Pro Tip: A UV-VIS spectroscopy reveals how protein is changed as it’s processed and shows the quality of protein emulsification.

Understanding what makes a good protein emulsifier is a difficult task for many ingredient designers. One tool that can help quantify how a process like extrusion or spray drying will impact these properties is ultraviolet-visible (UV-VIS) spectroscopy.

Spectrophotometers, including 96-well plate readers, measure how much light is absorbed by a protein. Depending on the colors of light that are absorbed or pass through, ingredient designers can gain insight on the protein structure and functionality. These machines can also be paired with chemicals that emit light if they are bound to a protein in particular regions, and these properties relate to a protein’s ability to form good emulsions. In general, these techniques take about two hours to prepare samples and run the equipment. 

A- wavelengths of different colors of light measured in visible light spectroscopy. Wave lengths lower than 380 nm (purple light) are considered ultra violet light.

B- Pea protein with hydrophobic surface area (purple) and hydrophilic surface area (blue) visualized. White area indicates a neutral surface area. ANS- probes bind to the purple areas in this protein.

C – Pea protein structure with tryptophan represented as red and purple spheres. Blue spheres are water molecules nearby theses residues. If there is more water near the residue, it turns redder, and if there is very little water around the tryptophan, it will appear more purple in UV-VIS spectroscopy.

Photo Credit: Thought Co.

One application for UV-VIS in plant-based protein is measuring the protein’s surface hydrophobicity. There is a correlation between the hydrophobic surface area and how good of an emulsion that protein will form. Using UV-VIS spectroscopy with ANS- probes, it is possible to measure the surface hydrophobicity. When the protein and ANS- probes are mixed, the probes bind to hydrophobic areas on the proteins’ surface, and when absorbance at 470 nm (purple light) is measured, it is directly related to the protein’s hydrophobicity (Panel B). In panel B, ANS- probes bind to the purple areas of this pea protein, and this is the protein’s hydrophobic surface area. When a protein unfolds, this area increases, which might make better emulsifiers. These measurements can be made before and after processing your plant-based proteins to help you engineer the exact characteristics you desire.

UV-VIS spectroscopy can also be used to see how a protein’s structure changes from processing. Tryptophan is an amino acid that naturally emits light in the ultraviolet range. If tryptophan is exposed to water, it will appear redder, and if it is not in water, it will appear more purple (Panel C). When a protein is in its native state, most of the tryptophan will be purple, but if the protein has unfolded, the tryptophan will be redder. This test can show how your processing changed the structure of the protein. If there is more exposed tryptophan, this indicates the protein denatured, and the protein might not behave like you expected. By testing proteins before and after processing, or at different processing steps, you can see exactly where and how your process changes the protein. This can help inform when temperature control is the most important, or how the chemicals used during processing impact the functionality of your protein. UV-VIS spectroscopy has many other applications, like measuring protein solubility or lipid oxidation, but by understanding how light and protein interact, you can quickly quantify how your process is impacting your proteins.

Harrison Helmick is a PhD candidate at Purdue University. Connect on LinkedIn and see his other baking tips at BakeSci.com.

His research is conducted with the support of Jozef Kokini, Andrea Liceaga, and Arun Bhunia.