Role of Intestinal Microbiota in Ulcerative Colitis

6. Methodology, considerations and choices
Methodology part
6. Methodology, considerations and choices
The methodologies used in the experimental part of this thesis were chosen in response to a
number of circumstances and carefully balanced in order to meet the requirements of the overall
project, this thesis is part of. The considerations and choices for the methods used are described in
the following chapter.
6.1. In vitro fermentation systems
The preferred methods for testing prebiotic properties of novel carbohydrates are animals studies
or best humans trials using microbiological and immunological analysis. However, if a high number
of carbohydrates is to be tested both economically and ethically, in vitro experiments can be used
for screening, giving first indications of prebiotic properties, even though the models have the
limitations such as lack of host‐bacterial interaction and absorptive processes (Rastall, 2007).
6.2. Batch static systems
In the present thesis, static batch systems were used to evaluate the prebiotic properties of novel
carbohydrates derived from sugar beet and potato pulp pectin. However, several experimental
parameters had to be determined prior to screening.
Reaction volume
The batch static system was chosen, since a large number of prebiotic candidates were to be
tested and, additionally only a low amount of the developed compounds was available. This also
meant that the reaction volume had to be scaled down to 2 ml (50 ml is often used (Barry et al.,
1995;Olano‐Martin et al., 2000;Karppinen et al., 2000)), and hence non‐pH controlled. Sanz et al.
(2005) has previously developed a non‐pH controlled microscaled batch system using 1 ml of
reaction volume, which was validated against a pH‐controlled batch system with larger reaction
volume (150 ml). The down scaling of volume allowed testing of experimental saccharides, which
were in limited supply.
Incubation time
An incubation time of 24 hours was used, based on preliminary growth experiments on FOS (5 g/l
and 10 g/l) using pure cultures of strains from the genera Lactobacillus, Bifidobacterium,
Clostridium and Bacteroides. The growth experiments showed that some of the species had a long
lag‐phase and needed a period of 24 hours to reach exponential growth. However, an incubation
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