Levan Enhances Associated Growth of Bacteroides, Escherichia, Streptococcus and Faecalibacterium in Fecal Microbiota

Abstract

The role of dietary fiber in supporting healthy gut microbiota and overall well-being of the host has been revealed in several studies. Here, we show the effect of a bacterial polyfructan levan on the growth dynamics and metabolism of fecal microbiota in vitro by using isothermal microcalorimetry. Eleven fecal samples from healthy donors were incubated in phosphate-buffered defined medium with or without levan supplementation and varying presence of amino acids. The generation of heat, changes in pH and microbiota composition, concentrations of produced and consumed metabolites during the growth were determined. The composition of fecal microbiota and profile of metabolites changed in response to substrate (levan and amino acids) availability. The main products of levan metabolism were acetic, lactic, butyric, propionic and succinic acids and carbon dioxide. Associated growth of levan-degrading (e.g. Bacteroides) and butyric acid-producing (e.g. Faecalibacterium) taxa was observed in levan-supplemented media. The study shows that the capacity of levan and possibly also other dietary fibers/prebiotics to modulate the composition and function of colon microbiota can be predicted by using isothermal microcalorimetry of fecal samples linked to metabolite and consortia analyses.

Fig 1. Bacterial taxa in individual fecal samples and their abundance after the growth in microcalorimeter.

(A) 22 bacterial taxa of fecal samples (FS1-11) with average abundance ≥ 1% are shown (sum of reads in relative scale, %). (B) abundance of genera (family/order level if the genus was not identified) per million cells (1 read = 1 cell) after growth on levan (red dots), levan plus amino acids (green triangles) or without added substrates (blue rectangles) in a logarithmic scale. All data points presented as average values of all fecal samples. *—statistical difference between the samples with levan and without added substrates (p-value < 0.05), **—statistical difference between the samples with levan and levan + amino acids (p-value < 0.05).

Fig 2. Growth characteristics of fecal consortia on media of varied composition.

(A) Growth of fecal cultures from group I (FS1-5) on medium without levan and amino acids. (B) Growth of fecal cultures from group II (FS6-11) on medium without levan and amino acids. (C) Growth of fecal cultures from group I on medium with levan. (D) Growth of fecal cultures from group II on medium with levan. μ - specific growth rate (h^-1), Q—heat accumulated (J) and P_max—maximum heat evolution rates (power, μW) achieved in different growth phases. Growth phase is defined as time between the power peak maxima, i.e. each power peak divides growth into two phases: before and after power peak maximum. Error bars indicate standard deviation of 2–4 biological replicates. Line graphs illustrate the heat evolution rate (P–power, μW) during the whole experiment. Note that line graphs on consortia growth with and without levan have different Y axis scale.

Fig 3. Production of organic acids, carbon dioxide and ethanol (A), biogenic amines and branched-chain fatty acids (B), and production/consumption of amino acids (C) (mmol/gDW) during growth of fecal consortia in media containing levan (in red), levan + amino acids (in green) or without added substrates (in blue).

The data presents the average of all fecal samples (n = 11 with 1–4 biological replicates). Dotted line indicates the zero level (no production or consumption). *—significant difference between levan and no-substrate conditions (p-value < 0.05), **—significant difference between levan and levan + amino acids (p-value < 0.05), ***—significant difference between levan and levan + amino acids and no substrate conditions (p-value < 0.05).

Fig 4. Proposed scheme for levan degradation and product formation on the basis of our experimental data and information from the literature.

Fig 5. Score plots of PLS-DA (A) and variable importance in projection (VIP) (B).

Plots derived from integrated analysis of microcalorimetric data (total heat accumulated (Q), maximal heat evolution rate (P_max), specific growth rate (μ)), consumption of substrates (levan, amino acids) and formation of products (organic acids, gases and ethanol), and the bacterial genera grown in microcalometry experiments. Analysis was done by MetaboAnalyst 3.0 program [25]. Levan+AA–medium containing levan and 20 amino acids. Groups I and II indicate the fecal samples of FS1-5 and FS6-11, respectively.