The protein fraction of cow milk consists of about 80% of caseins. Four different gene products are designated as α s1 - , α s2 - , β , and κ - caseins, which together form micellar structures of 20 nm to 500 nm by noncovalent aggregation (Swaisgood 1992 ).
Casein is a phosphoprotein, which precipitates from raw skim milk upon acidifi cation to pH 4.6 at 20 ° C.
Size Distribution of Casein Micelles
The casein micelle determines the colloidal stability of the polydisperse system in milk. The dimension and composition of casein micelles are of great importance for the coagulation process. Coagulation time varies with micelle size and reaches an optimum with small and medium - size micelles, which have higher κ - casein contents than the larger micelles (Ekstrand 1980 ). Smaller micelles give fi rmer curd than larger micelles at the same casein concentration (Grandison 1986 ). Published data on the state of the casein micelle structure in camel milk are very scarce. In these studies, different techniques were applied; therefore, the results are contradictory to some extent, but all of them showed that camel milk casein micelle is different from that of cow milk. A study used electron microscopy after solidifying milk with agar. The casein micelle ranged in size from 25 to more than 400 nm, where no clear iden- tifi cation of smaller micelles was specifi ed (Gouda et al. 1984 ).
Freeze - fractured samples of camel milk were examined by electron microscopy (Farah and Ruegg 1989 ), which showed that the distribution of casein
micelles is signifi cantly broader than in cow and human milk, with a greater number of large parti- cles. The particles in the lowest - size class with diameters smaller than 40 nm comprise about 80%
of the total number of particles but represent only 4 – 8% of the mass or volume of casein. The volume distribution curve of casein micelles in camel milk is broad and shows a maximum between 260 and 300 nm versus 100 – 140 nm for cow milk casein. In another study (El - Agamy 1983 ), the diameter of casein micelle was estimated at 956 Angstrom ( Å ) (905 – 1031 Å ) versus 823 Å , 801 Å , 716 Å , and 662 Å for buffalo, goat, sheep, and cow milk casein, respectively. This indicates that casein micelles of camel milk are bigger in diameter than those of other species.
Casein Fractionation
Whole camel milk caseins were separated on alka- line native polyacrylamide gel electrophoresis (Fig.
6.1 ) and compared with those of cow and human milk. Camel milk casein fractions were slower in
migration than those of cow and human milk. This feature reveals the differences among the three types of milk caseins in both types and density of the charges (El - Agamy et al. 2006 ).
Molecular weights of camel α s1 - CN and β - CN were estimated at 33 and 29.5 kDa, respectively (El - Agamy et al. 1997 ). In another study camel milk casein was also fractionated on ion exchange chro- matography and fractions were identifi ed by polyacr- ylamide gel electrophoresis (Larsson - Raznikiewicz, M. and Mohamed, M.A. 1986 ). Four casein fractions were identifi ed as α s1 - , α s2 - , β - , and κ - CN. Their corresponding molecular masses were 31, 25, and 27 kDa for α s1 - , α s2 - , and β - casein, respectively. The study showed also that α s1 - and β - casein were domi- nants, whereas α s2 - CN appeared as a diffuse band on the gel. While κ - CN band was absent from the gel, it was isolated by ion exchange and identifi ed by amino acid sequence as homologous to cow milk κ - CN. Furthermore, camel milk α s1 - and β - CN were phosphorylated to about the same extent as in cow milk, while α s2- CN was more heavily phosphor- ylated than that of cow milk casein ( Kappeler 1998 ).
Figure 6.1. Alkaline native - PAGE of acid camel, cow, and human milk caseins. Anode is toward bottom of photo (El - Agamy et al. 2006 ).
β-CN
αS1-CN
αS2-CN
Camel Cow Human
The amino acid compositions of camel milk caseins are similar to cow milk casein fractions (Eigel et al. 1984 ). Camel milk acid - casein was frac- tionated on reversed - phase HPLC chromatography (Kappeler 1998 ). Four fractions were well identifi ed as α s1 - CN, α s2 - CN, β - CN, and κ - CN in camel and cow milk as shown in Table 6.2 . It was found that the ratio of β - CN to κ - CN is lower in camel milk casein than in cow milk. This low ratio affects some of the processing characteristics, heat treatment, and enzymatic coagulation of casein micelles in camel milk. The same study revealed that the pH values of isoelectric points ( p I) of camel and bovine milk caseins were similar.
Primary Structure
Camel α s1 - CN and β - CN, similarly to bovine caseins, are devoid of cysteine residues, and α s2 - CN and κ - CN both contained only two cysteines. The proline content in camel caseins is slightly higher than in cow caseins, with 9.2% in α s1 - CN, 4.5% in α s2 - CN, 17.1% in β - CN, and 13.6% in κ - CN, compared to 8.5%, 4.8%, 16.7%, and 11.8% in cow caseins, respectively. This higher proline content in camel caseins may lead to destabilization of secondary structures in a more pronounced manner than it does in cow milk caseins (Kappeler 1998 ).
Secondary Structure
Limited pronounced structural differences were found between camel and cow milk caseins when
sequence comparison was made. Although α s1 - CN of camel and cow milk had a low percentage similarity in primary structure, similarities in the secondary structure (a series of α - helical regions followed by a C - terminus with little defi ned secondary structure) predominated. In camel milk α s1- CN hydrophilicity of the N - terminal end was slightly more pronounced. Similarly to cow milk, camel α s2 - CN was the most hydrophilic among the four caseins and had a high potential for secondary structures, mainly α - helices. The two cysteine residues also occurred at about position 40 (Kappeler 1998 ).
For camel κ - CN secondary structure, it was found that it is similar to that of cow milk κ - CN, with an N - terminal α - helix containing one cys followed by β - pleated sheets and a second cys. Both cys residues are at the positions similar to those in bovine milk κ - CN.
It is well known that in bovine κ - CN, the site of cleavage by chymosin is Phe 105 - Met 106, leaving a macropeptide of 6.707 kDa, 64 amino acids in length with a p I of the unmodifi ed peptide at pH 3.87, and the amino acid sequence from His 98 to Lys 112 is involved in binding and cleavage of bovine κ - CN by chymosin (Visser et al. 1987 ). In camel milk κ - CN, the site of cleavage by chymosin was found to be Phe 97 - Ile 98 leaving a macropeptide of 6.774 kDa, 65 amino acids in length with a p I of the unmodifi ed peptide at pH 4.13 (Kappeler 1998 ). As shown below, all protein residues are conserved in camel milk κ- CN and the bovine residue Leu 103 was replaced by Pro 95 .
Table 6.2. Physicochemical characteristics of camel and cow milk caseins (Eigel et al. 1984 ; Kappeler 1998 )
Species Casein Fraction Molecular Mass (kDa) p I
Relative Amount in Total Casein
Amino Acid Residues
Camel α s1 - CNA 24.755 4.41 22.0% 207
α s1 - CNB 24.668 4.40
Cow α s1 - CNB 22.975 4.26 38.0% 199
Camel α s2 - CN 21.993 4.58 9.5% 178
Cow α s2 - CNA 24.348 4.78 10.0% 207
Camel β - CN 24.900 4.66 65.0% 217
Cow β - CNA 2 23.583 4.49 39.0% 209
Camel κ - CN 22.294 4.11 3.5% 162
22.987
Cow κ - CNA 18.974 3.97 13.0% 169
Camel: Arg 90 – Pro – Arg – Pro – Arg – Pro – Ser – Phe 97 – Ile 98 – Ala – Ile – Pro – Pro – Lys – Lys 104
Cow: His 98 – Pro – His – Pro – His – Leu – Ser – Phe 105 – Met 106 – Ala – Ile – Pro – Pro – Lys – Lys 112
This additional proline residue is suggested to help the stabilization of the conformation of κ - CN in the active site cleft by camel chymosin and different to the conformation by cow milk κ - CN in the cleft of bovine chymosin. Moreover, histidine residues in the sequence His 98 to His 102 of cow milk κ - CN are replaced by more basic arginine residues in camel milk κ - CN. This leads to the fact that camel milk κ - CN backbone does not need to be bound as tightly to chymosin as it was shown for cow milk κ - CN (Plowman and Creamer 1995 ).
Whey Proteins
It is well known that the major whey proteins of bovine milk are β - LG with 55% of total whey protein, α - LA with 20.25%, and BSA with 6.6%. Other minor whey proteins as immunoglobulins and proteose peptone were also well characterized (Butler 1983 ).
Camel milk whey proteins (CMWPs) were isolated and well characterized by chromatographic, electro- phoretic, and immunochemical analyses (Beg et al.
1985, 1986a, 1987 ; Conti et al. 1985 ; Farah 1986 ; El - Agamy et al. 1997, 1998a ; Kappeler 1998 ).
CMWPs were fractionated on polyacrylamide gel electrophoresis using alkaline native - PAGE tech- nique and compared with those of cow and buffalo milk (El - Agamy et al. 1997 ) (Fig. 6.2 ). Electro- phoretic patterns of CMWPs showed a different electrophoretic behavior than those of other species.
Camel milk α - LA is slower but BSA is faster in migration than cow and buffalo milk proteins.
Similar to human milk, no distinguished band belonging to β - LG was detected in camel milk. This was confi rmed by the molecular study (Kappeler 1998 ). Two different isoforms of camel α - LA were detected (Conti et al. 1985 ; El - Agamy et al. 1997 ).
Camel milk proteins were characterized by the pres- ence of several minor peptides being lower in molecular weights compared to milk of other species.
These peptides may play an important role in the therapeutic value of camel milk (El - Agamy 1983,
2000a ; El - Agamy et al. 1997 ). Kappeler et al. ( 2003 ) reported that concentration of α - La in camel milk (3.5 g/L ) is closer to that of human milk (3.4 g/L), compared to bovine milk (1.26 g/L). El - Hatmi et al.
(2007) recently reported that serum albumin is the major whey protein present in camel milk with an average concentration of 10.8 g/L.
In another study (Beg et al. 1985 ) the primary structure of camel α - LA was determined by analysis of the intact protein, and of CNBr fragments and enzymatic peptides from the carboxymethylated protein chain. Results showed that camel α - LA has 123 residues and a molecular mass of 14.6 kDa. The amino acid sequence is homologous to other α - LAs, but also exhibits extensive differences: 39 residues differ in relation to the bovine protein and only 35 residues are like other known α - LAs. The molecular mass of CMWPs was estimated at 67, 15, and 13.2 kDa for BSA, α - LA variants A, and B, respec- tively, versus 66.2 kDa for BSA and 14.4 kDa for α - LA of cow milk (El - Agamy et al. 1997 ).
Two different unknown proteins were isolated from camel whey having molecular masses of 14 and 15 kDa. Protein of 14 kDa is rich in cysteine/
half - cystine; while that of 15 kDa has no cysteine.
No obvious structural similarities were noted between these proteins and other known milk pro- teins (Beg et al. 1984, 1986a, 1987 ).
Figure 6.2. Polyacrylamide gel electrophoresis (alkaline native - PAGE), 12.5% T of camel milk whey proteins compared with those of other species. Anode is toward bottom of photo (El - Agamy et al. 1997 ).
BSA α-LA
α-LA
BSA α-LA Minor β-LG
peptides
Camel Cow Buffalo
Recently, acid whey prepared from camel milk was separated by HPLC analysis. Three peaks were identifi ed by N - terminal sequencing as whey acidic protein, α- LA and lactophorin (Kappeler 1998 ).
Their ratios were 86.6, 11.5, and 1.9% for α - LA, lactophorin, and whey acidic protein, respectively.
SDS - PAGE of fractionated proteins showed that BSA and other proteins coeluted with α - LA as minor fractions. Table 6.3 summarizes the physico- chemical characteristics of camel milk whey pro- teins as compared with those of cow milk (Eigel et al. 1984 ; Hernandez et al. 1990 ; De Wit and Van Hooydonk 1996 ; Kappeler 1998 ). Camel milk lac- tophorin is a major protein in whey, whereas bovine lactophorin is a minor protein in whey. Camel milk lactophorin was not isolated from proteose peptone component 3 (PP 3 ) as it was in bovine milk protein.
Bovine PP 3 consisted of several proteins of which lactophorin was just the main fraction. The protein had 60.4% amino acid sequence identity to a prote- ose peptone component 3 protein from bovine whey and 30.3% identity to the glycosylation dependent cell adhesion molecule 1 in mice. The N terminal heterogeneity of the protein was a result of alterna- tive mRNA splicing. About 75% of the protein was expressed as a long variant A with 137 amino acid residues and a molecular mass of 15.7 kDa. About 25% was a short variant B with 122 amino acid residues and a molecular mass of 13.8 kDa. Both proteins are probably threefold phosphorylated. In contrast to the related proteins, no glycosylation was found in camel lactophorin. Because of this differ- ence, specifi c interaction with carbohydrate binding proteins, as reported for the murine protein, can be excluded, and a function of the protein other than cell recognition or rotaviral inhibition is proposed.
Pronounced similarities existed between the primary and secondary structures of bovine and camel pro- teins (Kappeler et al. 1999b ). Meanwhile, the percent sequence similarity of camel lactophorin to bovine and caprine lactophorin is much higher than to the rat and murine one (Kappeler 1998 ).
The concentration of lactophorin in camel milk was found to be about 3 times higher than the con- centration of the bovine homologue in bovine milk.
This could be of higher potential benefi t in milk processing, since lactophorin is an inhibitor of lipase (Glass et al. 1967 ).
It was reported that whey acidic protein, generally described as a major constituent of rodent milk, and peptidoglycan recognition protein, an intracellular protein binding to Gram - positive bacteria and pres- ently not known to be a milk constituent, were detected in major amounts in camel whey, both on the cDNA and protein level (Kappeler et al. 2003 ).
It was found that camel milk whey has an acidic protein (12.5 kDa) possessing a potential protease inhibitor (Beg et al. 1986b ). Depending on these fi ndings it is suggested that the higher level of natural preserving agents may bring about longer storage or shelf life of raw camel milk as compared with raw cow milk (El - Agamy 1983 ; Farah 1986 ).