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How plasmin proteases cause fat separation

Fat separation in UHT milk is a quality problem that rears its head sporadically and it can be difficult to predict and control. The most likely cause is that low levels of plasmin proteases have digested the main casein proteins that are stabilising the fat droplets, allowing them to coalesce and float to the surface.

Raw milk is a complex aqueous emulsion containing: lactose, whey proteins, casein “micelles” and fat globules that have a hydrophilic coating. For now, we’ll just focus on casein micelles and fat globules.

Milk fat globules have an average diameter of about 4 microns. Homogenisation breaks them down to 1/10th of that size. Before homogenisation the hydrophilic coating on the globules keeps them suspended, at least for the short term. Homogenisation is used to make the suspension permanent. The homogenisation process breaks the larger fat droplets into many smaller globules. This increases the total surface area of fat globules ten fold. With all the extra surface area, there isn’t enough of the hydrophilic coating to keep all the fat in suspension. This is where casein micelles come in handy. Casein protein from the micelles can supplement the fat droplets’ hydrophilic coating. Because of this, the homogenised milk fat globules are stable in suspension. In fact, because the droplets are smaller, they are much more stable than before.

Which is perfect, unless there is enough active plasmin protease to cause a problem.

Plasmin is a naturally occurring enzyme, from the cow, that passes unscathed through pasteurisation and ultra heat treatment. It chews up the main beta and alpha caseins that stabilise the droplets, leaving the droplets uncoated. Uncoated fat droplets aggregate and float to the surface of the milk. This is often the first visible sign of a protease problem and it is very apparent to consumers.

The following cartoon illustrates this process.

1. Milk contains large milk fat globules. The fats (yellow) are kept in suspension for a short time by a hydrophilic membrane coating (blue). There are also casein “micelles” (red), these are aggregates of casein proteins with calcium phosphate.

Homogenisation breaks down the large fat globules into much smaller ones and allows caseins to redistribute from casein micelles to exposed fat-water interfaces. Unlike unhomogenised milk, this system can be stable for 12 months or more.

2. When plasmin is present, however, it digests beta and alpha caseins, exposing the surfaces of the small fat globules

3. Over time, uncoated fat globules stick together, forming larger and larger globules until they become visible floating droplets.

For a more rigorous investigation of this phenomenon, see this open access paper from Zhang and co-authors of Wageningen University: