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Page 4 of 14 Kleerebezem et al. Microbiome Res Rep 2024;3:46 https://dx.doi.org/10.20517/mrr.2024.48

strain in the panel (Lp900) that could effectively grow on FOS or inulin was shown to contain a plasmid
encoding a cell-wall anchored extracellular β-fructosidase (FosE) as well as a fructose import system,
facilitating effective degradation and growth on FOS and inulin [11,12] . Besides the prebiotic substrates
mentioned above, the matchmaking study detected only marginal utilization of other candidate prebiotics
like arabinoxylan oligosaccharides (AXOS) and fucoidan . The latter finding does not exclude the
[29]
possibility that expansion of the L. plantarum strain panel could enable the identification of strains that are
able to utilize these substrates, particularly when L. plantarum isolates obtained from niches containing
these substrates would be included.

The finding that specific IMOS and GOS constituents could only be utilized by some strains offers
opportunities for further refinement of the synbiotic combinations that would more selectively stimulate
specific strains. For example, IMOS preparations commonly contain a substantial amount of isomaltose
(α-1,6-linked glucose-glucose disaccharide), which could be utilized by some but not all L. plantarum
strains. This capacity to utilize isomaltose perfectly correlated with the presence of a gene cluster that was
proposed to encode an (iso-)maltose phosphotransferase system (PTS), an (iso-)maltose-6’-phosphate
glucosidase, a β-phosphoglucomutase, and a transcriptional regulator. Accordingly, follow-up experiments
that used isomaltose as a sole carbon source for growth exclusively stimulated the growth of the subset of
[29]
strains that encoded these functions . Similarly, a subset of the strains was able to utilize the higher DP
isomaltose constituents of IMOS (estimated DP > 3), which could also be associated with a gene cluster
encoding several ABC-import systems annotated to import multiple sugars and several α-mannosidases,
which appears in agreement with the observed higher-DP IMOS utilization phenotype, and suggests that
these oligosaccharides are not degraded extracellularly but are imported and intracellularly hydrolyzed and
[29]
used to support growth . Further analysis of the GOS constituent utilization per strain revealed that the
strains could predominantly be divided into two utilization groups. The minority of the strains could utilize
several glucose-galactose disaccharides, including lactose (β-1,4-linked) and the β-1,2- and β-1,3-
disaccharides, but could not utilize the β-1,2 and β-1,3 galactose-galactose disaccharides. The majority of the
strains could utilize the latter galactose-galactose disaccharides, as well as some higher-DP oligosaccharide
[32]
constituents of GOS (estimated DP > 3), to varying extents . The strains with the extended GOS-
constituent utilization capacity encoded a lacAS operon with a divergently oriented transcription regulator
encoding gene (lacR) that was absent in the other group of strains. The lacAS operon was annotated to
encode a β-galactosidase (LacA) and a GPH-family permease (LacS) that is annotated to be involved in the
import of lactose and galactose. However, the introduction of a lacS mutant did not only result in reduced
growth on lactose, but also a complete loss of the higher DP-fraction constituent utilization, confirming the
[32]
role of the lac operon in the observed variability of the GOS-utilization phenotype . Similar to what was
found for IMOS, the results imply that the higher-DP constituents of these prebiotics are first imported into
the cell to be subsequently hydrolyzed and catabolized. Moreover, for both IMOS and GOS prebiotics,
further fractionation of these complex saccharide mixtures would enable the isolation of specific
constituents that would more exclusively stimulate the growth of a few L. plantarum strains, which opens
the door for precision-prebiotic substrates for highly selective synbiotic combinations that would stimulate
the growth of only few specific strains.

Establishing increased intestinal survival and persistence by prebiotic supplements
Following the identification of prebiotic substrates that could selectively stimulate the growth of specific
L. plantarum strains, subsequent experiments aimed to verify that the prebiotic substrate inulin was able to
stimulate the intestinal survival and persistence of L. plantarum Lp900 in situ in the intestine using a
preclinical rat model. As mentioned above, strain Lp900 contains a plasmid encoding a cell-wall anchored
extracellular β-fructosidase (FosE), as well as a fructose import system, facilitating effective degradation and
growth on inulin. The results obtained clearly established that inulin supplementation of the rats’ diet