Coalescence:
- an irreversible increase in the size of fat globules and a loss of identity of the coalescing globules;
Flocculation:
- a reversible (with minor energy input) agglomeration/clustering of fat globules with no loss of identity of the globules in the floc; the fat globules that flocculate ; they can be easily redispersed if they are held together by weak forces, or they might be harder to redisperse to they share part of their interfacial layers;
Partial coalescence:
- an irreversible agglomeration/clustering of fat globules, held together by a combination of fat crystals and liquid fat, and a retention of identity of individual globules as long as the crystal structure is maintained (i.e., temperature dependent, once the crystals melt, the cluster coalesces). They usually come together in a shear field, as in whipping, and it is envisioned that the crystals at the surface of the droplets are responsible for causing colliding globules to stick together, while the liquid fat partially flows between they and acts as the "cement". Partial coalescence dominates structure formation in whipped, aerated dairy emulsions, and it should be emphasized that crystals within the emulsion droplets are responsible for its occurrence.
A good reference for more information on fat globules can be found in Mulder and Walstra.
Milk Lipids - Functional Properties
Like all fats, milkfat provides lubrication. They impart a creamy mouth feel as opposed to a dry texture. Butter flavour is unique and is derived from low levels of short chain fatty acids. If too many short chain fatty acids are hydrolyzed (separated) from the triglycerides, however, the product will taste rancid. Butter fat also acts as a reservoir for other flavours, especially in aged cheese. Fat globules produce a 'shortening' effect in cheese by keeping the protein matrix extended to give a soft texture. Fat substitutes are designed to mimic the globular property of milk fat. The spreadable range of butter fat is 16-24° C. Unfortunately butter is not spreadable at refrigeration temperatures. Milk fat provides energy (1g = 9 cal.), and nutrients (essential fatty acids, fat soluble vitamins).
Milk Proteins
Introduction and Review
The primary structure of proteins consists of a polypeptide chain of amino acids residues joined together by peptide linkages, which may also be cross-linked by disulphide bridges. Amino acids contain both a weakly basic amino group, and a weakly acid carboxyl group both connected to a hydrocarbon chain, which is unique to different amino acids. The three-dimensional organization of proteins, or conformation, also involves secondary, tertiary, and quaternary structures. The secondary structure refers to the spatial arrangement of amino acid residues that are near one another in the linear sequence. The alpha-helix and ß -pleated sheat are examples of secondary structures arising from regular and periodic steric relationships. The tertiary structure refers to the spatial arrangement of amino acid residues that are far apart in the linear sequence, giving rise to further coiling and folding. If the protein is tightly coiled and folded into a somewhat spherical shape, it is called a globular protein. If the protein consists of long polypeptide chains which are intermolecularly linked, they are called fibrous proteins. Quaternary structure occurs when proteins with two or more polypeptide chain subunits are associated.
Milk Protein Fractionation
The nitrogen content of milk is distributed among caseins (76%), whey proteins (18%), and non-protein nitrogen (NPN) (6%). This does not include the minor proteins that are associated with the FGM. This nitrogen distribution can be determined by the Rowland fractionation method:
Milk Proteins
Introduction and Review
The primary structure of proteins consists of a polypeptide chain of amino acids residues joined together by peptide linkages, which may also be cross-linked by disulphide bridges. Amino acids contain both a weakly basic amino group, and a weakly acid carboxyl group both connected to a hydrocarbon chain, which is unique to different amino acids. The three-dimensional organization of proteins, or conformation, also involves secondary, tertiary, and quaternary structures. The secondary structure refers to the spatial arrangement of amino acid residues that are near one another in the linear sequence. The alpha-helix and ß -pleated sheat are examples of secondary structures arising from regular and periodic steric relationships. The tertiary structure refers to the spatial arrangement of amino acid residues that are far apart in the linear sequence, giving rise to further coiling and folding. If the protein is tightly coiled and folded into a somewhat spherical shape, it is called a globular protein. If the protein consists of long polypeptide chains which are intermolecularly linked, they are called fibrous proteins. Quaternary structure occurs when proteins with two or more polypeptide chain subunits are associated.
Milk Protein Fractionation
The nitrogen content of milk is distributed among caseins (76%), whey proteins (18%), and non-protein nitrogen (NPN) (6%). This does not include the minor proteins that are associated with the FGM. This nitrogen distribution can be determined by the Rowland fractionation method:
- Precipitation at pH 4.6 - separates caseins from whey nitrogen
- Precipitation with sodium acetate and acetic acid (pH 5.0) - separates total proteins from whey NPN
The concentration of proteins in milk is as follows:
Caseins, as well as their structural form - casein micelles, whey proteins, and milk enzymes will now be examined in further detail.
Caseins
The casein content of milk represents about 80% of milk proteins. The principal casein fractions are alpha(s1) and alpha(s2)-caseins, ß -casein, and kappa-casein. The distinguishing property of all caseins is their low solubility at pH 4.6. The common compositional factor is that caseins are conjugated proteins, most with phosphate group(s) esterified to serine residues. These phosphate groups are important to the structure of the casein micelle. Calcium binding by the individual caseins is proportional to the phosphate content.
The conformation of caseins is much like that of denatured globular proteins. The high number of proline residues in caseins causes particular bending of the protein chain and inhibits the formation of close-packed, ordered secondary structures. Caseins contain no disulfide bonds. As well, the lack of tertiary structure accounts for the stability of caseins against heat denaturation because there is very little structure to unfold. Without a tertiary structure there is considerable exposure of hydrophobic residues. This results in strong association reactions of the caseins and renders them insoluble in water.
Within the group of caseins, there are several distinguishing features based on their charge distribution and sensitivity to calcium precipitation:
- Two hydrophobic regions, containing all the proline residues, separated by a polar region, which contains all but one of eight phosphate groups. It can be precipitated at very low levels of calcium.
- Concentrated negative charges near N-terminus and positive charges near C-terminus. It can also be precipitated at very low levels of calcium.
- Highly charged N-terminal region and a hydrophobic C-terminal region. Very amphiphilic protein acts like a detergent molecule. Self association is temperature dependant; will form a large polymer at 20° C but not at 4° C. Less sensitive to calcium precipitation.
- Very resistant to calcium precipitation, stabilizing other caseins. Rennet cleavage at the Phe105-Met106 bond eliminates the stabilizing ability, leaving a hydrophobic portion, para-kappa-casein, and a hydrophilic portion called kappa-casein glycomacropeptide (GMP), or more accurately, caseinomacropeptide (CMP).
Structure: The Casein Micelle
Most, but not all, of the casein proteins exist in a colloidal particle known as the casein micelle. Its biological function is to carry large amounts of highly insoluble CaP to mammalian young in liquid form and to form a clot in the stomach for more efficient nutrition. Besides casein protein, calcium and phosphate, the micelle also contains citrate, minor ions, lipase and plasmin enzymes, and entrapped milk serum. These micelles are rather porous structures, occupying about 4 ml/g and 6-12% of the total volume fraction of milk.
The "casein sub-micelle" model has been prominent for the last several years, and is illustrated and described with the following link, but there is not universal acceptance of this model, and mounting research evidence to suggest that there is not a defined sub-micellar structure to the micelle at all. Another model of a more open structure is also defined with the following link.
In the submicelle model, it is thought that there are small aggregates of whole casein, containing 10 to 100 casein molecules, called submicelles. It is thought that there are two different kinds of submicelle; with and without kappa-casein. These submicelles contain a hydrophobic core and are covered by a hydrophilic coat which is at least partly comprised of the polar moieties of kappa-casein. The hydrophilic CMP of the kappa-casein exists as a flexible hair.
The open model also suggests there are more dense and less dense regions within the midelle, but there is less of a well-defined structure. In this model, calcium phosphate nanoclusters bind caseins and provide for the differences in density within the casein micelle.
Colloidal calcium phosphate (CCP) acts as a cement between the hundreds or even thousands of submicelles that form the casein micelle. Binding may be covalent or electrostatic. Submicelles rich in kappa-casein occupy a surface position, whereas those with less are buried in the interior. The resulting hairy layer, at least 7 nm thick, acts to prohibit further aggregation of submicelles by steric repulsion. The casein micelles are not static; there are three dynamic equilibria between the micelle and its surroundings:
- the free casein molecules and submicelles
- the free submicelles and micelles
- the dissoved colloidal calcium and phosphate
The following factors must be considered when assessing the stability of the casein micelle:
Role of Ca++:
- More than 90% of the calcium content of skim milk is associated in some way or another with the casein micelle. The removal of Ca++ leads to reversible dissociation of ß -casein without micellular disintegration. The addition of Ca++ leads to aggregation.
- Some occurs between the individual caseins in the micelle but not much because there is no secondary structure in casein proteins.
- alpha(s1) and ß-caseins do not have any cysteine residues. If any S-S bonds occur within the micelle, they are not the driving force for stabilization.
- Caseins are among the most hydrophobic proteins and there is some evidence to suggest they play a role in the stability of the micelle. It must be remembered that hydrophobic interactions are very temperature sensitive.
- Some of the subunit interactions may be the result of ionic bonding, but the overall micellar structure is very loose and open.
- No sucess in relating these forces to micellular stability.
- As already noted, the hairy layer interferes with interparticle approach.
There are several factors that will affect the stability of the casein micelle system:
Salt content:
- affects the calcium activity in the serum and calcium phosphate content of the micelles.
- lowering the pH leads to dissolution of calcium phosphate until, at the isoelectric point (pH 4.6), all phosphate is dissolved and the caseins precipitate.
- at 4° C, beta-casein begins to dissociate from the micelle, at 0° C, there is no micellar aggregation; freezing produces a precipitate called cryo-casein.
- whey proteins become adsorbed, altering the behaviour of the micelle.
- by ethanol, for example, leads to aggregation of the micelles.
- When two or more of these factors are applied together, the effect can also be additive.
- Caseins are able to aggregate if the surface of the micelle is reactive. The Schmidt model further illustrates this.
Although the casein micelle is fairly stable, there are four major ways in which aggregation can be induced:
- chymosin - rennet or other proteolytic enzymes as in Cheese manufacturing
- acid
- heat
- age gelation
Enzyme Coagulation
Chymosin, or rennet, is most often used for enzyme coagulation. During the primary stage, rennet cleaves the Phe(105)-Met(106) linkage of kappa-casein resulting in the formation of the soluble CMP which diffuses away from the micelle and para-kappa-casein, a distinctly hydrophobic peptide that remains on the micelle. The patch or reactive site, as illustrated in the above image, that is left on the micelles after enzymatic cleavage is necessary before aggregation of the paracasein micelles can begin.
During the secondary stage, the micelles aggregate. This is due to the loss of steric repulsion of the kappa-casein as well as the loss of electrostatic repulsion due to the decrease in pH. As the pH approaches its isoelectric point (pH 4.6), the caseins aggregate. The casein micelles also have a strong tendency to aggregate because of hydrophobic interactions. Calcium assists coagulation by creating isoelctric conditions and by acting as a bridge between micelles. The temperature at the time of coagulation is very important to both the primary and secondary stages. With an increase in temperature up to 40° C, the rate of the rennet reaction increases. During the secondary stage, increased temperatures increase the hydrophobic reaction. The tertiary stage of coagulation involves the rearrangement of micelles after a gel has formed. There is a loss of paracasein identity as the milk curd firms and syneresis begins.
Acidification causes the casein micelles to destabilize or aggregate by decreasing their electric charge to that of the isoelectric point. At the same time, the acidity of the medium increases the solubility of minerals so that organic calcium and phosphorus contained in the micelle gradually become soluble in the aqueous phase. Casein micelles disintegrate and casein precipitates. Aggregation occurs as a result of entropically driven hydrophobic interactions.
At temperatures above the boiling point casein micelles will irreversibly aggregate. On heating, the buffer capacity of milk salts change, carbon dioxide is released, organic acids are produced, and tricalcium phophate and casein phosphate may be precipitated with the release of hydrogen ions.
Age Gelation
Age gelation is an aggregation phenomenon that affects shelf-stable, sterilized dairy products, such as concentrated milk and UHT milk products. After weeks to months storage of these products, there is a sudden sharp increase in viscosity accompanied by visible gelation and irreversible aggregation of the micelles into long chains forming a three-dimensional network. The actual cause and mechanism is not yet clear, however, some theories exist:
- Proteolytic breakdown of the casein: bacterial or native plasmin enzymes that are resistant to heat treatment may lead to the formation of a gel
- Chemical reactions: polymerization of casein and whey proteins due to Maillard type or other chemical reactions
- Formation of kappa-casein-ß -lactoglobulin complexes
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