Harvesting Casein Micelles in their Native State
Authors: Ankita Hooda , Bimlesh Mann, Rajan Sharma, Ranvir Suvartan Gautam, Sulaxana Singh

Milk Proteins :

Normal bovine milk has roughly 3.5% protein. The natural functions of milk proteins is to supply neonates with the essential amino acids required for the development of muscular and other protein-containing tissues (Fox and McSweeney, 1998). To perfectly perform this function the milk proteins are designed in such a way that they form complexes with a comparatively large amount of calcium phosphate, which immediately coagulate in the stomach of the newborn.

MAJOR MILK PROTEIN : CASEINS

The major part of the milk proteins, together with calcium phosphate, occurs in the form of large colloidal particles, which are known as the casein micelles (Rollema, 1992). The properties of many dairy products depend directly on the properties of milk proteins, although the fat, lactose and especially the salts, are also very important. In addition, casein based products are entirely milk protein while the production of most cheese varieties is initiated through the specific alteration of proteins by proteolytic enzymes or isoelectric precipitation. The high heat treatments to which many of the dairy products are subjected are possible only because of the high heat stability of the principal milk proteins, the caseins (Fox and McSweeney, 1998). Because of their importance for the functional behaviour of milk products, casein micelles have been studied for a long time. The casein micelle occupies a important position among biological systems because of the various different models that have been proposed for its structure. This situation has probably developed due to the complication and the relatively large size of the casein micelles, which restrict a direct and explicit determination of the structure.

UNIQUE CHARACTERSTICS OF CASEIN:

• The unique characteristic of caseins is due to their post-translational modifications, resulting in the phosphorylation at seryl and infrequently threonyl residues (Swaisgood, 1992). Hence, caseins can be defined as phosphoproteins containing approximately 80% of the total protein content of milk proteins (Brunner, 1977). Caseins are made up of large number of components, and the main types are αs1- casein, αs2-casein, β-casein, and κ-casein (Walstra et al., 1999) as defined and validated through DNA sequences.
• There are trace amounts of γ- casein occurring naturally due to limited proteolysis of β-casein by plasmin (Swaisgood, 1992). The main casein components can have several genetic variants and contain variable numbers of phosphoseryl residues, especially αs2-casein exhibiting a large variability in phosphorylation. κ-Casein has only one phosphoseryl residue, and it is also glycosylated.
• Another unique feature of caseins is the large amount of propyl residues, especially in β-casein, which has great effect on the structure of caseins, because the proline residues disrupt the formation of α-helical and β-sheet (Swaisgood, 1992).
• In addition, all casein proteins have different hydrophobic and hydrophilic regions along with the protein chain.
• αs-Casein is the major casein proteins containing 8-10 seryl phosphate groups, while β- casein has about 5 phosphoserine residues, and it is more hydrophobic than αs-caseins and κ-casein. As αs-caseins and β-caseins are highly phosphorylated, these are very sensitive to the concentration of calcium salts, that is, they will precipitate with excess Ca2+ ions. Unlike rest of components of caseins, κ-caseins are glycoproteins, and they have only one phosphoserine group. Hence, these are stable in the presence of calcium ions, and also play an important role in protecting other caseins from precipitation and make casein micelles stable (Whitney, 1988; Walstra et al. , 1999).
• Casein is very heat stable; only temperatures up to or above 120ºC causes the casein to gradually become insoluble, whereas it is sensitive to pH and will precipitate at its isoelectric pH (Walstra et al., 1999).

TECHNIQUES TO HARVEST CASEIN MICELLES IN NATIVE STATE:

• Membrane Technology

Size of casein micelles vary from 50 nm to 500 nm, these could be separated from skim milk by membrane technology. Microfiltration processing membrane (pore size 0.1 to 1 micrometer) is suitable for harvesting casein micelles from skim milk, which are retained during microfiltration and all the other components pass in the permeate (including whey proteins). Because there is 10 to 100 fold difference in size between the CN micelles and whey proteins, microfiltration membranes which typically retain particles > 100 nm in diameter, can be used to separate whey protein from CN micelles. Level of concentration can be controlled to avoid physical changes in state of casein micelles.

Retentate from the MF of skim milk consists mainly of micellar CN; and called as Micellar Casein Concentrate (MCC). Micellar Casein Concentrate (MCC) would have a very lower concentration of lactose, SP, NPN and serum phase minerals compared to Skim Milk. MCC shows different properties as compared to skim milk because of this phenomenon. MCC has higher heat stability and shelf life.

• ULTRACENTRIFUGATION

Casein micelle occur as large aggregates, most (90-95%) of the casein in milk is sedimented by centrifugation at 100 000 g for 1 h. Sedimentation is complete at higher (30-37°C) than at low (2°C) temperature, at which some of the casein components disintegrate from the micelles and are non-sedimentable. Casein prepared by ultracentrifugation has its original level of colloidal calcium phosphate and can be redispersed as micelles with properties essentially similar to the original micelles.

• CENTRIFUGATION OF CALCIUM-SUPPLEMENTED MILK

Addition of calcium chloride, to about 0.2 M causes aggregation of the casein such that it can be readily removed by low-speed centrifugation. If calcium is added at 90"C, the casein forms coarse aggregates which readily precipitate. This principle is used in the commercial production of some 'casein co-precipitates' in which the whey proteins, denatured on heating milk at 90°C for ten minutes, co-precipitate with the casein. Such products tend to have very high ash content.

• ULTRAFILTRATION

The casein micelles are retained by fine-pore filters. Filtration through large-pore ceramic membranes is used to purify and concentrate casein on a laboratory scale. Ultrafiltration (UF) membranes retain both the caseins and whey proteins while lactose and soluble salts are permeable; total milk protein may be produced by this method. The casein micelles permeate the membranes used in microfiltration (pore size - 0.05-10 pm) but bacteria are retained by membranes with pores of less than OSpm, thus providing a method for removing more than 99.9% of the bacteria in milk without heat treatment; microfiltration is being used increasingly in several sectors of the dairy industry. Industrially, whey proteins are prepared by ultrafiltration or diafiltration of whey (to remove lactose and salts), followed by spray drying; these products, referred to as whey protein concentrates, contain 30-80% protein.



About Author / Additional Info:
I am currently pursuing PhD in Dairy Chemistry from National Dairy Research Institute Karnal.