JavaScript is disabled. Please enable to continue!
Print this page

Save this page as a PDF

Mobile search icon
Menu
Food Testing >> Resources >> Molecular Weight Distribution Analysis in Hydrolyzed Proteins for Pet Foods and Feed

Molecular Weight Distribution Analysis in Hydrolyzed Proteins for Pet Foods and Feed

Sidebar Image

Introduction

Hydrolyzed proteins play a crucial role in the pet food, animal nutrition, and feed industries, offering significant benefits in digestibility, allergen reduction, and bioavailability. This white paper explores the advantages of hydrolyzed proteins and their impact on product formulation. Additionally, it provides an overview of the analytical methods employed at Eurofins Nutrition Analysis Center (ENAC) to assess molecular weight distribution and optimize ingredient performance.

Hydrolyzed Proteins: Enhancing Digestion and Reducing Allergic Reactions

Proteins can come from plant-based sources, such as soy and wheat, or animal-derived sources, including milk, meat, bones, and even feathers. Sometimes proteins are allergenic, or they are difficult for the body to digest. To assist with these issues, proteins can be hydrolyzed into shorter peptide chains and amino acids using enzymes, heat, or acids.

Hydrolyzed proteins play a crucial role in reducing allergic reactions for both people and pets. By breaking them down into smaller peptides and amino acids, hydrolysis effectively removes many allergenic epitopes—the parts of proteins that can trigger immune responses (1). This makes hydrolyzed proteins an excellent option for individuals or animals with sensitivities to traditional protein sources.

 

Figure 1. Proteins are hydrolyzed into small peptides and amino acids by hydrolysis using enzymes, acids, bases, or heat.
Figure 1. Proteins are hydrolyzed into small peptides and amino acids by hydrolysis using enzymes, acids, bases, or heat. Photo credit: www.veterinary-practice.com/article/food-hypersensitivity-and-hydrolysed-protein-diets

Hydrolyzed proteins don’t only reduce potential for allergic reactions.  they also aid in digestion. Because the proteins go through hydrolysis, in a sense they are already pre-digested. This makes it easier and faster for the body to process, increasing bioavailability, and it is gentler on the digestive system. Their ability to enhance palatability enables manufacturers to reduce reliance on additives and fillers, aligning with industry trends toward cleaner labels and more natural ingredient sourcing. As demand for high-quality, digestible proteins continues to grow, hydrolyzed formulations present a valuable solution for animal nutrition.

Method

During the molecular weight distribution analysis, a sample is first analyzed for crude protein by Kjeldahl. This initially estimates the total amount of protein in the sample by measuring the amount of nitrogen present. The sample is then dissolved in an extraction solution for 1 hour and centrifuged to separate into the supernatant containing dissolved protein and the undissolved protein residue. The undissolved residue is dried, weighed, and analyzed again for crude protein by Kjeldahl. The dissolved portion is decanted and then analyzed by gel permeation chromatography (GPC) to determine the molecular weight distributions for the dissolved portion of the hydrolyzed proteins.

 

Figure 2. After extraction, the samples are centrifuged to collect the supernatant – dissolved protein and the residue – undissolved protein.
Figure 2. After extraction, the samples are centrifuged to collect the supernatant – dissolved protein and the residue – undissolved protein.

Gel Permeation Chromatography (GPC) is a type of size exclusion chromatography that separates molecules, such as the hydrolyzed peptides, based on their molecular weight. The column is filled with porous gel beads, which elute peptides according to their size relative to the bead pores. Larger peptides cannot enter the pores and elute first, while smaller peptides travel through the pores and elute later (2). As a result, peaks for larger molecular weights appear first, followed by smaller ones. For example, the chromatogram in Figure 4 illustrates one of our standard mixes, showing clean separated peaks with known molecular weights from 40 to 2kDa. For this method, we use six protein standards, giving us a range of 40 to 1kDa. With these standards, we can measure the molecular weight distribution relative to the calibration curve to give a molecular distribution profile of a hydrolyzed protein raw material.

Figure 3. A typical GPC column is filled with porous gel beads. Larger peptides cannot enter the pores and elute first, while smaller peptides travel through the pores and elute later.
Figure 3. A typical GPC column is filled with porous gel beads. Larger peptides cannot enter the pores and elute first, while smaller peptides travel through the pores and elute later. Photo credit: www.creative-biostructure.com/custom-size-exclusion- chromatography-service-259.htm
Figure 4. This chromatogram illustrates one of the standard mixes, showing clean separated peaks with known molecular weights of 43kDa for the first, 12kDa for the second, and 2.5kDa for the third.
Figure 4. This chromatogram illustrates one of the standard mixes, showing clean separated peaks with known molecular weights of 43kDa for the first, 12kDa for the second, and 2.5kDa for the third.

The known molecular weight standards also help us determine our molecular weight boundaries. Figure 5 is an example chromatogram of what a typical sample might look like. On Figure 5, the chromatogram is split into molecular weight markers of 40 to 1 kDa based on the previously mentioned standards. The GPC software calculates each area between the molecular markers as a percentage of the broad unknown peak that we analyze from the dissolved supernatant of the sample. The results are reported in %, where the sum equals 100%.

Figure 5. The left chromatogram is an example of what a typical sample might look like. On the right, the chromatogram is split into molecular weight markers of 40, 20, 10, 5, 2, and 1 kDa.
Figure 5. The left chromatogram is an example of what a typical sample might look like. On the right, the chromatogram is split into molecular weight markers of 40, 20, 10, 5, 2, and 1 kDa.

Conclusion

Molecular weight distribution profiles give clients a glimpse to the degree of protein hydrolysis in their raw ingredients intended for use in pet foods, human foods, and feeds. With the use of a wide molecular weight range and gel permeation chromatography, ENAC can give clients their profile information to help digestibility, reduce allergenicity, and increase palatability of their formulations. This approach supports improved product development and performance across various industries.

Reach out today to learn how ENAC's scientific expertise can assist in meeting your molecular weight analysis needs.

Connect with an expert.

 

About the Author

Emily McManus, Associate Method Development Scientist, Eurofins Nutritional Analysis Center, Des Moines, IA

Emily McManus is one of the scientists on the Method Development Team at Eurofins Nutrition Analysis Center (ENAC) in Des Moines, IA. Emily has spent over 7 years at Eurofins and began as a laboratory technician in the Vitamins department before transitioning as a scientist on the Method Development team in 2022. Emily’s expertise at ENAC has been with water soluble vitamins before expanding into new areas of analysis. Emily was the leading scientist for the development of the Molecular Weight Distribution Analysis verification study in Des Moines.


References

https://www.eurofinsus.com/food-testing