Probiotics are defined as living organisms that benefit the host when administered, and the use of
probiotics to prevent or treat intestinal disorders is supported by a large body of literature.
Probiotics enhance host health via multiple mechanisms, including stimulating immunity, inhibiting
epithelial invasion, producing antimicrobial substances, improving absorption of nutrients, and
protecting the host against pathogens (Rolfe 2000).
Saccharomyces cerevisiae var. boulardii (S. boulardii), a patented yeast preparation and the only
probiotic yeast available on the market, has been widely studied as a probiotic. The clinical activity
of S. boulardii is especially relevant to antibiotic-associated diarrhoea and recurrent Clostridium
difficile intestinal infections (Czerucka, Piche, and Rampal 2007), and this yeast is used in many
countries as both a preventive and therapeutic agent for diarrhoea and other gastrointestinal (GI)
disorders because it is not affected by the administration of antimicrobial agents. Furthermore, S.
boulardii possesses many properties of a probiotic organism. For example, it survives transit
through the GI tract, it can grow at a temperature of 37 °C, and it inhibits the growth of a number of
microbial pathogens.
Each probiotic has its own specific mode of action how it benefits its host. Since much is known
about S. boulardii and its specific mode of action as a probiotic, it is also wise to consider other
species of yeast to discover other yeast probiotic modes of action. In the current project, the main
organism studied was the yeast, Debaryomyces hansenii (D. hansenii). Previous studies examined
D. hansenii as a probiotic for improving the growth and immunity of fish (Andlid, Vázquez-Juárez,
and Gustafsson 1998; Tovar-Ramı́rez et al. 2004), however, very few studies explained the
mechanism by which D. hansenii could act as a probiotic. The present study aims to examine this
species of yeast as a probiotic for human use, and to investigate the mechanism by which this yeast
could act as a potential probiotic in vitro, from its survival in the human gastrointestinal tract to the
adhesion on gut epithelial cells and ability to modulate immune responses.
It is important to understand how yeasts respond when subjected to a series of stresses in the
gastrointestinal tract, because it could explain why some yeast cells survive better than others do.
One important measure of cell health is the intracellular pH (pHi), since changes in cell pHi greatly
affect the metabolism of the cell. Quantitative measurements of pHi can be achieved using
fluorescent indicators that switch on or off at sharply defined pH values (Han and Burgess 2010)
along with fluorescence ratio imaging microscopy (FRIM). FRIM utilizes fluorescence microscopy
and multi-wavelength spectroscopy principles to provide ion concentration measurements with a
high degree of selectivity and sensitivity (Martin and Jain 1993). Ratio imaging of fluorescent
indicators is an extremely powerful tool for studying dynamic intracellular biochemistry in multiple
individual cells. For example, the response of a single yeast cell can be studied overtime, which can
reveal information regarding the regulatory mechanism involved in stress response. The ability to
see how a graded average population response is built from discrete unitary responses is an
important factor for understanding chemical signals (Tsien and Poenie 1986).
The primary objective of this thesis is to obtain a deeper knowledge and understanding of D.
hansenii as a potential probiotic by studying its (i) survival in in vitro gastrointestinal conditions,
(ii) adhesion to epithelial cell model surfaces, (iii) ability to modulate an immunological response in
dendritic cells, (iv) intracellular pH response upon exposure to an in vitro gastrointestinal model,
and (v) intracellular pH regulation strategies of individual cells overtime under extreme stress
conditions.