Measurement of in vivo fermentation of resistant starch
Two approaches were used to assess resistant starch fermentation: 1) Measurement of plasma acetate and breath hydrogen. The major source of acetate in blood in the fed state is from colonic fermentation of carbohydrate. Plasma acetate has been used previously for studying fermentation of dietary fibre. Breath hydrogen is also commonly used but there are several disposal routes for hydrogen making it a poor marker for slowly fermented carbohydrates like RS. 2) Use of stable isotope tracers (13C). Although 13C enriched starch has been used to measure digestion, this is the first study to use 13C-labelled pea flour to measure fermentation in human adults. Fermentation of raw potato starch was monitored by serial plasma acetate and breath hydrogen. Five subjects were fed 100g raw potato starch (34; RS2) in the evening and breath hydrogen and plasma acetate were measured throughout the next day. Guar gum was used as a fermentable standard. Breath hydrogen and plasma acetate increased within approximately 11 hours, peaking within 14 hours compared with 4 and 6 hours after guar gum. There was much variation in breath hydrogen and plasma acetate responses. The rise in plasma acetate occurred at a very different time to breath hydrogen. In most, but not all subjects, the rise and peak of plasma acetate happened earlier than for breath hydrogen. This makes it difficult to use these measurements for studies of slowly fermented carbohydrates. An alternative approach using stable isotopes was therefore explored. Starchy foods had to be enriched with 13C during their starch deposition phase. Peas and potatoes were chosen because of their potential high RS and faster rate of growth. A high 13C enrichment of pea flour was gained by photosynthetic incorporation of 13CO2. Pea plants (Baccara variety) were grown and when pods began to form, placed in a 13CO2 enriched environment in polypropylene bags sealed air-tight. 250 ml of 13CO2 were added and the bags filled to capacity with room air. The plants were incubated for 6 days on two occasions separated by 1 week. Peas were allowed to ripen under normal conditions and were harvested and dried to form flour. The mean atom % excess of 13C in once labelled pea flour was 2.4% and for twice labelled peas was 8.64%. Chemical and enzymatic attempts to separate the components of the pea flour were not totally successful, but it was clear that the label was distributed throughout the pea flour. Potatoes were not successfully labelled (mean atom % excess of 13C 0.71%) because the plants could not tolerate a long time in the polypropylene bags. The digestibility of starch in the pea flour, measured using the Englyst method (in vitro model), was 14.4% rapidly digestible starch, 63.7% slowly digestible starch and 21.9% RS. 300 ms labelled-pea flour incorporated into biscuits was fed to six subjects and breath samples taken every 30 mins for up to 34 hours (with a short gap when subjects were asleep) and analysed for hydrogen and 13CO2 enrichment. The appearance of 13CO2 in breath showed a complex of three peaks. The first peak occurred over the first 6 hours and should correspond to digestion and absorption of rapidly digestible and slowly digestible starch fractions in the small intestine.