Graduate Studies


First Advisor

Dr. Devin Rose

Second Advisor

Dr. Robert Hutkins

Date of this Version



Car Reen Kok, "In vitro enrichment (IVE): a novel method for formulating synergistic synbiotics" (2018).


A THESIS Presented to the Faculty of The Graduate College at 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 Professors Devin J. Rose & Robert W. Hutkins. Lincoln, Nebraska: December 2018

Copyright (c) 2018 Car Reen Kok


Research on the role of diet on gut and systemic health has led to considerable interest in identifying novel therapeutic modulators of the gut microbiome. This includes the use of prebiotics, probiotics, and synbiotics. Although most of the attention has long focused on probiotics, varying host responses have been reported among many clinical trials. This can be attributed to the absence of functional or ecological niches within the host environment, as well as host-mediated constraints mediated via colonization resistance. To address these challenges, the use of synergistic synbiotics have been proposed. These are comprised of prebiotic-probiotic combinations, formulated such that the probiotic is supplemented with a prebiotic substrate that it can specifically ferment. This would provide the strain with a competitive advantage and enhance its establishment in vivo. However, there is a lack of effective strategies for rational formulation of synergistic synbiotics. In this research, we propose in vitro enrichment (IVE) as a novel method for isolating probiotics that can function as synergistic synbiotics. Our approach involved stepwise in vitro fecal fermentations to enrich for and isolate Bifidobacterium strains that could ferment a candidate prebiotic, xylooligosaccharide (XOS). To test for synergistic properties of potential isolates, the strains were re-introduced into fermention vessels containing different fecal substrates. Successful establishment was based on re-isolation of the test strains after multiple generations. We subsequently isolated a putative probiotic strain, B. longum subsp longum CR15 that was successfully established in 36% of fecal samples (n=5) and potentially established in another 60% (n=10) using a total sample size of 17. In addition, we observed an ecological relationship between B. longum subsp longum CR15 and a strain of Bifidobacterium pseudocatenulatum that were both enriched upon XOS supplementation. Our findings revealed that the IVE method is a convenient method for isolating potential synergistic probiotic strains from a fecal environment and that also have the genetic and biochemical ability to ferment specific prebiotic substrates. The IVE method can potentially be used to greatly increase throughput of probiotic selection and isolation prior to further characterization and in vivo testing.

Advisors: Devin J. Rose & Robert W. Hutkins