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Kleerebezem et al. Microbiome Res Rep 2024;3:46 https://dx.doi.org/10.20517/mrr.2024.48 Page 7 of 14

L. plantarum strains will play a prominent role in the in situ selectivity of the L. plantarum fitness
stimulation [37-40] . The L. plantarum intestinal fitness advantage induced by GOS or inulin supplementation
was again assessed in rats that were fed a high-calcium control diet or the same diet supplemented with
GOS or inulin. The mixture of the seven strains that was also used in vitro was gavaged (given once at the
start of the experiment) in these rats and the population dynamics of these strains were followed up to
7 days post gavage in fecal samples. Strikingly, these experiments did not reveal any enrichment for any of
the strains in the mixture, and the populations present in feces displayed a virtually identical strain
composition compared to the gavaged inoculum. Nevertheless, the intestinal persistence of the L. plantarum
community as a whole was significantly enhanced in rats that were fed a prebiotic-supplemented diet (GOS
or inulin) compared to the un-supplemented control diet. Notably, consistent with earlier results, the
prebiotic effects on persistence enhancement were more pronounced in high-calcium diets compared to
low-calcium diets in these experiments . These observations indicate that contrary to the strain-specific
[41]
fitness benefit observed in vitro, the intestinal fitness of L. plantarum was enhanced by prebiotics in a strain-
independent manner, irrespective of the prebiotic utilization phenotype of the individual strains.

Further investigation of the fecal samples obtained from these rats (prior- and post-gavage of the
L. plantarum strain mixture) illustrated the prominent confounding effects of prebiotic supplementation,
which elicited significant changes in the intestinal microbiome composition and increased the levels of fecal
[43]
short chain fatty acids (SCFA) and other organic acids . The effects of supplementation on the endogenous
microbiome were highly similar between inulin and GOS, both leading to a marked increase in the relative
abundance of Bifidobacterium. This increase was accompanied by a rise in the relative abundance of the
Faecalibaculum genus, but only in diets high in calcium, not in those low in calcium [41,43] . The species- and
strain-specific utilization of GOS and inulin has been well described in members of the Bifidobacterium
genus [44-47] . Furthermore, the public genomes of Faecalibaculum rodentium encode secreted proteins with
[48]
glycoside hydrolase (GH) family 32 domains that are typically involved in inulin utilization . These
genomes also encode lactose-PTS systems and genetically linked intracellular 6-P-β-galactosidases that
resemble lactose-PTS systems found in Lactobacillus gasseri strains that were described to grow on GOS but
lacked orthologues of the lactose/galactose-specific permease that is typically involved in lactose-
import [49,50] . These highly abundant endogenous microbes probably outcompete the administered
L. plantarum strains when it comes to prebiotic utilization as a substrate for growth. Nevertheless, the
resulting intestinal milieu is apparently more favorable for the colonization of L. plantarum, which is
probably related to the elevated levels of especially lactic and acetic acid in the high-calcium and prebiotic-
supplemented diets compared to control diets, an environmental condition that agrees very well with the
[41]
acidophilic character of L. plantarum .
The experiments assessing the L. plantarum intestinal persistence as a function of prebiotic utilization,
disrupts the synergistic synbiotic concept that was aimed for. The intestinal fitness advantage observed is
independent of a direct interaction between the substrate and the microorganisms, but is achieved through
the effect of the prebiotic substrate on the endogenous microbiome. Thereby, the synbiotic combinations of
L. plantarum and prebiotics actually fall under the concept of complementary synbiotics, illustrating the
complexity of accurately assessing the term “selectivity” in the synbiotic definition and discriminating
between truly synergistic vs. complementary synbiotic delivery systems.

PERSPECTIVE
The direct synergy between the probiotic and prebiotic components in a synergistic synbiotic represents a
highly attractive concept. This is particularly based on the direct interaction of these two ingredients that
enables their rational and experimentally verifiable design intended to adequately predict the in situ