Graduate Thesis Or Dissertation

“Red Light, Yellow Light!”: Evaluating the Anti-Listerial Potential of Bacillus Isolate Metabolites Using a High-Throughput Chromogenic Assay”

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  • Listeria monocytogenes contamination continues to pose challenges for the food industry and there is demand for effective methods of food preservation and protection that can also be considered clean label. A promising source of antilisterial compounds may be sourced from bacteria that produce novel byproducts. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are one class of bacterial byproducts that can have antilisterial applications in food systems. In the last decade, consumers have increasingly preferred products that have a clean label (i.e. a product with an ingredient list that is minimal, with naturally sourced ingredients that are easy to recognize) over more traditional preservatives to increase the safety and/or shelf life of their food. Non-pathogenic bacteria with the capability to produce RiPPs are easily cultured in food-grade ingredients like milk or dextrose and have recently gained the attention of the food industry. Lactic acid bacteria have been the primary focus of RiPPs studies due to their historical association with fermented foods and safe consumption; however, many other non-pathogenic bacteria are capable of producing RiPPs. The primary objective of this work was to identify bacterial isolates that produce antilisterial byproducts with the long-term goal of using them in food systems as a clean label additive. To approach this goal, a high-throughput chromogenic assay was adapted to screen approximately 450 bacterial strains previously isolated from dairy systems for their antilisterial potential. Twenty-seven isolates with antilisterial activity were identified, and whole genome sequencing was conducted and analyzed using BAGEL4 to identify potential RiPPs. Potential pathogenicity of isolates was evaluated using virulence factor databases. The influence of incubation conditions and growth medium on target bacteria and the antilisterial activity of candidate strain byproducts were explored. Strain-dependent sensitivity of Listeria spp. to 2,3,5-triphenyltetrazolium chloride (TTC) with and without the presence of buffering salt (K2HPO4) was investigated, and the antilisterial activity of candidates when cultured in UHT milk under high and low relative oxygen availability was determined. Preliminary studies of Bacillus-cultured UHT milk added to Mexican table cream was performed to investigate L. monocytogenes growth during refrigerated storage when exposed to potential Bacillus-produced byproducts in a food-grade substrate. 21 of the 27 isolates that demonstrated antimicrobial activity against multiple Listeria strains under the presented conditions were non-pathogenic Bacillus species identified as B. pumilus (11), B. licheniformis (2), B. paralicheniformis (4) B. amyloliquefaciens (3), and B. subtilis (1). Virulence factor databases confirmed the absence of pathogenic virulence factors on the candidates’ genomes. B. subtilis, B. pumilus, B. licheniformis, and B. amyloliquefaciens have at least one GRAS designation for use of the bacteria and/or their metabolites in food-processing. Analysis of RiPPs found on the Bacillus candidate genomes revealed the potential for the production of multiple distinct antilisterial byproducts (n = 34) with high degrees of variation and structure (e.g., geobacillin II, uviB, licheniciden, haloduracin, LCI, plantazolicin, sactipeptides, lasso peptides) previously reported in literature. Nearly all Bacillus candidates were found to have the potential to produce circular bacteriocins (i.e., pumilarin, closticin 574, enterocin Nkr-5-3B, circularin A, amylocyclicin, butyrivibriocin AR10), known for their resilience against extreme pH and temperature changes and are only recently expanded upon as a bacteriocin group. Other classes that have linear, globular, and two-component structures, showing potential for the presence of heterogenous antimicrobial peptides that may demonstrate effective antilisterial activity across a more neutral pH range. The production of antilisterial byproducts when Bacillus candidates were cultured in UHT milk was enhanced when incubated under higher oxygen availability (via rotisserie agitation), but candidates were not able to inhibit L. monocytogenes when cultured without this agitation. Cultured UHT milk was not able to inhibit L. monocytogenes when added to the formulation of a high-risk dairy product (Mexican table cream) and stored at refrigeration temperatures over the course of seven days. This research resulted in the discovery of non-pathogenic Bacillus isolates that demonstrate antilisterial activity against strains of L. innocua and L. monocytogenes and have the genomic potential to produce multiple heterogeneous inhibitive byproducts. The strains show promising potential for commercial applications as clean label additives in dairy systems. Additional research is needed to characterize potential byproducts produced in milk and optimize the culturing conditions necessary to maximize antimicrobial byproduct production by the Bacillus candidates.
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Peer Reviewed
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  • Intellectual Property (patent, etc.)
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  • 2024-04-10 to 2024-04-12



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