Date of Award:

12-2013

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Nutrition, Dietetics, and Food Sciences

Committee Chair(s)

Jeff Broadbent

Committee

Jeff Broadbent

Committee

Donald McMahon

Committee

Robert Ward

Committee

Charles Miller

Committee

Scott Ensign

Abstract

Probiotics are living organisms which exert a beneficial health effect when consumed in sufficient numbers. Consumer interest in probiotics has increased dramatically in recent years prompting an increase in production and development of functional foods. One major problem is the decreased viability of probiotic bacteria during functional food production and storage and subsequent digestion due to environmental stresses. The most common probiotic strains belong to the genus Lactobacillus or Bifidobacterium. Due to the anaerobic nature of these bacteria, they lack the required defense mechanisms for oxidative stress inherent in aerobic microorganisms. This study examined the oxidative stress responses of six strains of Bifidobacterium, which are commonly used as probiotics in functional foods

The first phase of the study investigated the innate and inducible hydrogen peroxide (H2O2) stress response of Bifidobacterium longum strains NCC2705 and D2957, Bifidobacterium longum ssp. infantis ATCC 15697, and Bifidobacterium animalis ssp. lactis strains BL-04, DSM10140 and RH-1. Strains were screened for survival at increasing concentrations of H2O2 and lethal and sublethal concentrations were determined for each. In the second phase, B. animalis ssp. lactis strains BL-04 and DSM10140 and B. longum strains NCC2705 and D2957 were treated with a sublethal H2O2 concentration and RNA samples were collected for transcriptome analysis after 5 min and either 20 or 60 min. Statistical analysis was performed to identify genes that increased or decreased in expression during H2O2 treatment compared to control cells.

Results showed that survival was species and strain dependent and that strains which naturally survived higher H2O2 concentrations had a larger number of differentially expressed genes early on during H2O2 exposure. Some of the protective genetic systems that were activated during H2O2 stress are mechanisms which perform basic cellular functions under normal conditions such as deoxuynucleotide synthesis. Under stress conditions, these systems can be used to detoxify oxidative free radicals. Also a number of genes involved in sugar transport and energy production for the cell showed increased expression, which reveals the increased energy needs of the cells during oxidative stress.

During testing, it was found that two B. animalis ssp. lactis strains, BL-04 and DSM10140, had differing levels of survival and gene expression during H2O2 exposure despite having almost identical genome sequences. It was determined that one possible cause of the differences was a genetic deletion in a gene that allows the cell to incorporate extracellular fatty acids into the cell membrane instead of synthesizing them.

Results from this project have increased the understanding of oxidative stress responses in bifidobacteria and highlighted possible methods to increase bacterial survival during food manufacture, storage, and human digestion.

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