Graduate Studies


First Advisor

Robert Hutkins

Second Advisor

Jennifer Auchtung

Third Advisor

Devin Rose

Date of this Version

Fall 7-27-2021

Document Type



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 & Technology, Under the Supervision of Professor Robert W. Hutkins. Lincoln, Nebraska: July, 2021

Copyright © 2021 David Fabian Gomez Quintero


Synbiotics, combinations of beneficial microbes and substrates to support their growth, are of considerable interest due to their potential ability to improve gastrointestinal health. However, formulating synbiotics remain challenging, due in part, to the absence of rational strategies to assess these products for synbiotic activities prior to clinical trials. The goal of this project was to design potential synergistic synbiotics using an in vitro enrichment (IVE) method. This method relies on dilution pressure to identify probiotic strains that persist during stepwise fermentations when specific prebiotic substrates are provided. Seven commercial probiotics were tested, including proprietary strains of Bacillus coagulans, Bifidobacterium animalis subsp. lactis, Bifidobacterium longum subsp. infantis, Leuconostoc mesenteroides subsp. mesenteroides, Lacticaseibacillus paracasei subsp. paracasei, Lactiplantibacillus plantarum, and Lacticaseibacillus rhamnosus. For the IVE fermentations, each probiotic was paired with one of six prebiotics, including fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, pectin, inulin and 2-Fucosyllactose. Each pair was tested using ten different fecal samples. Quantitative PCR (qPCR) was used to quantify cells at various time points up to 72h. Persister, non-persister, and intermediate persister phenotypes were based on log reductions between 24h and 72h samples. To assess other changes in the microbiota during fermentation, 16S rRNA gene sequencing was also performed. As hypothesized, results showed that persistence during the IVE fermentations was dependent on the strain and substrate. All Lactobacillus and Bifidobacterium strains had consistent persister or intermediate persister phenotypes whereas Bacillus coagulans and Leuconostoc mesenteroides subsp. mesenteroides were non-persisters. Subject variation was also observed, indicating that fecal samples’ microbial composition affected persistence. 16S rRNA gene sequencing results showed that all treatments reduced microbiota diversity and relative abundance of many members of the community composition. For most samples, abundances of ASV’s corresponding to the added probiotic increased, consistent with qPCR results. Pure culture experiments revealed that probiotic growth on the prebiotics was not always an indicator of persistence. Ultimately, the results from this research provide a high-throughput in vitro basis for identifying synbiotics with persistence phenotype.

Advisor: Robert W. Hutkins