Synergistic vs. complementary synbiotics: the complexity of discriminating synbiotic concepts using a Lactiplantibacillus plantarum exemplary study
0 
, Abstract
Synbiotics are defined as “a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host”. The definition discriminates between synergistic and complementary synbiotics. Synergistic synbiotics involve a direct interaction between the substrate and co-administered microbe(s), while complementary synbiotics act through independent mechanisms. Here, we evaluate the complexity of discrimination between these two synbiotic concepts using an exemplary study performed with a panel of Lactiplantibacillus plantarum (L. plantarum) strains to identify strain-specific synergistic synbiotics that eventually turned out to work via a complementary synbiotic mechanism. This study highlights that assessing the in situ selectivity of synergistic synbiotics in the intestinal tract is challenging due to the confounding effects of the substrate ingredient on the endogenous microbiome, thereby raising doubts about the added value of distinguishing between synergistic and complementary concepts in synbiotics.
Keywords
SYNBIOTICS
The term “synbiotic” refers to “synergy” and the Greek word for life “bios”, effectively meaning “synergistic life” and is an obvious nod to the terms prebiotic and probiotic and their potential synergy in the host’s digestive tract. Accordingly, the initial definition of synbiotics was “probiotics and prebiotics that beneficially affect the host”[1]. In 2020, an expert panel assembled by the International Scientific Association for Probiotics and Prebiotics (ISAPP) refined the synbiotics definition as “a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host”. The panel discriminated between complementary and synergistic synbiotics. In complementary synbiotics, the components non-cooperatively exert their synergistic health-promoting effects in the host, whereas in synergistic synbiotics, the substrate is designed to be selectively utilized by the co-administered microorganisms to enhance their health-promoting effects[2]. Notably, the latter concept does not exclude enrichment of other beneficial members of the endogenous microbiota, but identifies the co-administered microbe as the main target of the prebiotic substrate. Additionally, the synergy underlying the synbiotic concept intrinsically implies that these functional foods elicit a superior effect compared to those elicited by the separate administration of their constituents. However, due to the complications in scientifically establishing a synergistic effect on health, the consensus described by the expert panel[2] specifies that for synergistic synbiotics, it would be sufficient to demonstrate selective use of the prebiotic substrate by the
Health effects associated with the consumption of synbiotics include the general improvement of anthropometric, cardiometabolic, and inflammatory markers[3], as well as the eradication of Helicobacter pylori[4] and symptom improvement in various patient groups, including those suffering from atopic dermatitis[5], nonalcoholic fatty liver disease[6], and colorectal cancer[7]. Notably, in most of these studies, it remains unclear whether consumption of the synbiotic product has an added value over the consumption of its individual ingredients [i.e., the probiotic(s) or prebiotic(s)], and it is often not specified whether the synbiotic would classify as a complementary or synergistic synbiotic. Therefore, despite the refined definition of synbiotics, most synbiotic designs (i.e., the choice of pre- and probiotics that are combined) remain poorly rationalized and their health-promoting efficacy relative to their individual constituents often remains unclear.
Synbiotics can improve general health in newborns at risk
The most striking health impact associated with the administration of a synbiotic product has undoubtedly been described by Panigrahi et al., reporting on a large-scale, double-blind, placebo-controlled randomized synbiotic administration trial (N > 4,500) in infants recruited in Odisha, India, achieving significantly decreased neonatal sepsis and mortality in the synbiotic group compared to the placebo group[8]. Additionally, the synbiotic-treated group also displayed a significantly reduced incidence of lower respiratory tract infections and diarrhea, indicating that the overall health status of the synbiotic-treated children was improved compared to the placebo group[8]. The synbiotic product used in this landmark study was composed of Lactiplantibacillus plantarum (L. plantarum) ATCC202195 in combination with fructo-oligosaccharides (FOS), which was administered to newborns for 7 consecutive days starting 2-4 days after birth. Both this study[8] and earlier work[9] by the same group established that this synbiotic administration regimen succeeded in establishing long-term colonization (several months) of the L. plantarum strain in the intestines of these children. However, the synergistic interaction of the individual pre- and probiotic constituents in this study remains undetermined, and it was not reported whether the ATCC202195 strain could effectively utilize FOS as a substrate for growth. Analysis of the available genome sequence of this strain (NCBI: genome/GCF_004354995.1) indicates that it lacks a gene encoding an extracellular
In the section below, we will review some of the work performed with L. plantarum as an exemplary case for the design of strain-specific synergistic synbiotics and the exploration of their capacity to (selectively) enhance the intestinal fitness of L. plantarum.
DESIGNING SYNERGISTIC SYNBIOTICS
Strain-specific synergistic synbiotics for L. plantarum
Strains of the species L. plantarum have long been recognized as excellent probiotic candidates[14-17]. The species has a very wide ecological distribution ranging from the GI tract of humans and animals to decaying plant materials and fermented and non-fermented food products[18]. L. plantarum WCFS1, a single isolate of strain NCIMB8826, is among the most documented and extensively studied L. plantarum strains and has been instrumental in our understanding of the lifestyle of this species. The L. plantarum WCFS1 genome was the first genome of the lactobacilli to be published[19] and has since then been extensively analyzed, including a comprehensive overview of its predicted secretome[20,21], a genome-scale metabolic model[22], and a reconstruction of its regulatory network[23]. The availability of these tools rendered the WCFS1 strain a useful model for in-depth investigation of the molecular mechanisms that underlie probiotic function in lactobacilli[24]. More recently, comparative genomics of 54 L. plantarum strains[25] (later expanded to 611 genomes[26]) revealed a large pan-genome (> 7,000 orthologous groups), which did not approach saturation (i.e., also not with 611 genomes[26]), indicating that the genomic diversity of the species was still substantially larger than the 7,000 orthologous groups found in the 54 strains[25], which was expanded to more than 20,000 genes in 611 strains[26]. These findings agreed with the ecological flexibility and nomadic lifestyle of L. plantarum[25,27].
The genotypic and phenotypic diversity of L. plantarum strains is strongly reflected by the highly strain-specific carbohydrate utilization gene-repertoires, which is reflected by an array of genomic “lifestyle” islands that contain genes annotated as carbohydrate utilization cassettes, which appear to be clustered close to the origin of replication. Interestingly, no correlation between the diversity in these cassettes and the niche of isolation of these strains could be detected[18,25,28]. These strain-specific carbohydrate utilization gene repertoires offer an attractive starting point for the development of strain-specific synbiotics. Therefore, we developed a synbiotic matchmaking approach in our lab to identify prebiotic substrates that could differentially be utilized for growth by a panel of 77 L. plantarum strains[29]. Substantial variations in prebiotic utilization capacity were detected among the 77 strains for galacto-oligosaccharides (GOS) and isomalto-oligosaccharides (IMOS), whereas only a single strain (L. plantarum Lp900) isolated from ogi, a fermented sorghum pudding from Nigeria, was able to effectively utilize short- and long-chain fructo-oligosaccharides (FOS and inulin, respectively)[29]. Notably, it is well-established that the substrates that supported variable growth of the L. plantarum strains (i.e., GOS and IMOS) encompass a variety of distinct saccharide constituents that vary in degree of polymerization (DP) and glycosidic linkages[30,31]. Refinement of the matchmaking approach by determination of the strain-specific utilization capacity of individual GOS and IMOS constituents was able to explain the observed variations in the overall utilization (growth) of these prebiotics, which through gene-trait matching could be linked to specific L. plantarum genes that are involved in the metabolization of these IMOS and GOS constituents[29,32]. Similarly, the single L. plantarum 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[29]. The latter finding does not exclude the 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
Establishing increased intestinal survival and persistence by prebiotic supplements
Following the identification of prebiotic substrates that could selectively stimulate the growth of specific
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[41]). These simple in vitro competition experiments revealed that the growth of 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[41]. Notably, strain L. plantarum Lp900 (the inulin-degrading strain) 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 in vitro growth in a simple microbial consortium of strains of the same species. Notably, L. plantarum 299v and Heal19 were consistently among the most robust growing strains on various substrates, which may explain their success as cheaters in the inulin cultures and their apparent competitive advantage over SD5870 and Lp900 in High-DP GOS nutrient competition [Figure 1].
Figure 1. In vitro and in situ competitive fitness assessment of 7 L. plantarum strains. (A) The 7 L. plantarum strains used in this study. The strains were selected based on their discriminating prebiotic utilization phenotypes for GOS and inulin, combined with the possibility for high-throughput population dynamics based on strain-specific intergenic alleles that can be assessed using next-generation amplicon sequencing (for details, see ref[41,42]); (B) The strain-specific L. plantarum population shifts observed relative to the 7-strain inoculum mixture after 72 generations (generations are here defined as divisions of the overall seven strain population, and thus does not equal the number of generations of an individual strain in the seven strain mixture) of growth in a laboratory medium that contained GOS and inulin as a sole carbon source for growth, revealing considerable enrichment of at least some of the prebiotic utilizing L. plantarum strains by outcompeting non-utilizing strains; (C) The L. plantarum strain-specific population compositions observed in fecal samples obtained
Selective fitness advantages are further complicated when assessed in a complex microbial ecosystem such as the gut microbiome, where substantial redundancy for prebiotic utilization capacity can be expected in the members of the endogenous microbiome. In this context, not only the relative abundance of the endogenous microbiome members that can express such redundant functions and thereby compete for the same substrates, but also their substrate affinity and utilization rate relative to the administered
Further investigation of the fecal samples obtained from these rats (prior- and post-gavage of the
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 functionality when these products are delivered to the consuming host organism. However, the reviewed work above raises doubts about the reliability of such rational and experimentally driven design of synergistic synbiotics, which may eventually turn out to act as complementary synbiotics. Analogous to what is described for L. plantarum, competition experiments in GOS-fed gnotobiotic mice did not reveal a competitive advantage for Limisilactobacillus reuteri 6475 that is able to utilize this prebiotic substrate compared to its isogenic mutant-derivative that is unable to utilize GOS[51]. Nevertheless, irrespective of the precise nomenclature used for synbiotic concepts, the findings described above do support the usefulness of synbiotics in the intention to enhance the delivery of health-promoting microorganisms to their site of action (e.g., the human or animal intestinal tract). However, their selectivity in enhancing a specific microbial strain is unlikely to be achievable, especially when employing prebiotic substrates that are “commonly” degraded by endogenous microbiota members (e.g., GOS, FOS or inulin). Refinement of the substrate to chemically pure prebiotics rather than the current mixtures of prebiotic constituents such as IMOS and GOS (see above and ref[29,32]) may enhance the selectivity of such substrates in stimulating a specific strain or species. However, purification and/or chemical synthesis of these chemically pure prebiotics to generate more selective prebiotic substrates could be laborious and costly, which may compromise the economic feasibility of this scenario. Still, identifying more selective substrate compounds would provide more credible approaches to creating synergistic synbiotics compared to the canonically used prebiotics such as GOS, FOS, and inulin. In addition, selecting substrates that require an extensive enzymatic pathway for their degradation and utilization may contribute to the selectivity in stimulating only microorganisms that encode the entire enzyme repertoire required for substrate utilization. Besides the stimulation of the co-administered microorganism (i.e., the synergistic synbiotic concept), high specificity and selectivity substrates may also be very attractive compounds to stimulate an endogenous microbiota member in a precision-prebiotic approach. As an example, the endogenous Bifidobacterium population in the intestine was apparently selectively stimulated in healthy human volunteers by the consumption of synthetic human milk oligosaccharide (HMO) constituents 2’-fucosyllactose (2’FL) and/or Lacto-neotetraose (LNnT)[52]. However, and in view of the diversity of microbiome responses in individuals, one can question whether the Bifidobacterium stimulation is not simply the only common response in the participants rather than a truly selective response remains unclear, since additional microbiome responses may simply have remained undetected due to a lack of conservation among (i.e., distinct microbiome species reacting in individual participants prevents their detection in community-based microbiome response analyses) the participants. Extending this line of research toward chemically defined and pure prebiotics could determine to what extent certain single or mixed carbohydrate constituents of a prebiotic could selectively stimulate particular microbes in situ.
Irrespective of their use as part of a synergistic synbiotic mixture or as precision prebiotic, the proposed substrate compounds and their claims toward selectivity should be rigorously evaluated using robust in vivo nutrient-competition models to ensure that observed fitness enhancement effects are driven by direct interaction between substrate and microorganism. Experimental verification of the selectivity of synergistic synbiotics or the precision prebiotic requires that ecosystem interaction and nutrient competition concepts are taken into account and controlled for, e.g., “cooperative traits” and their role in generating “public goods”, the substrate monopolization as “personalized goods”, and the nutrient competition capacity redundancy and abundance, as well as the corresponding substrate affinity and utilization rates [Figure 2].
Figure 2. Competitive environmental fitness parameters that influence synbiotic mechanisms. (A) Exemplary prebiotic utilization strategies by a co-administered probiotic; left side of the cell: extracellular hydrolysis of the oligo- or polymeric substrate (e.g., FOS or inulin) with the subsequent import of released mono-saccharides or small oligosaccharides (i.e., sucrose and 1-kestose) that enter intracellular metabolism, with partial release of the substrate into the environment as “public goods”; right side of the cell: direct import of small oligosaccharides (e.g., tri- and tetra-saccharides in GOS) with subsequent intracellular hydrolysis and metabolization, i.e., “privatized goods”; (B) exemplary scenarios of endogenous microbiome members that can compete with the co-administered probiotic for the utilization of the prebiotic substrates. These competing capacities may be redundantly present in multiple members of the microbiome. Moreover, these redundant utilization pathways may have varying affinities and utilization rates for the prebiotic substrates, indicated by variations in arrow thickness; (C) In addition to the microbiome’s redundancy in prebiotic utilization capacities, their variable substrate affinities and utilization rates, the high relative abundance of competing microbes in comparison to the introduced probiotic favors complementary synbiotic effects rather than synergistic synbiotic effects, especially when the prebiotic substrate can readily be utilized by multiple members in the ecosystem and thus has poor species- or strain-specific selectivity. Consequently, the competing capacity of the dominating members of the microbiome (blue, pink, and orange) prevents the utilization of the prebiotic by, and the resulting growth stimulation of, the co-administered probiotic (green). FOS: Fructo-oligosaccharides; GOS: galacto-oligosaccharides.
How do the arguments raised above influence our perspective on the spectacular health effects of the administration in newborn infants of L. plantarum combined with FOS synbiotic, reported by Panigrahi et al.[8]? The administration regiment employed in that study provides this synbiotic to infants during their first weeks of life, which was demonstrated to achieve the engraftment of the L. plantarum strain in the intestine of these infants for a period of at least several months[8,9]. With such an administration regimen, the FOS prebiotics are highly unlikely to modify the intestinal milieu for such an extended period, implying that the engraftment observed is probably strongly related to the relatively empty niche of the GI tract of newborns that the L. plantarum strain is introduced into, which may explain the strain’s capacity to colonize and occupy this niche for an extended period. These arguments support the hypothesis that administration of the L. plantarum strain alone (without the prebiotic FOS) would likely achieve similar results. However, these studies did not include such a control group and, therefore, are unable to establish the importance of FOS in this approach. Notably, experiments that employed the simultaneous administration of L. plantarum Lp900 and inulin could enhance gastric survival (i.e., by 1-2 logs) but failed to affect intestinal persistence over an extended period[12]. Analogously, in vitro studies that stimulated conditions of the gastrointestinal tract demonstrated that L. plantarum survival during gastric passage (i.e., the acidity barrier of the stomach) could be significantly enhanced by the presence of glucose during the gastric transit (in concentrations as low as 0.05% w/w), but no such effect was achieved by the presence of the unfermentable enantiomer of glucose (i.e., L-glucose), indicating that the cellular energy state is crucial for this enhanced gastric stress tolerance[53]. These findings may imply that the co-administration of FOS with L. plantarum ATCC202195 employed by Panigrahi et al.[8] may have enhanced the survival of the probiotic strain during gastric passage by energizing the probiotic strain based on the use of the low-DP constituents of this prebiotic (i.e., fructose, sucrose, and 1-kestose, see above[12,29,41]), supporting more effective in situ delivery in the infant gut. Therefore, the administration of FOS could have influenced the outcome of the trial by improving the gastric survival and intestinal delivery of the probiotic, but this effect is probably not specific for FOS and is likely to be also achieved with a simple sugar such as glucose. In conclusion, it remains unclear to what extent this landmark study should be regarded as a true synbiotic study or could actually better be seen as a probiotic study, because it seems likely that the effect of the co-administered FOS simply depends on the energy state of the probiotic rather than any form of selective fitness advantage.
The above-mentioned concept of precision prebiotics is particularly relevant in view of the advances in our knowledge of the human intestinal microbiota. Traditional prebiotic applications have commonly targeted the stimulation of the endogenous populations of members of the Bifidobacterium genus or the Lactobacillaceae family based on their associations with health benefits. Precision prebiotics may offer opportunities to selectively stimulate the growth of other health-promoting members of the endogenous microbiome[54]. As an example, high abundance of Faecalibacterium prausnitzii (F. prausnitzii) has been inversely associated with flare incidence in inflammatory bowel disease (IBD) patients, and specific strains of the species (A2-165 and HTRF-F) were shown to alleviate inflammation in murine colitis models[55,56]. Moreover, F. prausnitzii is one of the main butyrate producers in the intestine[57], an essential short chain fatty acid involved in the regulation of a wide spectrum of health-promoting effects (e.g., trans-epithelial fluid transport, amelioration of mucosal inflammation, reinforcement of epithelial defense barriers, and intestinal motility)[58]. Strains of F. prausnitzii display a high degree of genetic diversity, particularly reflected in diverse carbohydrate utilization and immunomodulatory phenotypes[59], suggesting their strain-specific response to prebiotic supplementation. Extending the characterization of the genomic and phenotypic diversity of strains belonging to this species could offer new avenues toward the application of the species as candidates for next-generation probiotics[60]. Intriguingly, both FOS and inulin were shown to have stimulatory effects on F. prausnitzii in humans[61,62], and the capacity to utilize the substrate was confirmed for F. prausnitzii strain A2-165 in vitro[63]. Similarly, there is also evidence that GOS consumption can stimulate endogenous F. prausnitzii populations in humans[64,65], although GOS could not effectively support the growth of F. prausnitziii A2-165 in vitro, which led to the suggestion that GOS-mediated stimulation of F. prausnitzii would depend on bifidobacterial degradation of GOS and cross feeding[63,66]. However, the lack of growth of F. prausnitzii A2-165 is potentially meaningless in explaining the stimulatory effect of GOS supplementation on the relative abundance values of the endogenous populations of F. prausnitzii in humans, which may simply reflect the high diversity in carbohydrate utilization capacities in the strains of this species[60]. The genomic information available for this species is rapidly expanding, and at present, more than 700 F. prausnitzii genomes are available at NCBI [747 F. prausnitzii genome assemblies, including a large amount of metagenome-assembled genomes (MAGs), in July 2024]. Analogous to what is described above for L. plantarum, F. prausnitzii prebiotic matchmaking screening combined with detailed substrate utilization analysis by HPAEC-PAD and UPLC-MS can generate data that enable gene-trait matching to identify the genetic loci in F. prausnitzii that are required for the utilization of specific prebiotic constituents. Knowledge about the F. prausnitzii genes required for utilization of specific prebiotic constituents, offers the possibility of predicting which prebiotic substrates could selectively stimulate an individual’s endogenous F. prausnitzii populations by metagenome mining for its carbohydrate utilization genes. In addition, following the further development of F. prausnitzii strains as next-generation probiotics, the acquired knowledge could facilitate the design of synergistic synbiotics to enhance the delivery of these bacteria to the intestine in a viable and metabolically active form, which would stimulate their engraftment and persistence. Similar to what is described for L. plantarum, evidencing the selectivity of F. prausnitzii stimulation is crucial; this can be facilitated by the expansion of our knowledge about the specific genes involved in the utilization of specific substrates. Such efforts would build toward the concept of precision prebiotics, as well as next-generation probiotic-containing synergistic synbiotics.
CONCLUSION
Although the research track using L. plantarum started with the ambition to design synergistic synbiotics for specific strains of this species, it ended up with complementary synbiotics that could enhance the intestinal delivery and persistence of L. plantarum in a general rather than a strain-specific manner. The generation of convincing evidence for the selectivity of the direct interaction between the substrate and the microorganism that are combined in synergistic synbiotics requires dedicated in vivo competition experiments. Such experiments should include the analysis of potential confounding factors that may disrupt the “synergistic” characteristic of the synbiotic. Part of these confounding effects can be anticipated by taking ecosystem interaction and nutrient competition mechanisms [Figure 2] into account and specifically addressing them in the measurements included in the experimental design.
The concept of precision prebiotics using highly defined and pure prebiotic compounds (rather than the current substrate mixtures) combined with identification of the genes required for their utilization, offers a promising approach toward metagenomics-based, and thereby individualized selective stimulation of endogenous members of the microbiome that are associated with a healthy host. Moreover, a similar genome-based precision-substrate matching approach can support the design of truly selective synergistic synbiotics, containing either the classical Bifidobacterium and Lactobacillaceae probiotics or the emerging next-generation probiotics. A substantial amount of research will be required to progress our knowledge to a level that would enable these strategies. Additionally, it should be realized that profitable bulk production of the eventually identified precision prebiotic substrates in a chemically pure form may be challenging.
DECLARATIONS
Authors’ contributions
Design of this review, writing of the manuscript and the construction of the supporting figures: Kleerebezem M, Fuhren J
Both authors approved the submission of this version of the manuscript for publication.
Availability of data and materials
Not applicable.
Financial support and sponsorship
None.
Conflicts of interest
Both authors declared that there are no conflicts of interest.
Ethical approval and consent to participate
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Consent for publication
Not applicable.
Copyright
© The Author(s) 2024.
REFERENCES
1. Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr 1995;125:1401-12.
2. Swanson KS, Gibson GR, Hutkins R, et al. The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of synbiotics. Nat Rev Gastroenterol Hepatol 2020;17:687-701.
3. Teo YQJ, Chong B, Soong RY, Yong CL, Chew NW, Chew HSJ. Effects of probiotics, prebiotics and synbiotics on anthropometric, cardiometabolic and inflammatory markers: an umbrella review of meta-analyses. Clin Nutr 2024;43:1563-83.
4. Santacroce L, Topi S, Bottalico L, Charitos IA, Jirillo E. Current knowledge about gastric microbiota with special emphasis on helicobacter pylori-related gastric conditions. Curr Issues Mol Biol 2024;46:4991-5009.
5. Chang YS, Trivedi MK, Jha A, Lin YF, Dimaano L, García-Romero MT. Synbiotics for prevention and treatment of atopic dermatitis: a meta-analysis of randomized clinical trials. JAMA Pediatr 2016;170:236-42.
6. Liu L, Li P, Liu Y, Zhang Y. Efficacy of probiotics and synbiotics in patients with nonalcoholic fatty liver disease: a meta-analysis. Dig Dis Sci 2019;64:3402-12.
7. Moreira MM, Carriço M, Capelas ML, et al. The impact of pre-, pro- and synbiotics supplementation in colorectal cancer treatment: a systematic review. Front Oncol 2024;14:1395966.
8. Panigrahi P, Parida S, Nanda NC, et al. A randomized synbiotic trial to prevent sepsis among infants in rural India. Nature 2017;548:407-12.
9. Panigrahi P, Parida S, Pradhan L, et al. Long-term colonization of a Lactobacillus plantarum synbiotic preparation in the neonatal gut. J Pediatr Gastroenterol Nutr 2008;47:45-53.
10. Buntin N, Hongpattarakere T, Ritari J, et al. An inducible operon is involved in inulin utilization in Lactobacillus plantarum strains, as revealed by comparative proteogenomics and metabolic profiling. Appl Environ Microbiol 2017;83:e02402-16.
11. Fuhren J, Nijland R, Wels M, Boekhorst J, Kleerebezem M. Complete closed genome sequence of the inulin-utilizing Lactiplantibacillus plantarum strain Lp900, obtained using a hybrid nanopore and illumina assembly. Microbiol Resour Announc 2021;10:e00185-21.
12. Fuhren J, Schwalbe M, Rösch C, et al. Dietary inulin increases Lactiplantibacillus plantarum strain Lp900 persistence in rats depending on the dietary-calcium level. Appl Environ Microbiol 2021;87:e00122-21.
13. Saulnier DM, Molenaar D, de Vos WM, Gibson GR, Kolida S. Identification of prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1 through microarrays. Appl Environ Microbiol 2007;73:1753-65.
14. Fidanza M, Panigrahi P, Kollmann TR. Lactiplantibacillus plantarum - nomad and ideal probiotic. Front Microbiol 2021;12:712236.
15. Nordström EA, Teixeira C, Montelius C, Jeppsson B, Larsson N. Lactiplantibacillus plantarum 299v (LP299V®): three decades of research. Benef Microbes 2021;12:441-65.
16. Seddik HA, Bendali F, Gancel F, Fliss I, Spano G, Drider D. Lactobacillus plantarum and its probiotic and food potentialities. Probiotics Antimicrob Proteins 2017;9:111-22.
17. van den Nieuwboer M, van Hemert S, Claassen E, de Vos WM. Lactobacillus plantarum WCFS1 and its host interaction: a dozen years after the genome. Microb Biotechnol 2016;9:452-65.
18. Siezen RJ, Tzeneva VA, Castioni A, et al. Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches. Environ Microbiol 2010;12:758-73.
19. Kleerebezem M, Boekhorst J, van Kranenburg R, et al. Complete genome sequence of Lactobacillus plantarum WCFS1. Proc Natl Acad Sci U S A 2003;100:1990-5.
20. Boekhorst J, Wels M, Kleerebezem M, Siezen RJ. The predicted secretome of Lactobacillus plantarum WCFS1 sheds light on interactions with its environment. Microbiology 2006;152:3175-83.
21. Kleerebezem M, Hols P, Bernard E, et al. The extracellular biology of the lactobacilli. FEMS Microbiol Rev 2010;34:199-230.
22. Teusink B, Wiersma A, Molenaar D, et al. Analysis of growth of Lactobacillus plantarum WCFS1 on a complex medium using a genome-scale metabolic model. J Biol Chem 2006;281:40041-8.
23. Wels M, Overmars L, Francke C, Kleerebezem M, Siezen RJ. Reconstruction of the regulatory network of Lactobacillus plantarum WCFS1 on basis of correlated gene expression and conserved regulatory motifs. Microb Biotechnol 2011;4:333-44.
24. Lee IC, Tomita S, Kleerebezem M, Bron PA. The quest for probiotic effector molecules - unraveling strain specificity at the molecular level. Pharmacol Res 2013;69:61-74.
25. Martino ME, Bayjanov JR, Caffrey BE, et al. Nomadic lifestyle of Lactobacillus plantarum revealed by comparative genomics of 54 strains isolated from different habitats. Environ Microbiol 2016;18:4974-89.
26. Rajput A, Chauhan SM, Mohite OS, et al. Pangenome analysis reveals the genetic basis for taxonomic classification of the Lactobacillaceae family. Food Microbiol 2023;115:104334.
27. Duar RM, Lin XB, Zheng J, et al. Lifestyles in transition: evolution and natural history of the genus Lactobacillus. FEMS Microbiol Rev 2017;41:S27-48.
28. Molenaar D, Bringel F, Schuren FH, de Vos WM, Siezen RJ, Kleerebezem M. Exploring Lactobacillus plantarum genome diversity by using microarrays. J Bacteriol 2005;187:6119-27.
29. Fuhren J, Rösch C, Ten Napel M, Schols HA, Kleerebezem M. Synbiotic matchmaking in Lactobacillus plantarum: substrate screening and gene-trait matching to characterize strain-specific carbohydrate utilization. Appl Environ Microbiol 2020;86:e01081-20.
30. Logtenberg MJ, Akkerman R, Hobé RG, et al. Structure-specific fermentation of galacto-oligosaccharides, isomalto-oligosaccharides and isomalto/malto-polysaccharides by infant fecal microbiota and impact on dendritic cell cytokine responses. Mol Nutr Food Res 2021;65:e2001077.
31. Logtenberg MJ, Donners KMH, Vink JCM, et al. Touching the high complexity of prebiotic vivinal galacto-oligosaccharides using porous graphitic carbon ultra-high-performance liquid chromatography coupled to mass spectrometry. J Agric Food Chem 2020;68:7800-8.
32. Fuhren J, Schwalbe M, Peralta-Marzal L, Rösch C, Schols HA, Kleerebezem M. Phenotypic and genetic characterization of differential galacto-oligosaccharide utilization in Lactobacillus plantarum. Sci Rep 2020;10:21657.
33. Bovee-Oudenhoven IM, Wissink ML, Wouters JT, Van der Meer R. Dietary calcium phosphate stimulates intestinal lactobacilli and decreases the severity of a salmonella infection in rats. J Nutr 1999;129:607-12.
34. Govers MJ, Termont DS, Lapre JA, Kleibeuker JH, Vonk RJ, Van der Meer R. Calcium in milk products precipitates intestinal fatty acids and secondary bile acids and thus inhibits colonic cytotoxicity in humans. Cancer Res 1996;56:3270-5.
35. Govers MJ, Van der Meet R. Effects of dietary calcium and phosphate on the intestinal interactions between calcium, phosphate, fatty acids, and bile acids. Gut 1993;34:365-70.
36. Begley M, Gahan CG, Hill C. The interaction between bacteria and bile. FEMS Microbiol Rev 2005;29:625-51.
37. Bachmann H, Bruggeman FJ, Molenaar D, Branco Dos Santos F, Teusink B. Public goods and metabolic strategies. Curr Opin Microbiol 2016;31:109-15.
38. Cordero OX, Ventouras LA, DeLong EF, Polz MF. Public good dynamics drive evolution of iron acquisition strategies in natural bacterioplankton populations. Proc Natl Acad Sci U S A 2012;109:20059-64.
39. Gore J, Youk H, van Oudenaarden A. Snowdrift game dynamics and facultative cheating in yeast. Nature 2009;459:253-6.
40. Kümmerli R, Santorelli LA, Granato ET, et al. Co-evolutionary dynamics between public good producers and cheats in the bacterium Pseudomonas aeruginosa. J Evol Biol 2015;28:2264-74.
41. Fuhren J, Schwalbe M, Boekhorst J, Rösch C, Schols HA, Kleerebezem M. Prebiotic utilisation provides Lactiplantibacillus plantarum a competitive advantage in vitro, but is not reflected by an increased intestinal fitness. Gut Microbes 2024;16:2338946.
42. van Bokhorst-van de Veen H, van Swam I, Wels M, Bron PA, Kleerebezem M. Congruent strain specific intestinal persistence of Lactobacillus plantarum in an intestine-mimicking in vitro system and in human volunteers. PLoS One 2012;7:e44588.
43. Fuhren J, Schwalbe M, Boekhorst J, Rösch C, Schols HA, Kleerebezem M. Dietary calcium phosphate strongly impacts gut microbiome changes elicited by inulin and galacto-oligosaccharides consumption. Microbiome 2021;9:218.
44. Garrido D, Ruiz-Moyano S, Jimenez-Espinoza R, Eom HJ, Block DE, Mills DA. Utilization of galactooligosaccharides by Bifidobacterium longum subsp. infantis isolates. Food Microbiol 2013;33:262-70.
45. Motherway MOC, Kinsella M, Fitzgerald GF, van Sinderen D. Transcriptional and functional characterization of genetic elements involved in galacto-oligosaccharide utilization by Bifidobacterium breve UCC2003. Microb Biotechnol 2013;6:67-79.
46. Rossi M, Corradini C, Amaretti A, et al. Fermentation of fructooligosaccharides and inulin by bifidobacteria: a comparative study of pure and fecal cultures. Appl Environ Microbiol 2005;71:6150-8.
47. Viborg AH, Katayama T, Abou Hachem M, et al. Distinct substrate specificities of three glycoside hydrolase family 42
48. Lammens W, Le Roy K, Schroeven L, Van Laere A, Rabijns A, Van den Ende W. Structural insights into glycoside hydrolase family 32 and 68 enzymes: functional implications. J Exp Bot 2009;60:727-40.
49. Honda H, Nagaoka S, Kawai Y, et al. Purification and characterization of two phospho-β-galactosidases, LacG1 and LacG2, from Lactobacillus gasseri ATCC33323(T). J Gen Appl Microbiol 2012;58:11-7.
50. Maathuis AJH, van den Heuvel EG, Schoterman MHC, Venema K. Galacto-oligosaccharides have prebiotic activity in a dynamic in vitro colon model using a 13C-labeling technique. J Nutr 2012;142:1205-12.
51. Rattanaprasert M, van Pijkeren JP, Ramer-Tait AE, et al. Genes involved in galactooligosaccharide metabolism in lactobacillus reuteri and their ecological role in the gastrointestinal tract. Appl Environ Microbiol 2019;85:e01788-19.
52. Elison E, Vigsnaes LK, Rindom Krogsgaard L, et al. Oral supplementation of healthy adults with 2’-O-fucosyllactose and lacto-N-neotetraose is well tolerated and shifts the intestinal microbiota. Br J Nutr 2016;116:1356-68.
53. Van Bokhorst-van de Veen HB PA, Kleerebezem, M. Improving the digestive tract robustness of probiotic lactobaclli. In: Venema K, do Carmo AP, editors. Probiotics and prebiotics: current research and future trends. Norfolk, UK: Caister Academic Press; 2015. pp. 195-203.
54. Lordan C, Thapa D, Ross RP, Cotter PD. Potential for enriching next-generation health-promoting gut bacteria through prebiotics and other dietary components. Gut Microbes 2020;11:1-20.
55. Rossi O, Khan MT, Schwarzer M, et al. Faecalibacterium prausnitzii strain HTF-F and its extracellular polymeric matrix attenuate clinical parameters in DSS-induced colitis. PLoS One 2015;10:e0123013.
56. Sokol H, Pigneur B, Watterlot L, et al. Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc Natl Acad Sci U S A 2008;105:16731-6.
57. Louis P, Flint HJ. Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol Lett 2009;294:1-8.
58. Canani RB, Costanzo MD, Leone L, Pedata M, Meli R, Calignano A. Potential beneficial effects of butyrate in intestinal and extraintestinal diseases. World J Gastroenterol 2011;17:1519-28.
59. Fitzgerald CB, Shkoporov AN, Sutton TDS, et al. Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa. BMC Genomics 2018;19:931.
60. Martín R, Miquel S, Benevides L, et al. Functional characterization of novel Faecalibacterium prausnitzii strains isolated from healthy volunteers: a step forward in the use of F. prausnitzii as a next-generation probiotic. Front Microbiol 2017;8:1226.
61. Dewulf EM, Cani PD, Claus SP, et al. Insight into the prebiotic concept: lessons from an exploratory, double blind intervention study with inulin-type fructans in obese women. Gut 2013;62:1112-21.
62. Ramirez-Farias C, Slezak K, Fuller Z, Duncan A, Holtrop G, Louis P. Effect of inulin on the human gut microbiota: stimulation of Bifidobacterium adolescentis and Faecalibacterium prausnitzii. Br J Nutr 2009;101:541-50.
63. Scott KP, Martin JC, Duncan SH, Flint HJ. Prebiotic stimulation of human colonic butyrate-producing bacteria and bifidobacteria, in vitro. FEMS Microbiol Ecol 2014;87:30-40.
64. Davis LM, Martínez I, Walter J, Goin C, Hutkins RW. Barcoded pyrosequencing reveals that consumption of galactooligosaccharides results in a highly specific bifidogenic response in humans. PLoS One 2011;6:e25200.
65. Nyangale EP, Farmer S, Keller D, Chernoff D, Gibson GR. Effect of prebiotics on the fecal microbiota of elderly volunteers after dietary supplementation of Bacillus coagulans GBI-30, 6086. Anaerobe 2014;30:75-81.
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, Jori FührenHow to Cite
Kleerebezem, M.; Führen J. Synergistic vs. complementary synbiotics: the complexity of discriminating synbiotic concepts using a Lactiplantibacillus plantarum exemplary study. Microbiome. Res. Rep. 2024, 3, 46. http://dx.doi.org/10.20517/mrr.2024.48
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