Buckwheat (Fagopyrum esculentum) Hulls Are a Rich Source of Fermentable Dietary Fibre and Bioactive Phytochemicals

Abstract

Pseudo-cereals such as buckwheat (Fagopyrum esculentum) are valid candidates to promote diet biodiversity and nutrition security in an era of global climate change. Buckwheat hulls (BHs) are currently an unexplored source of dietary fibre and bioactive phytochemicals. This study assessed the effects of several bioprocessing treatments (using enzymes, yeast, and combinations of both) on BHs' nutrient and phytochemical content, their digestion and metabolism in vitro (using a gastrointestinal digestion model and mixed microbiota from human faeces). The metabolites were measured using targeted LC-MS/MS and GC analysis and 16S rRNA gene sequencing was used to detect the impact on microbiota composition. BHs are rich in insoluble fibre (31.09 ± 0.22% as non-starch polysaccharides), protocatechuic acid (390.71 ± 31.72 mg/kg), and syringaresinol (125.60 ± 6.76 mg/kg). The bioprocessing treatments significantly increased the extractability of gallic acid, vanillic acid, p-hydroxybenzoic acid, syringic acid, vanillin, syringaldehyde, p-coumaric acid, ferulic acid, caffeic acid, and syringaresinol in the alkaline-labile bound form, suggesting the bioaccessibility of these phytochemicals to the colon. Furthermore, one of the treatments, EC_2 treatment, increased significantly the in vitro upper gastrointestinal release of bioactive phytochemicals, especially for protocatechuic acid (p < 0.01). The BH fibre was fermentable, promoting the formation mainly of propionate and, to a lesser extent, butyrate formation. The EM_1 and EC_2 treatments effectively increased the content of insoluble fibre but had no effect on dietary fibre fermentation (p > 0.05). These findings promote the use of buckwheat hulls as a source of dietary fibre and phytochemicals to help meet dietary recommendations and needs.

Keywords: buckwheat (Fagopyrum esculentum) hulls; dietary fibre; enzyme bioprocessing; gut microbiota; in vitro digestion; in vitro fermentation; microbial metabolites; phytochemicals; short chain fatty acids.

Figure 1

Overview of experimental design: The bioprocessing treatments used in enzyme mixture I (EM), enzyme mixture II (EC), yeast fermentation (BY), and combinational treatments. The plant metabolites of the raw, first-bioprocessed, and second-bioprocessed buckwheat hulls were extracted into their free and bound forms and analysed by targeted LC-MS/MS; the monosaccharide compositions of soluble and insoluble non-starch polysaccharides were analysed with gas chromatography. The EM_1 (enzymes mixture I) and EC_2 (enzymes mixture II) treatments were selected for in vitro digestion and dialysis system (IVDG) and for in vitro fermentation studies. The concentrations of short chain fatty acids and other digestion and fermentation metabolites were measured using GC and LC-MS/MS analyses, respectively. The microbial composition analysis was performed with 16S rRNA gene sequencing.

Figure 2

Total content of plant metabolites in the free, alkaline-labile, and acid-bound forms of raw and bioprocessed buckwheat hulls, including total phytochemicals, phenolic acids and derivatives, flavonoids, and lignans (a). Total content is the cumulative sum of the content of individual plant metabolites measured LC-MS/MS in free form, alkaline form, or acid form. Individual phenolic acids and derivatives with a total content of free, alkaline, and acid forms of more than 10 mg/kg affected by both first and second bioprocessing technique, including derivatives of benzoic acid (b), benzaldehydes (c), and cinnamic acids (d). Individual flavonoids with a total content of free, alkaline, and acid forms of more than 10 mg/kg (e). The most abundant lignan compound (f). Within the first bioprocess: EM_1 = enzymes mixture I, BY_1 = Baker’s Yeast, EM+BY_1 = Baker’s yeast fermentation together with enzymes mixture I, within the second bioprocess: EM_2 = enzymes mixture I, EC_2 = enzymes mixture I with cellulase, BY_2 = Baker’s Yeast, EC+BY_2 = Baker’s yeast fermentation together with mixture I and cellulose. Data are represented with the mean ± S.D. Asterisks indicate the significance between the raw and bioprocessed samples, using One-Way ANOVA test: (*) p < 0.05 and (**) p < 0.01.

Figure 3

Heatmap showing the changes of plant metabolites from buckwheat hulls before (raw) and after bioprocesses during mixed microbiota incubations, including metabolites with consistent increase (a) and consistent decrease (b) during the in vitro colonic fermentation. Values were the mean of three donors, and each donor had three replicates. The concentration (log 10) was indicated by a colour gradient, where the darker represents the higher concentration. The multiple t-test with the Holm–Sidak method correction was used for statistical comparisons. Significance differences were * p < 0.05, ** p < 0.01, *** p < 0.001 when compared with 0 h samples. Between three donors, the consistently increased and decreased metabolites were marked with red and green, respectively. Principal component analysis (PCA) (unit variance (UV)-scaled) of average microbial metabolites measured from faecal incubations from three human donors (c) with baseline samples (0 h in green colour) for Raw, EM_1, and EC_2 treatments-EM and EC, IVDG-predigested samples, their blanks-B (0 and 72 h in pink colour); the metabolites measured at 24 h (bright blue colour), the microbial metabolites measured at 48 h (orange colour), and 72 h (dark blue colour).

Figure 3

Heatmap showing the changes of plant metabolites from buckwheat hulls before (raw) and after bioprocesses during mixed microbiota incubations, including metabolites with consistent increase (a) and consistent decrease (b) during the in vitro colonic fermentation. Values were the mean of three donors, and each donor had three replicates. The concentration (log 10) was indicated by a colour gradient, where the darker represents the higher concentration. The multiple t-test with the Holm–Sidak method correction was used for statistical comparisons. Significance differences were * p < 0.05, ** p < 0.01, *** p < 0.001 when compared with 0 h samples. Between three donors, the consistently increased and decreased metabolites were marked with red and green, respectively. Principal component analysis (PCA) (unit variance (UV)-scaled) of average microbial metabolites measured from faecal incubations from three human donors (c) with baseline samples (0 h in green colour) for Raw, EM_1, and EC_2 treatments-EM and EC, IVDG-predigested samples, their blanks-B (0 and 72 h in pink colour); the metabolites measured at 24 h (bright blue colour), the microbial metabolites measured at 48 h (orange colour), and 72 h (dark blue colour).

Figure 4

Short chain fatty acids (SCFAs) production over 72 h fermentation as incremental change (mM) in concentration, average ± SD (n = 3). The raw and enzyme-treated buckwheat hulls (EM_1 and EC_2), as well as their IVDG-predigested samples, were inoculated with faecal slurries from each of the 3 donors (donor 1, 2, and 3) and the faecal control samples representing the samples without the BH matrix. Values were calculated by subtracting the value of 0 h from the value of the fermented sample at 72 h for each sample. The SCFAs’ production across six matrixes were estimated by subtraction of the average values of faecal control samples from the average values of incremental changes in SCFA production for each matrix (values underneath plot).

Figure 5

Changes in microbial communities derived from human faecal inocula with raw buckwheat hulls as the sole added energy source or blank medium at 72 h time points. Four different measures of diversity (observed OTU richness, Chao estimate of total richness, Shannon diversity index, and inverse Simpson diversity index) are shown in (a). The relative abundance of top 10 bacterial families (b). Heatmap of the relative abundance of top 20 bacterial genera, the colour gradient (white–black) indicates the relative abundance of genus as labelled in each square (c). The abundance of selected species with sequence identity (d). D1, D2, D3 mean donor 1, donor 2, donor 3, respectively.