By Jon Hopkinson, PhD.
You can write a book about just one of the proteins found in milk. Like all things in nature, protein in milk is just downright complicated. Like a fingerprint, the type, amounts and specific properties of the protein in milk vary with the species that the milk comes from. Just for convenience let’s narrow the subject down to average bovine milk (from cows). There are two groups of protein; those that precipitate or have decreased solubility at pH = 4.6 and those that do not. Those precipitating at pH=4.6 are called casein protein and those that do not are called whey protein. In bovine milk, casein is the predominant group, comprising roughly 80 percent of the protein in the milk.
Casein in milk is a mixture of proteins that obviously have some properties in common. All of the casein molecules have at least one ester bound phosphate. None of the whey proteins have this. These molecules, in general, are not highly organized. The casein proteins are larger than the predominant whey proteins. A lot has been said in literature about the general hydrophobicity (water hating nature) of the casein proteins. However this is not truly the case. It is the distribution of charges along the protein’s chain of amino acids that differ. The caseins have a very uneven distribution of charges along the molecule while in the major whey proteins, the charges are more evenly distributed. This accounts for one of the major properties of the caseins in milk, their amphiphilic nature. Charged regions are, in general, water loving or hydrophilic and the uncharged regions along the molecule are more hydrophobic or water hating. Since these regions are unevenly spread along the molecule, these regions are more exposed. In proteins with even distributions, the hydrophilic and hydrophobic regions basically cancel each other out. It is due to the amphiphilic nature of the casein molecules that one of the most important properties of casein emerges, the casein micelle. Most of the caseins in milk are found in intricate loose aggregations called micelles. Micelles are rough spherical particles between 0.02 and 0.3μm in diameter with 20 – 150,000 casein molecules per micelle. These particles also contain some inorganic matter, mostly calcium phosphate. The casein micelle is composed of clusters of casein molecules, called submicelles and water. There is more water than casein in the micelle. The casein protein in the micelle is naturally organized such that the hydrophilic portions of the casein are orientated towards the bulk of the water and the more hydrophobic portions are orientated towards the center where the concentration of water is lowest.
In nature, the casein micelle is stabilized in two ways. First calcium phosphate is found in high concentrations in milk and within the casein micelle itself. The concentration is such that small aggregations of the phosphate form. Part of the phosphate is relatively free to pass into and out of the micelle and some of the phosphate is strongly bound to the protein. This bound phosphate is in tiny aggregations of undissolved (colloidal) calcium phosphate. These tend to cement the micelle together stabilizing its structure. The other stabilizing effect is due to a property of K-casein. K-casein has within its structure a carbohydrate group which is esterified to threonine in the protein. This carbohydrate group contains some negatively charged groups. The result is that K-casein is made more hydrophilic at one part of the molecule. In the micelle, K-casein and to some extent Bcasein self-locates as close to, or extending into the water interface of the micelle. This stabilizes the micelle by preventing aggregation of the micelles by steric repulsion (by preventing close approach between casein micelles) For K-Casein the peptide bond between the 105th and 106th amino acid is vulnerable to hydrolysis by proteolytic enzymes (microbial coagulator or rennet). This is the mechanism for curd formation (by removing the carbohydrate containing portion of the protein and thus reducing steric stability). Losing the protection of the K-casein allows the casein micelles to aggregate and form a coherent curd.
There are many ways that the casein micelles can aggregate and all have some effect on foods (both positive and negative) made with milk. The following table is a list showing the more common modes of aggregation, and whether or not the aggregation is reversible, and some remediation that may help prevent the formation of aggregations or gels.
In part from Walstra, Wouters and Geurts 2006 ‘Dairy Science and Technology second edition” pp-152
The two most common aggregations leading to gel formation in dairy products are the formation of a gel by adding rennet (or coagulator) and by slow acidification of the protein. Renneting is the process that forms cheese and slow acidification leads to fresh fermented (and acidified) milk products like yogurt and buttermilk. The two kinds of gel appear similar but have some important differences. The acid gel is more resistant to fracture, more elastic and it is less likely to synerese (loose water from the gel matrix). This property makes yogurt type products possible. The properties of rennet gel make cheese making possible by allowing for whey to drain or expel easily.
The most predominant casein in milk is αS1-casein at aproximately 36% of the total casein content. It is a loose flexible medium sized protein with a molecular weight at MW= 24000. It consists of two hydrophobic regions connected by a hydrophilic region. It has phosphate groups located in the hydrophilic region and most of the calcium associated with is exists at the phosphate goups.
αS2- casein is a protein with a concentration of negative charged regions at one end and positive ones at the other end. This protein strongly binds calcium. At neutral pH it associates with itself by bridging with calcium. αS2- casein contains two closely spaced cystine residues which can form disulfide bonds, though this is less likely due to their proximity.
B-casein is strongly negatively charged at one end of the polymer, the rest of the chain has nearly no net charge. This protein behaves like an anionic detergent or emulsifier. With the negatively charged end (hydrophilic) and a hydrophobic tail. ϒ-caseins are formed from the C-terminal end of the B-casein molecule. The clevage points are at the 28/29, 105/106 and 107/108 positions along the B-casein polymer. They are formed by the action of the enzyme plasmin. Depending on the chemistry of the fragment, these proteins can show up in the whey protein fraction or in the casein fraction of milk protein.
K- casein is perhaps the most economically important of the caseins, being the protein largly responsible for milk clotting and the action of rennet used in the cheese industry. Still it is only 12% of the total casein present in bovine milk. About 2/3rds of K-Casein does not have the carbohydrate that is largly responsible for its use in cheese making as noted above. K-casein is a smaller molecule than the other major caseins. It contains two widely seperated cystine protein residues. At temperatures above 60⁰ C (140⁰F) the sulfur groups can open to expose a reactive sulfur atom (CH2-S-) on the cystine monomer. This free sulfer group can redily combine with reactive sulfur from other casein molecules and with whey protein (which has many more cystine groups). Among the many reasons for using a high heat treated milk in yogurt and other similar dairy products, is to encourage these intermolecular bonds to form. This increases the average size of the protein complexes in the milk and thus increases viscosity. Similarly, this affect allows the K-casein – whey protein to precipitate allowing for an increased protein content in the cheese curd thus increasing yield.
It is important to anyone considering the casein fraction of milk to realize that there is considerable variation within the proteins. Each variation will have effects, some large and some small on the functionality of the protein in dairy foods. The species, subspecies, breed, individual and even which gland on the mammal that produces the milk can have considerable effect on the products made from it. The functionality of casein also varies with the stage of lactation, feed, ambient temperature, stress, season, and water quality. While milk in general is remarkably consistent when combined from many sources (comingled), it is important to recognize that differences do occur and these can be important in diagnosing problems with dairy products.