Genomic modification tools for probiotic lactic acid bacteria and their application in dairy fermentation

This thesis explores the development and application of advanced genomic modification tools for lactic acid bacteria (LAB), focusing on enhancing probiotic functionality and improving dairy fermentation processes. By addressing the molecular mechanisms underlying plasmid-host interactions, genome editing, and the influence of prebiotics and probiotics in yogurt, this work contributes to the broader understanding of microbial genetics in the context of functional food development. Chapter 2 describes the discovery and characterization of a novel plasmid, pTRK829. This plasmid was generated through the spontaneous insertion of an insertion sequence (IS) element, ISLrh, from Lacticaseibacillus rhamnosus M1 into plasmid pGK12. The insertion of ISLrh altered the plasmid’s host range, enabling it to replicate in LAB strains that were previously incompatible, including L. rhamnosus M1, L. rhamnosus GG, Lacticaseibacillus casei ATCC 393, and Lacticaseibacillus paracasei ATCC 25598. However, pTRK829 was found to be unstable, which led to the discovery of a smaller and more stable variant, pTRK830, following a spontaneous deletion event. Besides changing the host range, the insertion of ISLrh also influenced plasmid stability and copy number across different host strains. Furthermore, pTRK830 was successfully used as an expression vector for heterologous gene expression in several lactobacilli species. This study provides valuable insights into plasmid-IS element interactions, with potential applications in expanding the host range of plasmids for LAB research and biotechnological purposes. Building upon the findings of Chapter 2, Chapter 3 describes the development of a novel genome editing tool for LGG using its endogenous type II-A CRISPR-Cas9 system. This study first characterized and verified the activity of the native CRISPR-Cas9 system. Then, by leveraging the native CRISPR-Cas9 machinery, an artificial single guide RNA (sgRNA) was employed to target specific genomic loci, enabling precise genetic modifications in LGG. Moreover, this system was successfully used to construct a lactose-positive LGG strain, MJM570, which demonstrated improved growth in milk due to its ability to metabolize lactose. MJM570 also showed enhanced performance during yogurt fermentation and retained high viability during cold storage, making it a promising candidate for both probiotic and starter culture applications. This work underscores the potential of genome editing to enhance probiotic functionality in food production and illustrates how the integration of CRISPR technology can be harnessed for the creation of next-generation probiotics tailored to specific industrial needs. Chapter 4 shifts focus to the interaction between probiotics and prebiotics in yogurt fermentation. This chapter investigates the effects of adding 2’-fucosyllactose (2’-FL), a prebiotic, and different Bifidobacterium strains on yogurt properties, including pH, organic acid production, and bacterial viability during refrigerated storage. The results indicate that 2’-FL remained stable in yogurt for up to five weeks of cold storage, with no significant impact on fermentation parameters or the viability of starter cultures. However, the addition of Bifidobacterium strains negatively affected the survival rate of the yogurt starter cultures, and the presence of 2’-FL did not enhance the viability of bifidobacteria. These findings suggest that while 2’-FL is a promising prebiotic for inclusion in yogurt formulations, its benefits on bifidobacterial viability may be strain-dependent, and the interaction between bifidobacteria and yogurt starter cultures requires further investigation.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Food Science & Human Nutrition

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Xie, Zifan

Contributors dc:contributor

• Miller, Michael J
• Jin, Yong-Su
• Stasiewicz, Matthew J
• Cadwallader, Keith R

• Genome Editing
• Lactic Acid Bacteria
• Probiotic
• Fermentation
• Plasmid
• Dairy

Statement dc:rights

• Copyright 2024 Zifan Xie

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/127468

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F127468