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Kleerebezem et al. Microbiome Res Rep 2024;3:46 https://dx.doi.org/10.20517/mrr.2024.48 Page 5 of 14

significantly enhanced the intestinal delivery of L. plantarum Lp900 compared to rats that were fed a non-
[12]
supplemented diet . This result supports that the identified candidate synergistic synbiotics are able to
stimulate the in situ delivery of their matched L. plantarum strain. In the same study, it was recognized that
the stimulatory effect of inulin was much more pronounced when inulin was added to diets that had a high
calcium phosphate level compared to those with a low level, indicating that the background diet,
particularly its micronutrient levels, has a marked impact on the stimulatory efficacy of inulin
supplementation. These results aligned with previous studies showing that high dietary calcium phosphate
intake (primarily from dairy products) was associated with higher levels of endogenous lactobacilli in the
intestines compared to individuals with lower calcium phosphate intake . The mechanisms by which
[33]
dietary calcium (calcium phosphate) modulates the intestinal microbiota are not fully understood, but it has
been proposed to depend on the increased buffering capacity of the intestinal lumen and the ensuing
precipitation of cytotoxic surfactants like secondary bile acids [34,35] . The latter compounds are established
antimicrobials with particular effective inhibitory capacities against endogenous Gram-positive bacteria like
lactobacilli . At first sight (see also below), these results supported the synergistic mechanism of action of
[36]
the synbiotic combinations identified through the in vitro matchmaking and gene-trait matching approach
described above.

Competition in vitro or in situ in the gut: nutrient competition or environmental selection?
The final stage of this line of research on strain-specific L. plantarum synbiotic combinations intended to
investigate the term “selectively” in the synbiotic definition, because the inulin-mediated enhancement of
the intestinal fitness of L. plantarum Lp900 in rats fed an inulin-containing diet did not directly assess this
aspect.

In the context of substrate-mediated selective fitness stimulation, it is important to realize that this is
strongly influenced by the mechanism by which microbes utilize the substrate. The extracellular
degradation of inulin by L. plantarum Lp900 is a “cooperative” trait that liberates the fructose building
blocks of inulin as “public goods” in the environment of the cell. Consequently, the selectivity of inulin as a
substrate for the growth of L. plantarum Lp900 in a microbial ecosystem may suffer from so-called
“cheaters” that do not contribute to the cooperative trait of degrading the polymeric substrate, but capitalize
on the availability of the public goods. In contrast, the L. plantarum strains that can utilize the high-DP
constituents of IMOS and GOS by internalizing these substrates followed by intracellular degradation and
metabolization monopolize these substrates (i.e., “privatized goods”) and circumvent cheater-risks [37-40] .
These considerations should be taken into account when assessing substrate-induced competitive fitness
advantage, as the selectivity of such advantages may depend on the substrate and the mechanism of its
utilization. In view of these considerations, the relative selectivity of the fitness benefits associated with
specific prebiotic utilization capacities of individual L. plantarum strains was evaluated using a panel of
seven genetically distinguishable strains [41,42] , which differ in their inulin and GOS utilization capacities.
Initial experiments evaluated the population dynamics of the seven L. plantarum strains during
approximately 72 generations of in vitro growth in media containing inulin or GOS as a sole carbon source
(Figure 1, adapted from ref ). These simple in vitro competition experiments revealed that the growth of
[41]
the mixture of strains on both GOS and inulin as a substrate led to a significant enrichment of the strains
that are able to utilize these substrates . Notably, strain L. plantarum Lp900 (the inulin-degrading strain)
[41]
clearly accumulated in the population after 72 generations of growth on inulin, but this coincided with the
enrichment of two potential cheater strains (299v and Heal19). Additionally, after 72 generations of growth
on GOS, two of the strains - 299v and Heal19 - that could import and utilize High-DP GOS constituents
showed significant enrichment. In contrast, the remaining two strains displayed modest (SD5870) or no
(Lp900) enrichment under those conditions. These observations support that the prebiotic-matchmaking
results are able to at least partially predict the selective fitness advantage of these L. plantarum strains during