The influence of inulins with different average degree of polymerization (ranging from 3 to 25) on the metabolic activity of the human colonic microbiota with or without the addition of Clostridium difficile was investigated in vitro. The in vitro system used was a dynamic, computercontrolled model that simulates the conditions of the proximal part of the large intestine with peristaltic mixing, water absorption and absorption of fermentation products. The addition of inulin stimulated the formation of the total amount of short-chain fatty acids acetate, propionate and butyrate up to 50%, and lactate /10-fold for short-chain inulin, while the formation of ammonia and the branched-chain fatty acids iso -butyrate and iso -valerate was suppressed. Ammonia formation was suppressed by about 30% and that of iso -butyrate and isovalerate was almost completely suppressed. These effects became much more pronounced when C. difficile was present in the system. The introduction of C. difficile caused a stimulation of the production of the protein fermentative metabolites ammonia, branched-chain fatty acids and the phenolic compounds indole, phenol and p -cresol. This stimulatory effect of C. difficile was almost completely prevented by the addition of inulins. Thus, these results indicate a potential of inulins to shift the metabolic activity of the human colonic microbiota towards the production of less potentially toxic metabolites, both under normal conditions and under conditions with a disturbed microbiota (with a high level of C. difficile ).

The inherent and diverse capacity of dietary fibres, nondigestible oligosaccharides (NDOs) and prebiotics to modify the gut microbiota and markedly influence health status of the host has attracted rising interest. Research and collective initiatives to determine the composition and diversity of the human gut microbiota have increased over the past decade due to great advances in high-throughput technologies, particularly the 16S ribosomal RNA (rRNA) sequencing. Here we reviewed the application of 16S rRNA-based molecular technologies, both community wide (sequencing and phylogenetic microarrays) and targeted methodologies (quantitative PCR, fluorescent in situ hybridisation) to study the effect of chicory inulin-type fructans, NDOs and specific added fibres, such as resistant starches, on the human intestinal microbiota. Overall, such technologies facilitated the monitoring of microbiota shifts due to prebiotic/fibre consumption, though there are limited community-wide sequencing studies so far. Molecular studies confirmed the selective bifidogenic effect of fructans and galactooligosaccharides (GOS) in human intervention studies. Fructans only occasionally decreased relative abundance of Bacteroidetes or stimulated other groups. The sequencing studies for various resistant starches, polydextrose and beta-glucan showed broader effects with more and different types of gut microbial species being enhanced, often including phylotypes of Ruminococcaceae. There was substantial variation in terms of magnitude of response and in individual responses to a specific fibre or NDO which may be due to numerous factors, such as initial presence and relative abundance of a microbial type, diet, genetics of the host, and intervention parameters, such as intervention duration and fibre dose. The field will clearly benefit from a more systematic approach that will support defining the impact of prebiotics and fibres on the gut microbiome, identify biomarkers that link gut microbes to health, and address the personalised response of an individual’s microbiota to prebiotics and dietary fibres.

Analysis of the conservation of functional residues between yeast and Escherichia coli inorganic pyrophosphatases (PPases) suggested that Asp-97, Glu-98, Asp-102, and Lys-104 are important for the action of E. coli PPase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K., & Cooperman, B. S. (1990) Biochim. Biophys. Acta 1038, 338-345]. We replaced these four residues by oligonucleotide-directed mutagenesis, giving variant PPases DV97, DE97, EV98, DV102, DE102, KI104, and KR104. PPase variants DV97, DV102, and KI104 had no enzyme activity, whereas PPase variants DE97, EV98, DE102, and KR104 had 22%, 33%, 3%, and 3% of the wild-type PPase activity, respectively. This suggests that Asp-97, Asp-102, and Lys-104 are essential for the catalytic activity of E. coli PPase. PPase variants DV98 and KR104 also had an increased sensitivity to heat denaturation; incubation of these mutant PPases at 75 degrees C for 15 min in the presence of 5 mM magnesium ion decreased the activity to 20% and 1%, respectively, of the initial value while 74% of the activity was observed with wild-type PPase. Furthermore, these thermolabile mutant PPases displayed the most profound conformational changes of the PPase variants examined, as demonstrated by the binding of the fluorescent dye Nile red that monitors the hydrophobicity of protein surfaces. Accordingly, Glu-98 and Lys-104 seem to be important for the structural integrity of E. coli PPase.

The relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus alpha-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+ and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and temperature optima. Replacement of Asp-331 by Glu in the active site caused almost total inactivation. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling.

Inulin is a soluble dietary fibre, also classified as a prebiotic, extracted from chicory roots. The present study aimed to determine the effect of consumption of native chicory inulin on the stool frequency of middle-aged to older adults (40–75 years old) with uncomfortably but not clinically relevant low stool frequency, specified as two to four days without bowel movements per week. Two randomised, double blind, placebo-controlled crossover trials were conducted using similar protocols in differing populations. Trial A was conducted in Amsterdam, The Netherlands and subsequently Trial B was conducted in Newcastle, United Kingdom. Both trials involved supplementation for 5 weeks with 10 g per day of inulin or placebo, a washout period of 2 weeks, and then crossed over to receive the other treatment. In Trial B, faecal gut microbiota composition was assessed using 16S rRNA gene sequencing. In Trial A, which 10 volunteers completed, the stool frequency was significantly increased to an average 4.9 ± 0.23 (SEM) times per week during inulin periods versus 3.6 ± 0.25 in the periods with placebo (p = 0.01). In contrast, in Trial B which 20 volunteers completed, there was no significant effect of the inulin on stool frequency (7.5 ± 2.1 times per week with inulin, 8.1 ± 3.0 with placebo, p = 0.35). However, many subjects in Trial B had a stool frequency >5 per week also for the placebo period, in breach of the inclusion criteria. Combining the data of 16 low stool frequency subjects from Trials A and B showed a significant effect of inulin to increase stool frequency from 4.1 to 5.0 per week (p = 0.032). Regarding secondary outcomes, stool consistency was significantly softer with inulin treatment compared to placebo periods, it increased 0.29 on the Bristol stool scale (p = 0.008) when data from all subjects of Trials A and B were combined. No other differences in bowel habit parameters due to inulin consumption were significant. None of the differences in specific bacterial abundance, alpha or beta diversity were significant, however the trends were in directions consistent with published studies on other types of inulin. We conclude that 10 g per day of native chicory inulin can increase stool frequency in subjects with low stool frequency.