Oral supplementation of healthy adults with 2'-O-fucosyllactose and lacto-N-neotetraose is well tolerated and shifts the intestinal microbiota

Emma Elison, Louise K Vigsnaes, Laura Rindom Krogsgaard, Julie Rasmussen, Nikolaj Sørensen, Bruce McConnell, Thierry Hennet, Morten O A Sommer, Peter Bytzer, Emma Elison, Louise K Vigsnaes, Laura Rindom Krogsgaard, Julie Rasmussen, Nikolaj Sørensen, Bruce McConnell, Thierry Hennet, Morten O A Sommer, Peter Bytzer

Abstract

The gut microbiota has been established as an important player influencing many aspects of human physiology. Breast milk, the first diet for an infant, contains human milk oligosaccharides (HMO) that shape the infant's gut microbiota by selectively stimulating the growth of specific bacteria, especially bifidobacteria. In addition to their bifidogenic activity, the ability of HMO to modulate immune function and the gut barrier makes them prime candidates to restore a beneficial microbiota in dysbiotic adults and provide health benefits. We conducted a parallel, double-blind, randomised, placebo-controlled, HMO-supplementation study in 100 healthy, adult volunteers, consuming chemically produced 2'-O-fucosyllactose (2'FL) and/or lacto-N-neotetraose (LNnT) at various daily doses and mixes or placebo for 2 weeks. All participants completed the study without premature discontinuation. Supplementation of 2'FL and LNnT at daily doses up to 20 g was shown to be safe and well tolerated, as assessed using the gastrointestinal symptoms rating scale. 16S rRNA sequencing analysis showed that HMO supplementation specifically modified the adult gut microbiota with the primary impact being substantial increases in relative abundance of Actinobacteria and Bifidobacterium in particular and a reduction in relative abundance of Firmicutes and Proteobacteria. This study provides the first set of data on safety, tolerance and impact of HMO on the adult gut microbiota. Collectively, the results from this study show that supplementing the diet with HMO is a valuable strategy to shape the human gut microbiota and specifically promote the growth of beneficial bifidobacteria.

Keywords: 2′FL 2′-O-fucosyllactose; GI gastrointestinal; GSRS gastrointestinal symptom rating scale; HMO human milk oligosaccharide; LNnT lacto-N-neotetraose; OTU operational taxonomic units; 2′-O-Fucosyllactose; Clinical study; Lacto-N-neotetraose; Safety; Tolerance.

Figures

Fig. 1
Fig. 1
Flow chart of the study. A total of 110 healthy, adult volunteers were screened for eligibility to participate in the study; 100 of them were randomised to one of the following intervention groups: 2′-O-fucosyllactose (2′FL), lacto-N-neotetraose (LNnT) or 2:1 mix of 2′FL:LNnT, each in three daily doses of 5, 10 or 20 g, or 2 g glucose as placebo. GSRS, gastrointestinal symptom rating scale.
Fig. 2
Fig. 2
Gastrointestinal symptom rating scale (GSRS) scores at the end of the intervention. Scores ranged from 1 (no discomfort) to 7 (very severe discomfort). (a) 2′-O-fucosyllactose (2′FL) supplementation groups and placebo group; (b) lacto-N-neotetraose (LNnT) supplementation groups and placebo group; (c) 2′FL:LNnT (2:1) mix supplementation groups and placebo group. , 20 g, , 10 g, , 5 g, , placebo. GSRS scores at the end of intervention for placebo and the intervention group were compared using a two-way ANOVA and Bonferroni’s multiple comparisons correction. * Significantly different between the intervention group and the placebo group (P<0·05).
Fig. 3
Fig. 3
Principal coordinates analysis plot of generalised UniFrac distances for all samples collected. (a) Before intervention and (b) after intervention. Phyla abundances are overlaid in blue. Samples are divided into intervention groups with the label at the centre of gravity for each group. Before intervention, there is no clear pattern. After intervention, the human milk oligosaccharide supplementation groups followed an axis of increasing Actinobacteria and decreasing Firmicutes for increasing doses.
Fig. 4
Fig. 4
Relative abundance of faecal bacteria at the phylum level. (a and b) Phyla level in the three 2′-O-fucosyllactose (2′FL) groups receiving 5, 10 or 20 g and placebo before and after intervention; (c and d) phyla level in the three lacto-N-neotetraose (LNnT) groups receiving 5, 10 or 20 g and placebo before and after intervention; (e and f) phyla level in the three mix groups receiving 5, 10 or 20 g of 2′FL:LNnT (2:1) and placebo before and after intervention. * Significantly different between before and after intervention (P<0·05). , Actinobacteria; , Bacteroidetes; , Firmicutes; , Proteobacteria; , others.
Fig. 5
Fig. 5
Change in sequence abundance of Actinobacteria (a) andBifidobacterium (b). The box represents the median and the 25th to 75th percentiles. The whiskers represent the smallest and largest changes observed. * Significantly different between the intervention group and the placebo group (P<0·05). 2′FL, 2′-O-fucosyllactose; LNnT, lacto-N-neotetraose; Mix, 2′FL:LNnT (2:1).
Fig. 6
Fig. 6
Change in sequence abundance of three operational taxonomic units (OTU) showing high similarity to the described Bifidobacterium species,B. adolescentis, B. longum and B. bifidum. The box represents the median and the 25th to 75th percentiles. The whiskers represent the smallest and largest changes observed. * Significantly different between the intervention group and the placebo group (P<0·05). 2′FL, 2′-O-fucosyllactose; LNnT, lacto-N-neotetraose; Mix, 2′FL:LNnT (2:1). ,s1_r64; , s1_r2031; , s1_r379.
Fig. 7
Fig. 7
Relative abundance of faecal bacteria at the genus level from before and after intervention. The eighteen genera, Bifidobacterium,Bacteroides, Barnesiella,Parabacteroides, Prevotella,Alistipes, Lactobacillus,Eubacterium, Blautia,Coprococcus, Dorea, Lachnospiracea incertae sedis, Roseburia,Faecalibacterium, Ruminococcus,Dialister, Escherichia/Shigella andAkkermansia, selected have been associated with obesity, irritable bowel syndrome or inflammatory bowel disease. (a) The mean of relative abundance of eighteen genera from the three intervention groups given 10 g of human milk oligosaccharide (HMO). (b) Relative abundance of eighteen genera from the placebo. Values are means, with their standard errors represented by vertical bars. Multiple t test was performed followed by a calculation of false discovery rate indicated as an adjusted P-value. * Significantly different between the groups (P<0·05). Lachn_inc_sedis,Lachnospiracea incertae sedis; , Before; , after.
Fig. 8
Fig. 8
SCFA in faeces. Concentrations are given as mm/g faeces of acetate (a), butyrate (b) and propionate (c) in samples from each intervention group and placebo before () and after () intervention. The box represents the median and the 25th to 75th percentiles. The whiskers represent the smallest and largest concentrations measured. 2′FL, 2′-O-fucosyllactose; LNnT, lacto-N-neotetraose; Mix, 2′FL:LNnT (2:1).

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