1. Introduction
Over the past decades, it became clear that the human gut microbiome constitutes an overlooked system that makes a significant contribution to human biology and development [
1]. Observational studies applying metabolomics and metagenomics have largely broadened our understanding on how the human gut microbiota is associated with, among other things, obesity-related diseases [
2], liver diseases [
3], inflammatory bowel disease (IBD) [
4], and colorectal cancer [
5]. A key function of the gut microbiome consists in the fermentation of carbohydrates in the colon, resulting among other things in the production of short-chain fatty acids (SCFA) [
6], mainly including acetate, propionate, and butyrate, which have each been related to particular health benefits as reviewed elsewhere [
7,
8,
9,
10]. Among the hundreds of gut microbes,
Bacteroides species display diverse and versatile glycan metabolising capabilities, allowing them to ferment extremely complex glycans [
11,
12,
13,
14], with other members such as
Bifidobacterium species growing on the polysaccharide degradation products and metabolites [
15]. In contrast to the beneficial effect of fiber fermentation, fermentation of amino acids results in the formation of potentially detrimental compounds (e.g., phenols, cresol, and hydrogen sulfide), contributing to IBD or colon cancer [
16]. Due to the purported role of dietary fiber and plant-based foods to inhibit such detrimental effects, there is a good rationale for developing functional foods that improve gut health via their impact on the gut microbiome [
1].
Besides probiotics and polyphenols, prebiotic carbohydrates that are defined as substrates that are selectively utilized by beneficial host microorganisms [
17] are widely studied for their health-promoting properties. While fructans such as fructo-oligosaccharides [
18] and inulin [
19] have well-established prebiotic effects, novel classes of prebiotics include, amongst others, human milk oligosaccharides [
20], arabinoxylans [
21], and a specific class of pectin-derived polysaccharides such as rhamnogalacturonan-1 (RG-I) [
22,
23,
24]. The backbone of RG-I consists of repeated units of the disaccharide [-4)-α-D-galacturonic acid-(1,2)-α-L-rhamnose-(1] with sidechains comprising mainly D-galactose (~galactans), L-arabinose (~arabinans), and D-xylose (~xylans) branching off the rhamnose residues [
23]. These sidechains, varying in composition and length, offer RG-I a structural complexity that requires the concerted action of different enzymes provided by a microbial consortium to be fermented [
25]. The development and demonstration of functionality of a particular source of RG-I relevant for the current paper, i.e., carrot-derived RG-I (cRG-I), was recently reviewed by McKay et al. [
26]. A clear immunostimulatory activity could be attributed to cRG-I both in vitro and in vivo, while in vitro studies demonstrated its capacity to modulate the human gut microbiota [
27].
The lack of access to the in vivo site of activity combined with SCFA being rapidly absorbed [
28] makes it very difficult to draw meaningful conclusions on SCFA production in vivo unless complex techniques such as a stable-isotope dilution method are applied [
29]. The use of in vitro models has been recognized as an established solution to overcome this issue [
30]. These models range from fecal batch incubation strategies [
27,
31] to pH-controlled reactor systems [
32,
33,
34,
35], each having their distinct benefits and potential drawbacks. An important aspect to take into account in gut microbiome research is the currently well-established considerable inter-individual differences among donors [
36], which can greatly impact the results of dietary interventions [
37] or in vitro outcomes of fiber fermentation [
31]. In vitro studies should thus ideally include several test donors.
This study aimed to investigate the impact of repeated administration of cRG-I on the luminal and mucosal gut microbiota, while addressing potential interindividual differences among subjects. This complements the data of an earlier study where cRG-I treatment was evaluated over 2 days in batch fermentations [
27]. cRG-I has earlier been shown to display immune modulation properties in vitro and to stimulate the innate immune response in healthy human subjects [
26]. The data described in this paper thus support that cRG-I exerts a dual mode of action by impacting both the host immune system and gut microbiota.
4. Discussion
Overall, the proper operation of the SHIME
® model during the current study followed from the stability of metabolic markers throughout the two-week control period that preceded the treatment period (
Figure 2). In absence of a parallel control, such stability was required to ascribe changes during the treatment period by cRG-I treatment. On the final day of the control period, there were marked interindividual differences among donors, both at metabolic (
Figure 2) and community composition level (
Figure 6). The microbiota of donors 1/2 was enriched with
Bifidobacteriaceae/
Lachnospiraceae (
Table S1) and produced higher amounts of butyrate (
Figure S2). While
Bifidobacterium sp. do not produce butyrate as such, they stimulate butyrate production by, e.g.,
Lachnospiraceae members via cross-feeding mechanisms [
53,
54]. In contrast, the microbiota of donors 3/4 was enriched with
Veillonellaceae (
Table S1) and produced high amounts of propionate.
Veillonella species are indeed known to convert lactate to acetate and propionate as main end-metabolites [
55]. The four donors under investigation thus allowed addressing the hypothesis whether differences in initial microbiota composition affected the outcomes of cRG-I treatment. Remarkably, cRG-I consistently stimulated microbial activity across the four donors tested with increases in average levels of acetate (+21.1 mM), propionate (+17.6 mM), and to a lesser extent also butyrate (+4.1 mM) despite baseline interindividual variability. This finding is consistent with a recent in vitro batch fermentation study that demonstrated acetate and propionate as main end-metabolites upon fermentation of cRG-I [
27]. We deliberately averaged the individual microbiota data to assess if we could identify overarching changes in the microbiota composition independently of the baseline starting point and found that this was indeed the case. The consistent modulation of microbial activity and composition thus suggests that cRG-I is fermented by a specific microbial consortium.
First, cRG-I consistently and strongly stimulated OTUs related to
B. dorei and
Prevotella sp. This specific increase of OTUs related to
B. dorei and
Prevotella sp. was also observed previously with the same compound [
27]. Interestingly, when dosing pectin to in vitro fermenters, Chung et al. also identified a specific increase of
B. dorei (detected as an OTU combined with
B. vulgatus) amongst other members of a mixed microbiota [
35]. The genera
Bacteroides and
Prevotella are indeed known as primary pectin degraders, possessing carbohydrate-active enzymes (CAZymes) within their polysaccharide utilization loci (PUL) [
12,
56]. Specifically for RG-I fermentation,
Bacteroides thetaiotaomicron has been demonstrated to ferment RG-I via several PULs, releasing polysaccharide breakdown products in the culture medium, making them available for other microbes [
25]. While several
Bacteroides species might be capable of fermenting cRG-I,
B. dorei seems the most capable species in the presence of a competitive background microbiota. This is supported by the genetic potential of
B. dorei containing over 50 predicted enzymes related to pectin degradation [
57]. At metabolic level, as
Bacteroides sp. are major producers of acetate and propionate [
58], the marked increase of both metabolites upon cRG-I treatment further corroborated the involvement of
Bacteroides sp. Besides the well-described health benefits of both SCFA as reviewed by Rivière et al. [
7], the presence of
B. dorei as such could contribute to other health benefits. Inhibition of atherosclerotic lesion formation, lower endotoxemia, and suppression of proinflammatory responses have been observed in atherosclerosis-prone mice upon co-administration of live
B. vulgatus and
B. dorei to mice [
59].
Upon cRG-I fermentation by primary degraders, polysaccharide breakdown products likely become available for other microbial species. Microbial members benefiting from such polysaccharide breakdown products included
Bifidobacterium longum of which a related OTU strongly increased upon cRG-I treatment (+1.32 log
10(cells/mL)). Even though the overall
Bifidobacteriaceae family did not increase in this experiment, there was a clear stimulation of
B. longum as previously observed in batch fermentations with cRG-I [
27]. Additional growth experiments indicated that monocultures of
Bifidobacterium species are unable to grow on cRG-I as a sole carbon source but are part of the bacterial consortium needed to ferment the complex cRG-I structure. This is in line with Chung et al., who did not identify any predicted enzymes concerned with pectin degradation in
Bifidobacteriaceae sp. [
57]. Moreover, this is also in agreement with the conclusion of Kelly et al. that the complexity of pectin is likely too high for
Bifidobacteriaceae, rendering them dependent on the initial degradation of these large polymers by
Bacteroides sp. [
15]. The released oligo- and monosaccharides could further be then scavenged by
Bifidobacteriaceae that indeed are capable of fermenting arabinans and galactans [
60,
61], both side chains of cRG-I [
26]. While likely not being the primary degraders, the consistent increase of
Bifidobacteriaceae species in this or preceding studies [
27], along with the well-documented health effects of members of this family [
15], support the health-promoting potential of cRG-I fermentation by the human gut microbiota.
Another microbial member that is likely part of the cRG-I fermenting consortium is
Phascolarctobacterium faecium as an OTU related to this species increased consistently over the four donors tested (+0.47 log
10(cells/mL)). The stimulation of this species likely followed from the succinate production of aforementioned
Prevotella sp. [
62] or
Bacteroides sp. [
58], thus boosting
Phascolarctobacterium faecium, an abundant colonizer [
63] that is able to convert succinate into propionate [
64]. Besides further contributing to health effects ascribed to propionate such as exerting anti-inflammatory effects, promoting satiety, lowering blood cholesterol, decreasing liver lipogenesis, and improving insulin sensitivity (as reviewed by Rivière et al. [
7]), the presence of
Phascolarctobacterium faecium has for instance also been linked to a positive mood [
65], further supporting the health-promoting potential of cRG-I fermentation by the human gut microbiota.
Other microbial families that consistently increased across the four donors tested included
Erysipelotrichaceae (DC),
Lachnospiraceae (PC and DC), and
Ruminococcaceae (PC). These changes correlated with the modest increases in butyrate levels that were observed upon cRG-I treatment, butyrate being indeed produced by members belonging to these families [
58]. Overall, the consistency of the changes across these families further supports the contribution of a specialized consortium in cRG-I fermentation.
As mentioned above, the first cRG-I fecal fermentation experiment was conducted in a 2-day batch incubation model [
27]. Interestingly, results obtained in the batch and quad-M-SHIME in vitro models with cRG-I were consistent at metabolic and compositional levels, resulting in high levels of acetate and propionate via the involvement of
Bacteroides dorei,
Prevotella sp., and
Bifidobacterium longum-related OTUs. Both studies also indicated the involvement of succinate scavengers, i.e.,
Phasolarctobacterium faecium (quad-M-SHIME) and
Dialister succinatiphilus (batch incubation). A difference between both models was that the batch incubation strategy allowed the observation of effects of cRG-I treatment on a broader range of microbes. It was shown that cRG-I also stimulated other
Bacteroides sp. beyond
B. dorei, i.e.,
B. ovatus,
B. plebeius, and
B. xylanisolvens, while also boosting two
F. prausnitzii OTUs and an OTU related to
R. hominis that profoundly increased in mucus from 9.8% in the blank up to 64.3% upon cRG-I treatment (27). That the batch incubation strategy allowed to observe effects on a broader range of microbes is likely explained by the fact that in batch fermentations, in vivo-derived fecal microbiota in its maximal diversity is used as direct inoculum, an approach of which the biorelevance is supported by the minor spatial differences along the colon according to recent in vivo studies [
66]. In contrast, long-term in vitro experiments involve a pre-growth of the in vivo-derived inoculum under fixed laboratory conditions for several days up to weeks (as in the current study). While this results in a stable microbiota at the start of the treatment, such pre-growth decreases the diversity by around 50%, mostly due to washout of
Clostridium cluster IV and XIVa members (including
F. prausnitzii and
R. hominis) [
67]. This likely explains why the treatment effects of cRG-I on
F. prausnitzii and
R. hominis observed in batch fermentations were not recapitulated in the quad-M-SHIME. While
Clostridium cluster IV and XIVa members can be more optimally maintained in in vitro gut models by including a simulation of the mucosal environment, as was shown during 3-day experiments [
38], frequent replacement of the mucosal beads results in loss of species-specific colonization of the mucus layer [
39], likely due to limited cross-spreading between old and new mucin beads or due to physical opening of reactors to replace mucin beads. This was confirmed in the current study where despite inclusion of a mucosal microbiota simulation, no OTUs related to, e.g.,
R. hominis were detected in any of the donors at the start of the treatment (after 4 weeks of pre-growth), impairing the observation of treatment effects on such mucosal microbes. The washout of true mucosal microbes upon long pre-growth periods could potentially also explain why treatment effects on lumen and mucus were relatively similar during the current study. Despite such differences between both in vitro models, many findings were consistent and provided confidence in the robustness of (i) the observed impact of cRG-I on the human gut microbiota and (ii) the use of both types of in vitro gut models in gut microbiome research. While the added value of in vitro gastro-intestinal models is currently well-recognized [
68], it is acknowledged that they mimic only partly the complexity of the gut ecosystem. Nevertheless, confidence in both in vitro gut models is further provided by the observation of similar shifts in microbial community composition during a recent clinical trial in which RG-I was dosed at 0.3 or 1.5 g/day to the daily diet (manuscript in preparation).
Finally, a critical remark on the limitation of the applied experimental design is that the reference to which treatment effects were compared consisted of a preceding control period. Such longitudinal control is common in a SHIME setup [
69,
70,
71], and the stability of in vitro communities upon applying a 2-week stabilization period has previously been demonstrated [
67]. It would have been ideal to follow the stability of the microbial communities during the entire duration of the current study by means of incorporating four additional untreated parallel control arms. As the purpose of the present study was to assess the consistency of the impact of cRG-I on individual fecal samples, such a control would not optimally be performed with a single pooled fecal sample. It was therefore important to start with well-characterized and stable individual microbial communities.