Casein

The caseins behave differently than most proteins. They hace extremely flexible structures and for some time they have deen described as being essentially random. There is no crystal data for the caseins because the molecules do not form crystals. This has been cited as evidence that they do not have well defined three-dimensional structures. The best evidence for seconday and tertiary structure is obtained from x-ray difraction studies of crystalline molecules. It is possible to make estimated of secondary structure from molecules in solution.

Recently, workers at the USDA Eastern Reagional Research Center in Philadelphia have studied the caseins using Raman spectroscopy. They have been able to make estimates of the amount of secondary structure in the various caseins.

The structure resolved for a s-casein is presented in figure 1.

Figure 1. Three-dimensional molecular model for a s-casein.

These researchers describe the structure as a short hydrophillic segment on the right of the molecule connected to a hydrophobic b-shhet region. This is connected to the region that contains the phospahte groups which is connected to a shory alpha-helical segment. This is connected to the very hydrophobic carboxy-terminal domain which contains extended beta strands. This mode suggests that a s-casien contains approximately 15% alpha helical structure, 22% beta structure, 45% turns and 18% that can not be specified. This is probably a reasonable approximation of the conformation of the molecule in solution and is in reasonable agreement with estimates made by other methods.

The fact that it is impossible to obtain crystals of the molecule suggests that this secondary structure is not as permanent as in a more typical protein and that considerbale variation is possible.

The proposed structure for k-casein is presented in figure 2.

Figure 2. Three-dimensional model for kappa casein.

The authors have described this as a "horse and rider" model. The amino terminal section of the molecule makes up the "horse" and the c-terminal section the "rider". The two legs of the horse are made up of b-structure. This mode suggests that k-casien contains approximately 16% alpha helical structure, 27% beta structure, 37% turns and 20% that can not be specified. The "leg" sections are very hydrophobic and the authors have postulated that his may be the area of interaction with other caseins.

Casein Micelles

In milk, the caseins exist in large colloidal particles called micelles. Thes are large aggregates with diameters of from 90 to 150 nm. Evidence from electron microscopy and other means, suggests that the micelles are composed of smaller units called submicelles having diameters of from 10 to 20 nm. A number of observations have been made regarding the properties of casein micelles. Any model that proposes to explain the structure of these micelles must be tested against hw well it can explain these observations. Characteristics of micelles include:

1. Precipitation by the enzyme rennin. Rennin is known to specifically cleave the bond between phenylalaine 105 and methionine 106 of k casein. The k casein must be accesible to the enzyme.
2. The contnet of calcium and phosphorous of milk is much higher than the solubility of calcium phosphate at pH 6.7. Much of this phosphate in present in an insoluble colloidal state and will precipitate with the micelles uopn high speed centrifugation.
3. Milks that contain relativley more k casein have smaller average micelle diameters than do milks with less kcasein.
4. If the casein micelles are disrupted and then reformed, the size distribution of the fianl micceles will be very similar to those of the initial micelles. Reformed micelles from milk that originally had a large average micelle diameter will be larger than those reformed from milk that had small average micelle diameters. This property has been called memory.
5. Micelles are highly solvated and contain approximately 3.7 g water per gram of protein.
6. The addition of extra k casein to a mixture of casein micelles will result in a decreae in the average micelle diameter of the mixture.
7. Kappa casein will stabilize a s-casein from precipitation by calcium. There is complete stabilization at alpha s/kappa ratios of 10 and some stability at higher ratios.
8. Micelle fromation requires the presence of calcium at concentrations greater than are required to precipitate a s-casein.
9. At low temperatures, some of the b casein is able to leave the micelle and becomes soluble. As the temperature is increased, the beta casein returns to the micelle.

The first generally accepted model for casein micelle structure was proposed by Waugh in the mid 1960s. The essentail elements of this model are a hydrophobic association of a s and b caseins that are rouhgly spherical . These aggregates are coated with a monolayer of k casein. This model has been described as a core-coat model beacause of its hydrophonic core that is stabilized by the k casein coat. A scematic representation of this model is presented in figue 3.

Figure 3. Model of casein micelles proposed by Waugh.

This model is able to explain a number of the observations listed above. The accesibility of k casein to rennin is obvious. The final size of the miceels would presumably depend on the amount of k casein available to form a coating. If there were relatively less k casein, the average micelle diameter would have to be increased to ensure complete coverage. The model does not directly address the loaction of calcium and phosphate, but the colloidal metrail would presumably be associated with the phosphate clusters on the a and caseins. These are indicated by the rings at one end of the molecule.

Following a number of observations of the composition of casein follwing ultracentrifgual fractionation, Morr proposed the model shown in figure 4 a few years later.

Figure 4. Model for casein micelles proposed by Morr.

This model is best viewed as a variation of the model proposed by Waugh. The hydrophobic a and b caseins are located with the dotted circles and are coated by a layer of b casein. The colloidal calcium phosphate is represented by the S in the diagram that connect the sub micelles. This is a very porous molecule and would accomodate a large amount of water. The diagram suggests that soluble casein could enter and leave the micelle, but the nature of the interaction is not specified. The relatively uniform size of the submicelles doeas not readily explain the differences in size of micelles with differing b casein contents.

Figure 5. Conceptual model of a casein micelle containing about 40 subunits. The lighter surface represents As and b casein polymers ( hydrophobic area ). The darker patches cover about 20% of the surface area and represent associated k casein polymers ( hydrophillic area ). The model provides for open channels through the micelle. Further growth is impeded by the extensive hydrophillic peripheral surface. Adapted from Slattery and Evard.

Properties of casein

The open and flexible nature of the caseins makes them unusual. While they may have a prefered secondary and tertiary structure, they are often in other conformations. These other structures must expose hydrophobic groups to contact with water. The structures attained by the caseins can accomodate this contact. For most proteins, unfolding and exposure of hydrophobic groups to water results in unstable structures. The proteins must refold to lower the contact with watre and precipitation often results. Becuase the caseins exist in open structures to begin with, they are not as sensitve to structural alterations.

For example, the caseins are very stable to heating. They maybe exposed to boiling for extended periods of time and remain totally soluble. This is an extremely useful property for food products that will be subjected to severe heat treatment such as UHT processing. Caseins also find application where flexibility is required for functionality.

Most proteins that contain significant amounts of strong secondary and tertiary structure require time to unfold at air or oil interfaces. The time required depends upon the flexibility of the protein. The most stable air cells and lipid droplets result from proteins that are able to quickly rearrange their structures and lower the interfacial free energy. Caseins function very well in these applications and there is very little lag between the time the molecules arrive at the interface and they exert their full functional affect.

High solution viscosity is a result of the very open, nearly random, structures of casein molecules. Sodium caseinate finds application in products were high viscosity is required. The lack of solubility in the presence of calcium changes the behavior of casein in its presence. As calcium is added to a solution of sodium caseinate, a number of changes are evident. The calcium will cause aggregation of casein into structures that resemble micelles. As these aggregates increase in size and number, the viscosity of the solution will decrease. The solution will also become turbid as the particles become large enough to scatter light. This, if a clear solution with high viscosity is required, sodium caseinate is a good choice. Calcium caseinate should be selected when a solution of relatively low viscosity and high turbidity (milky appearance) is desired.

Casein also exhibits melting properties that are unique among proteins. Following limited proteolysis, casein will become thermoplastic and will flow upon heating. A similar affect can be achieved by chelation of some of the calcium ions present. These phenomena are the basis for the melting of natural cheeses and the production of process or imitation process cheese. Structure must exist before a substance can be said to melt. With caseins this structure may be obtained by precipitation with calcium, acid or the addition of rennin. Casein does not form thermal gels and has little functionality in applications that require temperature set. High heat stability and the ability to melt are the two properties of caseinates that make them difficult to replace in many food applications.