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2.3. Faecal batch culture fermentation
The fermentation profile of the freeze dried digested different
sized oat flakes, the prebiotic oligofructose (Raftilose P95 ORAFTI)
and the lowly fermented control carbohydrate cellulose (OXOID)
was determined using anaerobic, pH controlled faecal batch
cultures. Sterile stirred batch culture fermentation vessels (200 mL
working volume) were prepared and aseptically filled with 180 mL
of sterile basal nutrientmedium. Basalmedium contained per litre:
2 g Peptone, 2 g Yeast extract, 0.1 g NaCl, 0.04 g K2HPO4, 0.04 g
KH2PO4, 0.01 g MgSO47H20, 0.01 g CaCl26H2O, 2 g NaHCO3,2ml
Tween 80, 0.05 g Hemin dissolved in 1 ml of 4 M NaOH 10 ml
Vitamen K (SIGMA), 0.5 g L-Cysteine HCL, and 0.5 g Bile Salts
(sodium glycocholate and sodium taurocholate). The medium was
adjusted to pH 7.0 and 4 mL of 0.025% (w/v) resazurin solution
added prior to autoclaving. Once in the fermentation vessels, the
sterilemediumwas sparged with O2-free N2 (15mL/min) overnight
to maintain anaerobic conditions. The following day test substrates
were allowed to dissolve in the basal medium to give a final
concentration of 1% (w/v) before inoculation of the vesselswith 10%
(w/v) faecal slurry, which was prepared with pre-reduced sterile
phosphate-buffered saline (PBS, pH 7.0). Faecal samples were
collected fromthree healthy faecal donors. All donors were healthy
males aged between 23 and 31, and had not received antibiotic
treatment for at least 3 months prior to stool collection, had not
consumed pre- or probiotic supplements immediately prior to
experimentation, and had no history of bowel disorders. The temperature of the fermentation vessels was held at 37 C by use of
a circulating water bath, pH values were held between 6.7 and 6.8
by the addition of 0.5 M NaOH or HCl to the vessels, pH was
controlled via pH meter controllers (Electrolab260, UK) and
anaerobic conditions weremaintained by sparging the vessels with
O2-free N2 (15 mL/min). A 5 ml sample was taken from each vessel
immediately for analysis similarly samples were taken at 5, 10, and
24 h for analysis of SCFA by gas chromatography (GC) and for
analysis of bacterial populations by fluorescence in situ hybrid-
isation (FISH). This experiment was performed in triplicate.
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