Food Science and Technology Department


First Advisor

Dr. Devin J. Rose

Date of this Version



Behounek, M. (2022). Differential Effects of Protein Isolates on the Gut Microbiome under High and Low Fiber Conditions. (Master Thesis, University of Nebraska- Lincoln, Lincoln, NE, United States).


A THESIS Presented to the Faculty of The Graduate College of the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Food Science and Technology, Under the Supervision of Professor Devin J. Rose. Lincoln, Nebraska: July, 2022

Copyright © 2022 Marissa Behounek


Protein isolates are a growing market share in the food industry both as food ingredients and as supplements. All dietary habits can influence and alter the gut microbiome; however, little is known about how protein isolates from different sources will change the composition and function of the gut microbiota under high and low fiber conditions. The study aims to determine the microbiome response to plant and animal protein isolates under high- and low-dietary fiber (H/LDF) conditions. Six commercially available protein isolates (beef, egg white, milk, pea, and two soy protein isolates) were subjected to in vitro enzymatic digestion and dialysis followed by in vitro fermentation with four microbiomes differing in dietary history. Two fermentation media containing 0.1% and 1% fermentable carbohydrate simulated LDF and HDF conditions, respectively. Plant protein isolates, which were all from legumes, had similar amino acid profiles, while the animal protein isolates had very different amino acid profiles depending on source. Under the HDF condition, the microbiome was primarily saccharolytic and there were minimal differences in fermentation properties among the different digested protein isolates. In contrast, under the LDF condition, the microbiome was proteolytic, as evidenced by decreases in peptide concentrations during fermentation and unique shifts in microbiome composition and function during fermentation of the digested protein isolates. Under the LDF condition, digested milk protein isolate increased the abundance of bacteria in the Clostridia class and the Firmicutes phylum with concomitant increases in butyrate production. Flavonifractor and Intestinimonas, genera with butyrate-producing pathways, were identified as differentially abundant genera associated with digested milk protein isolate after 24 h of fermentation. Soy proteins also resulted in high butyrate production, but induced increases in Uncl_Lachnospiraceae, Lachnoclostridium, and Butyricicoccus genera, suggesting a different pathway for butyrate production compared with digested milk protein isolate. Although digested milk protein and soy protein isolates resulted in high butyrate production, they also led to the highest concentrations of undesirable protein fermentation metabolites, ammonia and cadaverine, during fermentation. Several amino acids were found to be significantly correlated to metabolite production under the LDF condition, with glutamate and proline having a significantly positive correlation with butyrate production. In conclusion, digested protein isolates have differential effects on the gut microbiome, but only under conditions where dietary fiber is limited. Notably, digested milk and soy protein isolates were highly butyrogenic and increased abundance of some beneficial gut microbial taxa, but also led to high concentration of deleterious protein fermentation metabolites.

Advisor: Devin J. Rose