Solubility of dairy proteins is measured by determining the quantity of protein nitrogen that is water soluble versus the quantity of protein nitrogen that is water insoluble. Proteins are long chains of amino acids. Nitrogen is part of the backbone of every amino acid. Therefore, to measure protein solubility, one needs only to measure the solubility on the amino acid nitrogen in the protein. There are many slightly different methods for measuring protein solubility. Each one, because of the slightly different analytical procedures, will yield differing results with some yielding higher solubility values and some yielding lower solubility values for the same protein sample.

Therefore, when measuring dairy protein solubility, it is important to analyze all protein powders using the same analytical methodology in order to yield results that show relative solubility values. While there are many methods, most of them involve the following steps:

Protein Solubility (Nitrogen Solubility Index)

  1. Mixing a known quantity of protein powder into water.
  2. While keeping the dispersion temperature below that which would cause protein denaturation (usually below 130°F and usually around room temperature), allow the protein to hydrate for a specified amount of time.
  3. A sample of the aqueous protein dispersion is removed and analyzed for total protein content (total nitrogen content).
  4. Another portion of the same protein dispersion is centrifuged for a specified period of time at a specified centrifugal force. Centrifuging should yield a precipitate at the bottom of the centrifuge tube and a clear supernatant.
  5. Just to be sure, the supernatant is usually filtered through fine filter paper to remove any insoluble protein fines that may still be floating in the supernatant.
  6. The centrifuged and filtered supernatant is then analyzed for protein content (nitrogen content). The supernatant contains the water soluble protein.
  7. NSI is calculated by dividing the supernatant protein content by the total protein content and multiplying by 100.

Whey Protein Solubility (Whey Protein Nitrogen Index)

The steps are pretty much the same as for NSI, with the exception that one must first remove all casein nitrogen from the milk or MPC sample without causing damage to the whey proteins. This is usually accomplished by addition of certain chemical agents that will cause a complete precipitation of the casein as a curd but will not cause damage to the whey proteins in between steps 3 and 4 above (after a sample of the hydrated protein dispersion is removed for total protein nitrogen analysis). Once the casein curd has been precipitated out, it is filtered away from the resultant whey and the whey is subjected to the same procedure as for NSI determination. The whey contains the soluble portion of the whey protein. That portion of the whey that was denatured and insoluble is removed with the casein precipitate. WPNI is calculated by dividing the protein content of the clarified whey by the total protein content (before the casein and insoluble whey protein were removed) and multiplying by 100.

Of course, there are more recently developed methods involving the use of chromatographs to determine both casein content and whey content of samples. Most companies, however, rely on the centrifugation method to determine dairy protein solubility.