Coagulation

Milk Structure

Structural elements of milkRaw milk quality provided an introduction to milk chemistry. Now we look briefly at milk physics to help understand how milk coagulation works. Refer to the figure on the right and review the following facts:

  • Milk is an emulsion with fat particles (globules) dispersed in an aqueous (watery) environment.
  • The fat globules do not coalesce and form a separate layer (oil off or churn) because they are protected by a membrane layer which keeps the fat particles separate from the water phase.
  • The principal group of milk proteins, the caseins, are not soluble in water and exist in milk as small particles (<300 nm) called micelles.

We can now define the following terms:

Milk: a dispersion of milk fat globules (fat particles) and casein micelles (protein particles) in a continuous phase of water, sugar (lactose), whey proteins, and minerals. 

Milk Plasma: what is left after you separate the fat globules; equivalent to skim milk for practical purposes.

Milk Serum: what is left after you take away both fat globules and casein micelles; equivalent to cheese whey for most practical purposes

Milk permeate: what is left after you take away fat globules, casein micelles, and whey proteins.

Coagulation is what happens when the casein micelles stick together. Because casein particles are hydrophobic (they hate water) their natural tendency is to aggregate (clump together). In normal milk, aggregation is prevented by two factors. If one of these factors is eliminated the micelles will aggregate and form a gel something like jello.

  • The first stabilizing factor is a 'hairy' layer of surface active protein, called kappa-casein (-casein), on the surface of the micelle. This layer helps prevent the micelles from getting close enough to stick together.
  • The second factor is a negative charge on the micelles. At the pH of milk the micelles are negatively charged so they repel each other.

So, basically there are two ways to coagulate milk; one is to remove the hairy layer from the micelles. That's called enzymic coagulation. The other is to neutralize the negative charge on the micelle. That can be accomplished by acidification or a combination of high temperature and acidification.

Enzymic Coagulation of Milk

The three stages of enzymic coagulation

(1) Primary Stage

In the first stage, the enzyme (rennet) cuts off a specific fragment of one of the caseins, namely, -casein. At the natural pH of milk, about 80% of -casein must be cleaved to permit aggregation of the micelles to proceed.

(2) Secondary Stage

The next stage is the physical process of aggregation of casein particles (micelles) to form a gel. After losing its water soluble tail, -casein can no longer keep the casein particles separated, so they begin to form chains and clusters. The clusters continue to grow until they form a continuous, three dimensional network which traps water inside, and forms a gel, something like Jell-o.

(3) The third stage refers to an ongoing development of the gel network. For some cheese the gel is cut as soon as it is firm enough to do so. For others, like soft ripened cheese, cutting is delayed while the gel continues to become firmer.

Effects of processing parameters on enzymic coagulation

Because rennet coagulation takes place in stages, it is necessary to understand the effect of processing on each stage. We will focus mainly on only the first and second stages.

Effect of pH. Lower pH increases enzyme activity and neutralizes charge repulsion between micelles. Therefore, both primary and secondary stages of coagulation proceed more quickly at lower pH.

Effect of Calcium . Calcium is not required for the primary stage (i.e., enzyme hydrolysis of -casein) but is essential to aggregation of the casein micelles. At low levels of calcium the primary phase goes to completion. Subsequently, instantaneous coagulation can be induced by adding sufficient calcium chloride.

Effect of temperature. The optimum coagulation temperature for most cheese is 30-32C, the exception is Swiss which is set at 37C.

  • At temperature less than 30C the gel is weak and difficult to cut without excessive yield loss due to fines.
  • At temperatures less than 20C coagulation does not occur, but the primary stage goes to completion and the milk will then coagulate quickly when warmed.

Effects of heat treatments.

  • Mild heat treatment such as pasteurization decreases the rate of the secondary stage. During heat treatment calcium and phosphate move from soluble to colloidal (insoluble) form, so there is less calcium available to assist with coagulation. This effect is reversed by cold storage or the addition of calcium chloride
  • Heat treatment in excess of pasteurization results in increased clotting time and a weak gel. High heat treatments cause absorption of whey proteins onto the casein particles. The casein particles are then unable to form a strong gel.

Effects of Homogenization: The following effects occur if the cheese milk is homogenized in its entirety. As noted in Treatment of milk for cheese making, some of these results may be different if only the cream is homogenized and then added back to the skim milk. Homogenization primarily affects the secondary phase of aggregation. Some cheese quality effects are also noted.

  • Reduced aggregation of casein particles
  • Decreased syneresis
  • Finer gel network due to smaller fat globules
  • Improved texture of soft cheese
  • Fat recovery (i.e., percent transfer from milk to cheese) is increased (Note: the same is true for acid and heat/acid coagulated cheese).
  • Hard cheese becomes rubbery
  • Makes cheese whiter because the yellow fat is masked by the artificial protein membranes on the homogenized fat globules.

Coagulating Enzymes

The traditional enzyme is rennet (chymosin) which is derived from the abomasum of the milk fed calf. The practice of cheese making probably began when somebody discovered that milk stored in bags made from calf stomachs formed a sweet curd.

Other proteases which have been used for cheese making include:

  • Pepsins from the pig, cow and chicken
  • Microbial proteases (Mucor miehi, Mucor pusillus, and Endothia parasitica).
  • Synthetic chymosin by recombinant DNA techniques using strains of Eshericia coli or Klaveromyces lactis or Aspergillus niger as host organism is now available. The transferred genetic material exists in the host cell in the form of a plasmid and is used as a template for the production of an enzyme identical to chymosin.

Figure 8.3 Manufacture of chymosin (calf rennet) and fermentation produced recombinant chymosin

Requirements of suitable coagulating enzymes

  • Suitable ratio of clotting to proteolytic activity (C/P). This ratio is dependent on the specificity of the enzyme for the Phe105-Met106 bond of -casein. Most rennet substitutes are more proteolytic than rennet (i.e., low C/P) and cause diminished yields of casein and fat, and bitterness during ripening
  • Proteolytic specificity. Structure and flavour of ripened cheese depends on the type of proteolysis caused by the coagulant during cheese curing. The exception is in cheese such as Swiss or Parmesan where most of the rennet activity is destroyed by the high cooking temperature. During ripening chymosin breaks down one of the caseins, namely s1-casein much more than other caseins.
  • High pH optimum. Rennet activity is stable and able to coagulate milk at the normal pH of milk although its activity increases with decreasing pH. Most pepsins and microbial proteases are denatured at the pH of milk which has been a major difficulty in developing rennet substitutes.
  • Denaturation temperature is important for two reasons:
    • Ripening due to coagulating enzymes is not desirable in cooked cheese such as Swiss and Italian types. Rennet is eliminated during the high temperature cook in these cheeses but microbial coagulants are not.
    • The coagulant must be eliminated by pasteurization to prevent proteolysis in products made from whey. Some microbial rennets survive pasteurization.
  • Distribution between curd and whey. Only 0-15% of rennet remains in the curd, but small amounts of residual rennet are significant to ripening of aged cheese. The most important factors which determine rennet retention are:
  • Cooking treatments.
    • As noted above, rennet does not survive in high temperature cooked cheese varieties.
    • In low cooked cheese such as Cheddar, variations in cooking temperature and time influence rennet activity during aging.
  • The pH at draining. Rennet is more soluble at low pH and, therefore, the amount retained in the curd increases with decreasing pH at draining. Retention of microbial rennets in the curd is independent of pH at draining.
  • Changing rennet sources may also influence rennet retention and cheese ripening. Different rennets with the same coagulating properties may have different thermal tolerances and different proteolytic characteristics.
  • Standard and consistent activity. Single strength rennet is standardized so that 200 ml coagulates 1,000 kg of milk in 30 - 40 min. Typical commercial rennet preparations are about 50% chymosin (calf rennet) and 50% bovine pepsin, so there is much opportunity for variation. Commercial calf rennet preparations are about 94-96% chymosin. Using recombinant rennet it should be possible to produce commercial rennet preparations which are more consistent with respect to all of the properties listed above, including proteolytic specificity and heat tolerance. 

Acid coagulation

Acid milk gels can be formed by lactic bacteria or the use of acidifying agents such as glucono-delta-lactone (GDL is slowly hydrolysed to gluconic acid in the presence of water). Acid coagulation is used in the production of cottage cheese, bakers cheese and quark as well as other fermented milk products such as yoghurt, commercial butter milk, kefir etc. In the case of cottage cheese and quark a small amount of chymosin may be used (2 ml/1,000 hl) to make the curd more elastic and less subject to breakage (dusting).

Heat-Acid coagulation

This process permits recovery of caseins and whey proteins in a single step. The basic principle is that whey proteins which are normally acid stable, become sensitive to acid coagulation after heat treatment. This principle is exploited in the manufacture of ricotta cheese, Paneer and Channa, and in the manufacture of "co-precipitated" milk protein concentrates. The basic process for heat-acid coagulation is:

  • Heat milk or milk-whey blends to at least 80C for at least five minutes to completely denature (unfold) the whey proteins and encourage association of whey proteins with casein micelles.
  • Continue heating and acidify slowly with gentle agitation. The caseins and whey proteins will coagulate together and form either sinking or floating curds.