains soluble and is evacuated more tent in raw bovine milk compared to that of rapidly so that AA delivery to the gut is more rapid. human milk as reported by Agostoni et al. (2000). As a result, casein provokes a smaller postprandial Human milk contains five times the amount of increase in plasma AA compared with the non- FAAs in bovine milk and is thus more easily coagulating whey protein. To describe the digestion metabolised by the newborn. and absorption of casein and whey proteins, Boirie et al. (1997) introduced the concept of ‘slow’ 16.4.2 Nutritional Quality, Digestibility (slowly digested and absorbed) and ‘fast’ (rapidly and Utilisation digested and absorbed) proteins. These authors reported that slow proteins, such as casein, sustain a The nutritional quality of milk proteins, as well better N utilisation than fast proteins and are less as that of its two main fractions (i.e. casein and satiating. Deglaire et al. (2008) recorded a similar whey protein), has been evaluated by several nutritional value for intact casein (a slow protein) authors using different indices. The measured and hydrolysed casein (a fast protein), in agreement values are compared with those obtained for other with the N balance studies of other authors (Sales dietary proteins in Table 16.6. With respect to et al., 1995) but apparently in contradiction with the grains and legumes, milk proteins are character- results of Boirie et al. (1997). Indeed, the data ised by higher BV values, suggesting a better reported by Deglaire et al. (2008) suggest that the utilisation of the absorbed dietary N, and higher correlation between digestion/absorption rates and NPU values, which reflect true digestibility. net protein retention is more complex than that Furthermore, all of these indices confirm the based on the slow/fast protein concept. Lacroix higher nutritive value of the whey protein frac- et al. (2006) investigated the postprandial kinetics tion with respect to casein. of dietary N after the ingestion of different milk pro- tein fractions in humans. The authors demonstrated Milk proteins are characterised by a high that the stimulation of peripheral anabolism under NPPU in humans, that is, the amount of dietary N the examined conditions is unlikely to occur after retained in the body after milk protein ingestion, ingestion of whey protein isolate, despite its high showing that milk proteins are characterised by a leucine content (Table 16.4). A synergistic effect between casein and whey protein in terms of meta- bolic utilisation was observed. Indeed, Lacroix et al.
16 Nutritional Quality of Milk Proteins 525 Table 16.5 Average free amino acid content (mg/L) in bovine and in human milk Human milkb (n = 40) 24.4 Amino acid Bovine milka (n = 12) 11.6 28.8 Aspartic acid 3.3 Threonine 1.4 n.d. Serine 1.0 174.2 Asparagine 0.1 41.6 Glutamic acid 40.0 9.4 Glutamine 1.4 20.3 Glycine 6.3 Alanine 3.5 8.5 Valine 3.2 1.3 Methionine 0.1 4.4 Isoleucine 0.5 7.3 Leucine 0.8 0.5 Tyrosine 1.0 3.9 5.7 Phenylalanine 0.5 1.2 Lysine 3.0 6.2 Histidine 0.4 7.4 Arginine 2.7 356.7 Proline 2.1 Total free amino acids 71.3 Sources: aPersonal data and badapted from Agostoni et al. (2000) Table 16.6 In vivo indices of protein quality in milk and other food sources Protein source PER BV % NPU % NPPU % humans Milk 2.5a 91a–93b 77c–82c,a 75d–81d,c Casein 2.5a 75b–77a 76a–79c 71d Whey protein 3.2a 100b 92a–95c 64d Others 3.9a 100b 94a Egg Beef 2.9a 75b–80a 73a 73d Soy 72d Wheat 2.2a 74a 61a 66d 0.8a 64a 67a Sources: aHoffman and Falvo (2004) and FAO-WHO (1973), bNational Research Council (1989), cBos et al. (1999), and dAFSSA (2007) PER protein efficiency ratio; BV biological valve; NPU net protein utilisation; NPPU net postprandial protein utilisation Table 16.7 Values of protein quality indices of selected milk protein fractions Protein source PER NPR RPER (%) RNPR (%) Skim milk 3.7 4.7 77 82 Casein 3.9 4.8 80 84 Casein + met 4.8 5.7 100 100 Lactalbumin 4.3 5.2 89 91 Sources: Sarwar (1997) and Gilani and Sepehr (2003) PER protein efficiency ratio; NPR net protein ratio; RPER relative protein efficiency ratio; RNPR relative net protein ratio
526 L. Pellegrino et al. (2006) measured that the dietary N utilisation in a In vitro studies (Syndayikengera and Xia, 2006) milk-based meal was better, or at least identical, to on sodium caseinate and whey protein concen- that in the casein and whey protein meals. The trate showed an improved enzymatic digestibility higher value of the milk-based meal was suggested (by pepsin/pancreatin) of both hydrolysed forms. by the authors to be due to the combination of an Specifically, in terms of AA composition, CS and early metabolic and hormonal stimulation by the BV, whey protein concentrate and its hydrolysate whey protein fraction and a sustained delivery of have higher nutritional values than do sodium AAs from casein. A better NPPU in terms of muscle caseinate and its hydrolysate. Boza et al. (1994), protein accretion seems to be induced by whey pro- comparing the nutritional values of whey protein teins compared to either casein or casein hydro- and casein with their respective hydrolysates, lysate in healthy older men (Pennings et al., 2011) concluded that enzymatic hydrolysis per se does and athletes (Phillips, 2011). Such results have led not significantly decrease the nutritional value of to whey proteins becoming an important nutritional milk proteins. Actually, hydrolysis seemed to and functional food ingredient, with extensive use improve the NPU and BV of whey proteins. in such food applications as sport beverages, meat replacement products and energy bars. 16.4.3 Effects of Processing Fouillet et al. (2002) developed a compartmen- Nowadays, milk proteins are introduced in the tal model mimicking dietary N absorption, elimi- human diet almost exclusively via processed milk nation and distribution throughout the body after products. Thus, evaluation of the nutritional qual- the ingestion of a meal containing either milk or ity of milk proteins must consider the effects of soy. A lower whole-body N retention of soy pro- processing treatments on the bioavailability of teins compared to milk proteins was observed. The both N and AA. NPPU values 8 h after the meal were 80% and 72% for milk and soy, respectively. In addition, the This section deals with the impact of the most kinetics of dietary N absorption and subsequent common technological treatments employed in transfer to peripheral and splanchnic tissues was milk processing (Table 16.8) on the nutritional differentially affected by the protein source. Milk quality of milk proteins in the finished products. caused lower ileal losses and splanchnic oxidation in comparison to soy. According to the predictions 16.4.3.1 Heat Treatments of the model, 8 h after a meal, dietary N incorpo- Essentially all milk products are submitted to rated into proteins differed significantly for milk heat treatments during processing, with the aim and soy meals, being 26% and 19% of the ingested of ensuring safety and stability, as well as for N, respectively. Actually, Fouillet et al. (2002) many other technological reasons. Moreover, in ascribed the higher efficiency of peripheral protein addition to heating during processing, storage synthesis after milk ingestion to the higher propor- may be regarded as an additional heat treatment, tion of BCAAs. Similarly, Kimball and Jefferson especially for those milk products (e.g. sterilised (2001) hypothesised a higher amount of BCAAs, or powdered milk, infant formulae) usually which are known to be stimulators of muscle pro- stored at room temperature for an extended tein anabolism, in the peripheral area after milk period of time. ingestion. Bos et al. (2003) concluded that the dif- ferences in the NPPU of milk and soy proteins are The heating of milk promotes several reactions mainly due to differences in digestion kinetics but that induce protein modifications, which may that AA composition may also play a role. affect the nutritional quality of milk. From a nutri- tional point of view, the most important changes Milk protein hydrolysates are largely used in induced by heat are related to (1) protein denatur- the formulation of clinical diets in the case of ation, (2) protein glycation, (3) b-elimination reac- food allergies caused by intact protein epitopes. tions, (4) AA racemisation and (5) formation of In vivo studies demonstrated that hydrolysis does isopeptide bonds (Pellegrino et al., 1995). not affect the nutritional quality of milk proteins.
16 Nutritional Quality of Milk Proteins 527 Table 16.8 Main processes applied in dairy industry humans. This study revealed that pasteurised milk had a higher NPPU value than that mea- Milk product Process sured in UHT milk (76.2% vs. 68.3%). On the Drinking milk Pasteurisation other hand, Singh and Creamer (1993) high- UHT lighted an easier proteolysis of whey proteins Cheese In-bottle sterilisation when they lose the native globular structure. It Homogenisation has been reported (Hiller and Lorenzen, 2010) Processed cheese Pasteurisation of cheese that glycation by complex sugars (i.e. dextran) Fermented milk milk may facilitate the unfolding of b-lg, resulting Powdered milk products Coagulation in easier access to peptide bonds by prote- Fermentation olytic enzymes. Lacroix et al. (2008) observed Ripening that the digestive kinetics are more rapid for Melting UHT milk than for pasteurised milk, leading Sterilisation to an increased transfer of dietary N into Heat treatment of milk plasma protein or urea. Indeed, interaction Fermentation between whey protein and casein upon heat Heat treatment treatment results in a softer casein coagulum Concentration by and in a higher susceptibility to proteolytic evaporation enzymes, with a consequent acceleration of Drying protein digestion. Moreover, a large increase in very small easily digestible casein micelles 1. Among milk proteins, thermal denaturation in UHT milk could explain the improvement affects mainly whey proteins. No structural of protein digestibility. The improving effect modifications of casein occur upon heating at of heat treatment on milk protein digestibility the conditions usually applied in industrial was also pointed out by Kim et al. (2007b). processing as the protein lacks secondary and 2. Upon heating, milk protein lactosylation tertiary structures. Upon heating, the native induced by the Maillard reaction (MR) may globular structure of whey proteins undergoes occur. The MR is considered the most impor- unfolding, exposure of thiols and consequent tant mechanism impairing the quality of milk formation of both whey protein-whey protein proteins during the different technological treat- and whey protein-casein aggregates. The ments. The main nutritional consequence of the effect of heat-induced denaturation on the MR in milk is the loss of bioavailable IAAs, digestibility of milk proteins has been evalu- among which lysine is the most affected, due to ated in milk products submitted to different both its abundance in milk proteins and the high heat treatments (pasteurisation, UHT sterilisa- reactivity of its e-amino group towards lactose. tion, in-bottle sterilisation) by many researches During heat treatment of milk, lysine is blocked using different criteria and conditions. This in the Amadori compound (lactulosyl-lysine in may partly explain why contradictory results milk) deriving from the condensation reaction are reported in the literature. Some authors between the e-amino group of lysine and the (Rudloff and Lonnerdal, 1992; Carbonaro glucose moiety of lactose. Characteristic levels et al., 1996; Alkanhal et al., 2001) reported of blocked lysine in heated milk and in dairy that the formation of protein aggregates via products are reported in Table 16.9. These data direct covalent interactions and the consequent show that the loss of bioavailable lysine can be loss of solubility seem to decrease protein considered negligible in pasteurised or UHT digestibility, mainly because digestive milk, whereas higher levels with some nutri- enzymes may no longer recognise their tional significance can be encountered in in- specific substrate or cannot reach their site of bottle sterilised milk, in processed cheese and in action. Lacroix et al. (2008) evaluated the powdered milk products. In particular, Cattaneo effect of pasteurisation and UHT treatment on the protein digestibility of skimmed milk in
528 L. Pellegrino et al. Table 16.9 Levels of blocked lysine in some dairy prod- compound were found in sterilised milk prod- ucts (Pellegrino et al., unpublished) ucts, whereas higher amounts were detected in roasted coffee and bakery products (Henle Type of product/process Blocked lysine (% total et al., 1997). A recent study (Hiller and lysine) Lorenzen, 2010) reported the formation of Drinking milk highly cross-linked protein/sugar complexes pasteurised 0.1–0.2 that sterically shield peptide bonds from pro- UHT 3.0–6.5 teolysis. This phenomenon could explain the in-bottle sterilised 11.0–13.5 impaired in vitro digestibility of sodium caseinate submitted to heat treatment. Cheese 0.2–0.3 3. Along with cross-linking phenomena deriving Fresh 6.5–8.7 from the MR, several cross-linkers are reported Processed 7.2–9.8 to form via b-elimination reactions mainly involving phosphoserine and cysteine with the Skim milk powder 11.3–12.5 formation of the key intermediate compound Milk based infant formulas 18.5–31.2 dehydroalanine. Further condensation reactions 3.5–4.9 lead to formation of AAs, which do not occur liquid naturally, such as lysinoalanine (LAL), lanthio- powder nine, ornithinoalanine and histidinoalanine. Yogurt Among these, LAL has been studied exten- sively (Friedman, 1999a) in food proteins et al. (2009) demonstrated that by adopting a because of its suspected role in inducing neph- conventional UHT treatment and optimising the rocytomegaly in rats. However, up to now, no processing conditions for liquid infant formu- evidence of this effect has been observed in lae, the loss of available lysine can be dramati- primates. The presence of LAL has been cally reduced. It is noteworthy that FAAs can demonstrated in processed cheese (Pellegrino also be potentially involved in the MR. Thus, et al., 1996), in drinking milk (Faist et al., 2000; the extent of blockage of IAAs should be evalu- Cattaneo et al., 2008a) and in liquid infant ated taking into account the levels of FAAs. In formulae (Cattaneo et al., 2009). this regard, Rerat et al. (2002) showed that the decreased bioavailability of IAA other than The effect of cross-linking arising from lysine plays a minor role in the loss of the nutri- b-elimination reactions on the nutritional tional value of milk, infant formulae and other quality of protein content can be considered foods subjected to heat treatments. similar to that of cross-linkers formed via the MR. In this regard, Gilani and Sepehr (2003) Besides the loss of IAAs, the nutritional reported that the digestibility of a-la sensi- quality of milk proteins may be reduced by tively decreased when it was submitted to newly formed molecules arising from the alkalinisation followed by a heat treatment advanced MR. Some of these compounds have promoting the formation of significant been demonstrated to inhibit in vitro digestion amounts of LAL. However, some authors (de by carboxypeptidase A and aminopeptidase M Vrese et al., 2000) reported that the decrease (Oste et al., 1987), thus affecting the utilisa- in casein and b-lg digestibility subsequent to tion of dietary protein. In this regard, Gilani alkalinisation and heat treatment was mainly and Sepehr (2003) suggested that the presence due to AA racemisation to the D form and of Maillard products reduced the protein only partially due to LAL formation. Moreover, digestibility of skim milk powder. it was demonstrated (Pellegrino et al., 1999) that compounds other than LAL and pentosi- In addition, protein digestibility may be dine are responsible for heat-induced b-casein reduced by polymerisation induced by the covalent cross-linking. MR. Although the polymerisation phenom- ena of milk proteins have been extensively studied and related to the formation of dicar- bonyl compounds, until now the only identified cross-linker arising from the MR in food was pentosidine. However, only traces of this
16 Nutritional Quality of Milk Proteins 529 Fig. 16.4 Accumulation of furosine (¿) and lysinoalanine ( ) in liquid milk-based infant formulae during storage at 20°C (adapted from Cattaneo et al., 2009) For a comprehensive evaluation of the of Alkanhal et al. (2001), who observed a nutritional significance of the presence of decrease in the nutritional quality of proteins LAL in food, it is useful to consider that the in UHT milk after a 3-month storage at formation of LAL results in a loss of such ambient temperature. IAAs as lysine, cysteine and threonine, 4. During heat treatment of protein, especially although, as far as conventional dairy products at alkaline pH, AA residues may undergo are concerned, the levels of lysine involved in racemisation from l- to d-enantiomers. The LAL formation are significantly lower than presence of d-AAs lowers the nutritional those blocked in the early formed MR quality of proteins; in fact, the hydrolysis compounds. rates of d-AA-containing proteins are slower than those of unmodified proteins because Prolonged storage of several dairy prod- peptide bonds involving d-AAs are not easily ucts at ambient temperature also affects the cleaved by proteolytic enzymes. Moreover, nutritional quality of their protein content. racemisation can lead to formation of non- During storage, further protein modifications metabolisable and biologically non-utilisable occur as demonstrated by the progressive forms of AAs, and nutritionally antagonistic accumulation of some molecular markers of and toxic compounds (Friedman, 1999b). heat load, such as furosine (arising from Unlike most mammalians, humans lack early MR) and LAL in milk-based infant for- specific biochemical pathways for converting mulae reported in Fig. 16.4. These products some d-AAs into the corresponding are usually stored at room temperature for l-enantiomers (Meade et al., 2005). Raw 8–12 months if liquid or up to 24 months if milk contains some d-AAs (d-alanine, powdered, and thus, their nutritional quality d-aspartic acid, d-glutamic acid, d-lysine and may be greatly impaired by adverse storage d-serine), derived from lactic acid bacterial conditions (Pellegrino et al., 2011). The cell walls and from cow rumen microorgan- same behaviour of the above-mentioned isms (Marchelli et al., 2008). The content of heat-load molecular markers was observed these AAs is not considered important from a by Cattaneo et al. (2008a) during storage of nutritional point of view, and even milk pas- conventional UHT milk. Results arising teurisation and UHT treatment does not from this research may support the findings
530 L. Pellegrino et al. Fig. 16.5 Levels of soluble N and free amino acids in selected cheeses (Pellegrino et al., unpublished) result in a significant increase in the level of compounds, such as small peptides and FAAs, d-AAs (Csapó et al., 2007). which are readily absorbed through the small 5. Severe heat treatments of milk may also lead intestine. For this reason, proteolysis occurring to the formation of peptide bonds between the during cheese ripening can be regarded as a sort e-amino group of lysine and the amino group of predigestion and some varieties of cheese of asparagine or glutamine. The isopeptides show a higher protein digestibility than whole involving glutamine and lysine are fully milk. In Fig. 16.5, levels of FAAs and soluble N bioavailable, and their formation prevents fractions in selected cheeses are reported. lysine from reacting via the aforementioned reactions that may impair the nutritional quality Free d-AAs are often reported to be present in of milk proteins (Friedman, 1999a). long-ripened cheeses, and because, as previously reported, the presence of d-AAs depresses the 16.4.3.2 Cheesemaking nutritional quality of proteins, they should not be Following acid or rennet coagulation, about 95% of considered in the total amount of FAAs. Gouda, casein is transferred from milk to cheese and about Emmental, Grana Padano and Parmigiano- 95% of whey protein passes from milk to whey. Reggiano are reported to contain relatively high Because whey proteins have a higher BV than casein, amounts of d-valine, d-isoleucine, d-serine, d-al- mainly due to the presence of SAAs, cheesemaking anine, d-glutamic acid and d-asparagine implies a slight decrease in the nutritional value of (Friedman, 1999b; Marchelli et al., 2008). The milk proteins in terms of AA composition. From this level of d-AAs in ripened cheese seems to be point of view, the nutritional value of cheese protein related to the presence of racemases derived from can be improved by applying processing technolo- lysis of bacterial cell walls in both the retardation gies capable of increasing whey protein retention in and the death phases (Csapó et al., 2007). the curd (e.g. ultrafiltration of cheese milk). It is noteworthy that, during cheese ripening, An overall evaluation of the nutritional quality after their liberation from casein, several FAAs of milk proteins in cheese should also consider may undergo degradation reactions that lead to their increased digestibility. During cheese the formation of nonprotein AA (Krause et al., ripening, a progressive degradation of casein 1997; Cattaneo et al., 2008b), which play impor- occurs as a result of the alternate activity of tant roles in diverse physiological processes. proteolytic enzymes deriving from milk, rennet Arginine may be degraded into ornithine and then and starter and non-starter bacteria. Indeed, in transformed into citrulline, an immediate precur- ripened cheeses, the water-insoluble casein is sor for arginine synthesis in virtually all cell types partly already converted into water-soluble N (Wu and Morris, 1998). Decarboxylation of glu- tamate produces g-aminobutyric acid (GABA),
16 Nutritional Quality of Milk Proteins 531 an inhibitory neurotransmitter in the central ner- 1985). However, Gaudichon et al. (1995), measur- vous system that has been shown to reduce blood ing endogenous N secretion after ingestion of pressure (Nomura et al., 1998). Like arginine yogurt and milk, concluded that fermentation degradation, glutamate decarboxylation has been tends to delay the gastric emptying rate. interpreted as a response of lactic acid bacteria to high acidity (Cotter and Hill, 2003). The content Homogenisation is usually applied in the pro- of GABA determined by Nomura et al. (1998) in cessing of drinking milk to assure stability of fat several commercial cheese types ranged from emulsion during storage. Despite the fact that a 4 mg/kg cheese for Edam to 177 mg/kg cheese in reduction in fat globule size seems to improve Gouda, representing 0.1% and 6%, respectively, milk fat digestibility, the high pressures (10– of the total FAA content. In Grana Padano and 25 MPa) applied in milk homogenisation induce Parmigiano-Reggiano cheeses, GABA does not protein aggregation (Meade et al., 2005) and exceed 0.5% of the total FAA content, roughly adsorption of casein micelles and whey protein to corresponding to 400 mg/kg cheese, and higher the milk fat globule surface, and the impact of levels indicate the presence of such defects as late this phenomenon on protein digestibility is far blowing (Cattaneo et al., 2008b). Furthermore, in from clear (Michalski and Januel, 2006). the natural whey cultures commonly used as starters in the processing of hard cheeses, free 16.5 Nutritional and Physiological GABA contents of up to 80 mg/L were observed, Activities of Milk Proteins representing up to 17% of the total FAA content (personal data, 2011). It remains questionable, The nutritional role of milk proteins has been gen- however, if supplementation of GABA from milk eralised by studying the whole protein supply pro- or dairy products may affect GABA levels in the vided by milk. In the last several decades, simple brain because this AA is not transported efficiently and cost-effective industrial-scale methods have across the blood–brain barrier. Therefore, most been developed for separation, fractionation and of the GABA found in the brain is produced there isolation of the individual proteins occurring in (Petroff, 2002). bovine milk and colostrum. These methods have been addressed to maintain some of their biologi- Decarboxylation of FAAs occurring in some cal effects and digestibility. As a consequence, long-ripened or moulded cheeses may lead to the milk proteins are nowadays available for specific formation of amines, among which biogenic applications in different multifunctionality forms, amines have been demonstrated to be biologi- characterised by specific technological properties cally active, including either hypertensive (e.g. water solubility) and nutritional or sensorial (tyramine and phenylethylamine) or hypotensive characteristics. These facts have shifted research (histamine) effects on blood pressure (Krause towards the study and exploitation of the physio- et al., 1997). Healthy persons, however, rapidly logical health benefits of individual milk proteins, metabolise ingested biogenic amines by convert- some of which are of no significance in the princi- ing them into aldehydes and carboxylic acids pal milk products. Despite many nutritional issues (Renner, 1993). remaining unanswered, milk proteins provide a wide range of important functional and biological 16.4.3.3 Other Milk Treatments properties that are continuously being discovered The digestibility of the proteins in fermented milk and for which scientific data are being accumu- products is enhanced by (1) the partial proteolysis lated. For this reason, some milk proteins have performed by proteases from starter bacteria lead- already found applications in the development and ing to the release of peptides and AA and (2) the manufacturing of novel foods designed to provide presence of acid-coagulated protein that implies health-promoting functions, including preven- the formation of a softer clot in the stomach than tion of diseases and improvement of consumers’ that deriving from liquid milk ingestion. This wellbeing. In this regard, the potential biological coagulum is more easily accessed by gastric pro- activities relating to absorption of nutrients, teases during digestion (Hewitt and Bancroft,
532 L. Pellegrino et al. mineral binding, immune system modulation, stomach, whereas whey proteins are rapidly antimicrobial and antiviral actions and anticarci- digested resulting in high concentrations of AAs nogenic features have been extensively studied. in the blood stream (Lacroix et al., 2006). Nonetheless, different whey proteins are quite The nutritional role of caseins has been refer- resistant to digestion and are still detected in the enced in terms of their capacity to carry calcium and intestinal lumen following milk ingestion (Mahé phosphate. Whey proteins were also suspected to et al., 1991; Roos et al., 1995). Moreover, transcy- play a role in mineral absorption (Pantako et al., tosis of the intact form of some of these proteins 1992; Barth and Behnke, 1997) because several of has been demonstrated, as well as their presence in them can bind minerals and vitamins. For instance, the blood stream after ingestion (Caillard and b-lg contains a hydrophobic pocket that, in vitro, Tomé, 1994). can bind to and act as a carrier of different sub- strates, including retinol, vitamin A and vitamin D, There is now a substantial body of evidence thus simplifying their absorption (Wang et al., 1997; suggesting that the major components of bovine Beaulieu et al., 2006). The ability of b-1g and a-la milk, as well as several highly purified individual to bind cations has been reported as well (Baumy constituents or subfractions, can regulate immune and Brule, 1988; Jeyarajah and Allen, 1994; Simons function in nonruminant as well as ruminant spe- et al., 2002). Lactoferrin is believed to play a role as cies (Cross and Gill, 2000). Whey proteins have an iron-binding protein that could be involved in been reported as capable of modulating non- iron transport (Bos et al., 2000; Iyer and Lonnerdal, specific and specific immune responses, in both 1993). Other minor whey proteins are responsible in vivo and in vitro experiments (Gomez et al., for binding and transporting folate, vitamin D, 2002). On the contrary, Brix et al. (2003) demon- cobalamin and riboflavin (Fox and Kelly, 2003). strated that b-lg per se did not possess immuno- modulatory activity. Eventually, the effect was As far as physiological activities are concerned, found to be caused by the endotoxin contamina- milk proteins are suspected to modulate differen- tion present in some commercial b-lg prepara- tially the release of gastrointestinal hormones that tions. Whey proteins are SAA-rich proteins and control both gastric and intestinal motilities, as have been reported to possess anticarcinogenic well as gastric and biliopancreatic secretions (Hara properties. Indeed, through provision of methion- et al., 1992). Moreover, whey proteins and caseins ine and cysteine, they have a positive influence can affect the synthesis of metabolic components on cellular methylation, and, by stabilising DNA, (glutathione, serotonin) or different factors they retard the development of colon tumours involved in the regulation of the cellular signalling and tumour precursors (McIntosh et al., 1995). in different tissues and organs (McIntosh et al., The anticarcinogenic activity of whey proteins 1998). As a consequence of these activities, milk and their individual components is well docu- proteins influence food and energy intake, glucose mented (Badger et al., 2001; McIntosh et al., metabolism, fatty acid and adipose tissue metabo- 1995; Tsuda et al., 2000), and these proteins offer lism or insulin secretion and sensitivity. However, protection against colon and mammary cancers these effects are likely due to the presence of (Hakkak et al., 2000). a-La may also exert anti- specific bioactive peptides released during diges- ulcerative properties (Mezzaroba et al., 2006). tion of milk proteins and acting either at the lumi- Indeed, animal studies using rats demonstrated nal level or after absorption (Daniel et al., 1990; that a-la increases the gastric luminal pH and EFSA, 2009). In this regard, the rate at which milk gastric fluid secretion and delays gastric empty- proteins are digested in the stomach could explain ing (Ushida et al., 2003). Serum albumin, a-la why casein stimulates endogenous N secretions and lactoferrin are rich sources of the dipeptide more efficiently than whey protein, as observed by g-glutamylcysteine, which is an excellent source different authors (Mahé et al., 1995, 1996). As of dietary cysteine for cellular synthesis of gluta- previously mentioned, the rate at which peptides thione, which protects against free radical dam- and AA are released during digestion may differ age, pollution, toxins and infection and contributes among the milk proteins (Boirie et al., 1997). to defence mechanisms against cancer (Rogelj, Casein forms a clot that slowly empties from the
16 Nutritional Quality of Milk Proteins 533 2000). An increase in cellular glutathione stimu- (Tomita et al., 1994). The antibacterial property of lates immunity, thus increasing antitumour effects lactoferrin and lactoferricin protects against sev- in tumour cells (Bounous, 2000). Lactoferrin eral bacterial strains and yeast (Tomita et al., binds iron, which is potentially procarcinogenic 1994). Besides this action, lactoferrin and lactofer- thereby preventing intestinal damage. Indeed, the ricin present anti-inflammatory, immunosuppres- preventive effects of lactoferrin and lactoferricin sive or immunostimulatory activities, promoting on chemically induced colon carcinogenesis in lymphocyte proliferation in vitro (Tomita et al., rats and on transplanted carcinoma cell metasta- 1994). Both lactoferrin and lactoferricin exhibit sis in mice have been demonstrated (Tsuda et al., antiviral activity against hepatitis C (Iwasa et al., 2006). Bovine serum albumin is another whey 2002; Kaito et al., 2007), human papillomavirus protein that may have anticancer properties, as it (Mistry et al., 2007) and herpes simplex virus has been shown to inhibit growth of the human (Jenssen, 2005). Recombinant human and bovine breast cancer (Laursen et al., 1990). lactoferrins are now available for development into nutraceutical and pharmaceutical products Milk contains substances that provide passive (Weinberg, 2007). protection against infection in the intestinal lumen. This protection mainly involves immunoglobulins, Many cytokines and chemokines have been lysozyme, lactoperoxidase and lactoferrin. Indeed, discovered in cows’ milk, including interleukins, the antimicrobial activity of a-la and b-lg has been growth factors and chemokines that produce vari- attributed mainly to peptides deriving from their ous effects, which contribute to the development enzymatic hydrolysis (Hartmann and Meisel, and function of new tissues and of the immune 2007; Madureira et al., 2007). Both intact a-la and system. In particular, several growth factors have b-lg do not present significant antibacterial activ- been found in cows’ milk at trace levels, includ- ity (Pellegrini et al., 1999; Pihlanto-Leppala et al., ing epidermal growth factor (EGF) and insulin 1999). The antiviral activity of both a-la and b-lg growth factors 1 and 2 (IGF1, IGF2). They are has also been reported by Chatterton et al. (2006). very sensitive and easily destroyed by heating. Immunoglobulins mainly consist of secretory IgA Their effects on intestinal mucosal growth have in human milk and IgG in cow milk. In particular, been demonstrated, as shown by their ability to the main immunoglobulin present in cows’ milk is stimulate intestinal enzyme expression and matu- IgG1 with lower amounts of IgM, IgA and IgG2, ration of intestinal function in newborns (Ma and and they have been shown to partially resist degra- Xu, 1997; Young et al., 1990). The highest dation in the intestinal lumen (Roos et al., 1995). amount of these proteins is found in colostrum These proteins are responsible for protection during the first days of lactation. Colostral growth against microbial pathogens, activation of comple- factors (IGF-1, EGF and others) in addition to the ment, stimulation of phagocytosis, prevention of availability of dietary AA affect protein synthesis the adhesion of microbes and neutralisation of in neonates (Burrin et al., 1992), and, along with viruses and toxins (Mehra et al., 2006). They also hormones (insulin, growth hormone and others), increase the intracellular levels of glutathione, they contribute to the initiation of normal diges- which is a key cellular antioxidant (McIntosh tive function in newborn calves (Baumrucker et al., 1998). Lactoferrin is a major protein in et al., 1994; Hammon and Blum, 1998). human whey, being present only at a low level in cow milk. The defence against a broad range of References microbial infections has been historically ascribed to lactoferrin (Farnaud and Evans, 2003), and this AFSSA. (2007). Apport en protéins: consommation, qual- defence depends on its concentration and on the ité, besoins et recommandations. Agence Française de degree of iron saturation. Some, if not all, of this Sécurité Sanitaire des Aliments. http://www.afssa.fr/ activity results from lactoferricin, the bactericidal Documents/NUT-Ra-EtiquetageEN.pdf. peptide formed during the peptic digestion of lactoferrin, whose antimicrobial activity originates Agostoni, C., Carratù, B., Boniglia, C., Riva, E. and from a direct interaction with the bacterial surface Sanzini, E. (2000). Free amino acid content in stan- dard infant formula: comparison with human milk. J. Am. Coll. Nutr. 19, 434–438.
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Index A Aspergillus ACE. See Angiotensin-converting enzyme (ACE) A. awamori, 298 Acid phosphatase (AcP) A. nidulans, 298 assay methods, 360 Atherosclerosis, 344 isolation and characterisation, 360 significance, 361 B Adipophilin, 20, 418 Bacterial peptidases, 119 Adulteration, of dairy products, 121–122 Bile salts-stimulated lipase, 351 Aldolase, indigenous enzymes, 369 Binding proteins Alkaline phosphatase, indigenous enzymes assay methods FBPs, 324 riboflavin, 325 chemiluminescent assay, 357 vitamin B12, 325 ELISA, 357 vitamin D, 324–325 fluorometric methods, 357 Biosensors, 116–117 isolation and characterisation, 355–356 b-Lactoglobulin (BLG) reactivation of, 357–358 amino acid residues, 406 significance, 358–359 cell culture tests, 407 American Dairy Science Association (ADSA), 56 genomic organisation, 405 Amino acid polymorphic variants, 407 transport quantitative effects, 407 Na+ dependent transport mechanisms, BLG. See b-lactoglobulin (BLG) Blood serum albumin (BSA), 62 448–449 BML. See Bovine milk lysozyme (BML) Na+ independent transport mechanisms, BMPs. See Bone morphogenetic proteins (BMPs) Bone morphogenetic proteins (BMPs), 12 449–450 Boulengerula taitanus, 4 peptides, 451 Bovine milk angiogenin, 319 transport and metabolism, 450 Bovine milk lysozyme (BML), 365, 366 volume-activated amino acid transport, 450 Bovine milk proteins whey proteins as1-casein, 468–469 comparison with casein, 49–50 as2-casein, 471, 472 as sources, 27 b-casein, 468, 470 Amniotes, 5–6 k-casein, 471–472 Amphibian skin glands, 4 a-lactoglobulin, 474, 475 Amylase, indigenous enzymes, 367–368 b-lactoglobulin, 472–474 Angiotensin-converting enzyme (ACE), Bovine mucin 15 (MUC15), 328 Bovine serum albumin (BSA), 118, 328 319–320 Breast cancer resistance protein (BRCP), 495 Anti-adipogenic effects, lactoferrin, 305 BSA. See Bovine serum albumin (BSA) Anticancer effects, lactoferrin, 302–303 Butyrophilins, 19–20, 413–415 Antigen-binding fragments (Fab), 276 Antiviral effects, lactoferrin, 303 Apocrine glands, 10–12 Apo-pilo-sebaceous unit (APSU), 11–12 539 P.L.H. McSweeney and P.F. Fox (eds.), Advanced Dairy Chemistry: Volume 1A: Proteins: Basic Aspects, 4th Edition, DOI 10.1007/978-1-4614-4714-6, © Springer Science+Business Media New York 2013
540 Index C cations, 149 Caecilians, 5 genetic polymorphism, 145, 147 Capillary electrophoresis (CE), 101 interactions with calcium, 149 Capillary zone electrophoresis (CZE), 107 primary structure, 144–146 Caprine milk proteins secondary structure, 147 composition and nomenclature, 135–136 k-casein, 482, 484 k-casein as1-Casein, 474–479 association behavior, 154 as2-casein, 480, 482, 483 disulphide-bonding patterns, 153 b-casein, 479–480 genetic variation, 151–152 a-lactoglobulin, 484 glycosylation, 152–153 b-lactoglobulin, 484 interactions with calcium, 154–155 as1-Casein, 392 primary structure, 149–151 bovine milk proteins, 468–469 secondary structure, 153 genetic variation, 139 as1-casein hydrophobic dimers and oligomers genetic variation, 139 interactions with calcium, 140–141 double ribbon structure, 167 primary structure, 136–138 fragments f136–196, 166–168 secondary structure, 139 weight-average molecular weights, 166 self association, 139–140 interactions with calcium, 140–141 as2-casein ovine milk proteins, 484, 486–487 association properties, 144 primary structure, 136–138 genetic polymorphism, 142, 143 secondary structure, 139 interactions with calcium, 144 self association, 139–140 primary structure, 141–143 three-dimensional molecular models, 163 secondary structure, 143–144 as2-Casein, 395–397 Casein-encoding genes association properties, 144 gene cluster, 432–433 bovine milk proteins, 471, 472 individual gene structures, 433–434 genetic polymorphism, 142, 143 Casein micelles (CM) interactions with calcium, 144 calcium phosphate, 193–194 molecular modeling, 173–174 casein polymerization, 190–193 ovine milk proteins, 487, 489–490 characteristics of, 68 primary structure, 141–142 microstructural imaging secondary structure, 143–144 bovine milk, 195 b-Casein stereo pairs, 195, 196 amino acid sequence, 394 TEM, 194 association properties, 148 modeling bovine milk proteins, 468, 470 polyelectrolyte brush model, 188 cations, 149 subunit model, 188 exon skipping, 395 X-ray scattering, 189 gene encoding, 395 physical properties, 189–190 genetic polymorphism, 145, 147 stability, 69–70 interactions with calcium, 149 structure molecular modeling, 168–170 dual-bonding model, 73 ovine milk proteins, 487 principal features, 70–71 primary structure, 144–145 sub-micelle model, 71–73 secondary structure, 147 supramolecule k-Casein, 397 acidification of milk, 199–202 association behavior, 154 calcium phosphate nanoclusters, 197, 198 bovine milk proteins, 471–472 calcium sequestration, 202–203 disulphide-bonding patterns, 153 cooling of milk, 198–199 genetic variation, 151–152 ethanol, 204–205 glycosylation, 152–153 heating of milk, 203–204 interactions with calcium, 154–155 interlocked lattice, 196–198 molecular modeling, 170–173 pH, 201–202 ovine milk proteins, 490 thermodynamic forces, 190 primary structure, 149–151 Casein nitrogen, 91 secondary structure, 153 Caseinomacropeptide (CMP), 397 Caseinates preparation, 49 Casein protein preparation Casein chemistry caseinates preparation, 49 b-casein association properties, 148
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