Synbiotic matchmaking in Lactobacillus plantarum for enhanced in situ delivery in the intestinal tract

Probiotic are defined as “live micro-organisms that, when administered in adequate amounts, confer a health benefit to the host”, while prebiotics are defined as “substrates that are selectively utilised by host micro-organisms conferring a health benefit” and are often oligo- and poly- saccharides. Synbiotics combine probiotics and prebiotics to synergistically elicit a health effect in the consumer. This synergy can be interpreted in several ways. In a synbiotic, the prebiotic can directly impact the survival, persistence and/or activity of the probiotic. Alternatively, it can modulate the host microbiota, resulting in an environment that favours the co-administrated probiotics. Finally, the elicited health benefits of the probiotic and prebiotic constituents may not depend on their interaction but could exert their synergistic health effects through independent host interactions that synergistically achieve a health effect. The main goal of this thesis was to identify probiotic and prebiotic matches that could enhance the intestinal persistence of the probiotic in situ. An enhanced persistence can effectively increase the impact of consumed probiotics to elicit their health beneficial effects for a longer period of time. In this thesis we describe the search for synbiotic matches in Lactobacillus plantarum, a species of lactic acid bacteria with demonstrated probiotic properties and a strong genetic and phenotypic diversity in carbohydrate utilisation.

We describe the setup of a high-throughput screening model that demonstrates the strain-specific diversity in prebiotic utilisation within L. plantarum. A collection of 77 L. plantarum strains were screened for their capacity to utilise specific prebiotic fibres, revealing variable and strain-specific growth efficiencies on isomalto-oligosaccharides (IMO) and galacto-oligosaccharides (GOS). Only a single strain in the L. plantarum panel tested, could effectively utilise inulin and fructo-oligosaccharides (FOS). We further analysed the strain-specific growth of L. plantarum on IMO using high performance anion exchange chromatography (HPAEC) and revealed distinct utilisation phenotypes within the strain-panel used. These distinct phenotypes could be linked to the genomes of the strains through gene-trait matching to identify the genetic clusters involved in membrane transport and utilisation of constituents of isomaltose and other (unidentified) oligosaccharides in the IMO substrate. The same approach is described for GOS, identifying candidate genes involved in distinct GOS utilisation phenotypes in the L. plantarum strain panel. Two GOS utilisation phenotypes distinguished that could be linked to an import system and intracellular hydrolyse, of which the involvement in GOS utilisation was confirmed by the construction mutant derivatives that lack these functions.

Based on the outcomes of the initial screening, GOS and inulin were chosen to use as dietary prebiotic supplements in animal experiments with the goal to investigate the use of prebiotics for the enhancement of intestinal persistence of L. plantarum strains. Because previous research has shown that dietary calcium can affect endogenous Lactobacillus populations, distinct dietary calcium levels were used in the experimental diets that reflect habitual dietary calcium intake in humans in different parts of the world. The initial effects of a two-week period of dietary prebiotic supplementation (GOS or inulin) in high and low calcium contexts on the endogenous microbiota and fermentation outputs were assessed, revealing that prebiotic supplementation (i.e., GOS or inulin) and dietary calcium levels profoundly impacted the endogenous microbiota composition. The modulatory effects of inulin and GOS were congruent, but displayed a strong interaction dependent on the calcium levels of the diets. Furthermore, we characterised the inulin utilising capacities of L. plantarum Lp900, and confirmed the role of a plasmid-encoded extracellular β-fructosidase in the utilisation of inulin through heterologous expression experiments. The intestinal persistence of L. plantarum Lp900 was subsequently investigated in rats that were fed on diets with distinct calcium levels with or without inulin supplementation, showing that inulin supplementation strongly increased the gut-persistence of Lp900. Furthermore, in a separate experiment, rats that were fed a high or low calcium diet without prebiotic supplementation, were given a single dosage of only L. plantarum Lp900 (probiotic), or this bacterial dosage in combination with inulin (synbiotic), followed by the analysis of bacterial persistence over time. When provided in combination with inulin (i.e., as a synbiotic), L. plantarum Lp900 initial intestinal survival appeared to be improved, but this led to only moderately increased persistence in rats fed the low-calcium diet. 

We also describe the strain-specific, competitive intestinal persistence of seven L. plantarum strains with varying capacities to utilise GOS and inulin. The rats in this experiment were fed a high or low calcium diet, with or without prebiotic supplementation (GOS or inulin), and the L. plantarum strains were given to these rats in a mixture. In parallel, these mixtures were inoculated in in vitro culturing experiments with GOS or inulin as sole carbon source, which showed that the capability to utilise the prebiotic substrate (either GOS or inulin) provided a strong competitive advantage in vitro leading to the rapid enrichment of utilising strains at the expense of non-utilising strains. However, such competitive growth-advantage or strain-enrichment was not present in the in vivo intestinal persistence experiments in rats fed the prebiotic-supplemented diets. Although the high-calcium diet supplementation with GOS or inulin clearly increased the L. plantarum intestinal persistence in rats, this increase in persistence was observed for all 7 strains in the L. plantarum mixture irrespective of their prebiotic utilisation capacities. These results demonstrated that increased intestinal persistence of L. plantarum by dietary prebiotic supplementation does not depend on the strain’s ability to utilize the prebiotic for growth, but likely results from changes in the endogenous microbiota or the physico-chemical conditions in the gut that are elicited by prebiotic supplementation.

nhancing persistence, survival and/or activity of probiotics would be an improvement in the world of functional foods, but is highly determined and co-modulated by (a range of) dietary macro- and micro-nutrients, warranting further research into the complexity of the syntrophic food webs within the host-microbiota to understand how (functional) foods can modulate these complex ecosystems to elicit specific effects on host health.