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International Journal of Molecular Sciences logoLink to International Journal of Molecular Sciences
. 2026 Feb 10;27(4):1698. doi: 10.3390/ijms27041698

Improving Growth Dynamics of Faecalibacterium prausnitzii by Exposure to Prebiotics

Sadia Ahmed 1, Kyle D Roberts 1, Thomas S McCormick 1, Mahmoud A Ghannoum 1,2,*
Editors: Baojun Xu, Matteo Bordiga
PMCID: PMC12940339  PMID: 41751835

Abstract

Faecalibacterium prausnitzii, a major butyrate-producing gut commensal with anti-inflammatory activity, is extremely oxygen-sensitive, limiting its use as a probiotic. Dietary prebiotics may enhance its growth and resilience, thereby influencing host immune responses. This study examined how distinct classes of prebiotics including oligosaccharides (fructooligosaccharides, arabinoxylan), nondigestible polysaccharides (inulin, pectin, resistant starch, golden kiwi fiber), and the vitamin riboflavin affect the growth kinetics, bile tolerance, and immunomodulatory properties of F. prausnitzii. Doubling times were quantified in MRS medium supplemented with 0–2% prebiotics, bile tolerance was assessed under 0–0.5% bile salts, and immunomodulatory response was evaluated by measuring TNF-α expression in monocytic THP-1 cells exposed to bacterial supernatants. All prebiotics significantly reduced doubling times compared with controls, with FOSs, inulin, pectin, resistant starch, and riboflavin showing clear dose-dependent stimulation. Prebiotics also mitigated bile-induced growth delays, though with substrate-specific patterns; pectin and FOSs conferred the strongest protection. Culture supernatants significantly altered TNF-α expression, with pectin inducing the greatest response, followed by arabinoxylan, FOSs, and golden kiwi fiber. Overall, prebiotics enhanced F. prausnitzii growth, increased stress resilience, and differentially modulated immune-related metabolites. Pectin emerged as a particularly effective substrate for promoting microbial function and host-relevant immunomodulation.

Keywords: probiotic, prebiotic, bile tolerance, immunomodulation

1. Introduction

Human intestinal microbiota plays a crucial role in maintaining overall health through influencing numerous physiological functions including immune response, metabolic processes, nutrient absorption, barrier function, energy production, protection against pathogens, and even neurological health. Three microbial phyla dominate the healthy human gut: Firmicutes, Bacteroidetes and Actinobacteria. Among the many microbial species found in the gut, Faecalibacterium prausnitzii is one of the most abundant Firmicutes, contributing up to 15% of the total fecal microbiota and playing a significant role in gut homeostasis [1]. This Gram-positive, obligate anaerobic bacterium is classified as a beneficial microbe based on its production of butyrate, a short-chain fatty acid (SCFA) that provides maintenance of the intestinal lining, inhibition of harmful bacteria, regulation of blood sugar levels [2], provision of energy to colonocytes and inhibition of inflammation [3]. These properties make F. prausnitzii an ideal candidate as a next-generation probiotic [4,5] aimed at supporting gastrointestinal and metabolic disorders such as inflammatory bowel disease (IBD) [6,7], Crohn’s disease (CD) [8,9], and obesity [10]. However, the proper function and viability of F. prausnitzii can be significantly affected by stressful conditions such as increased bile acid levels in the human gut [11]. F. prausnitzii has been demonstrated to be particularly sensitive to changes in bile salt concentrations, with growth being inhibited at levels as low as 0.5% (wt/vol) [12,13]. This sensitivity may explain the reduced abundance of F. prausnitzii in CD patients, who often have elevated bilirubin levels and bile acids due to bile acid malabsorption, a sign of a compromised small intestine in CD patients [14]. Bile acids, produced in the liver and stored in the gallbladder, are essential for digesting and absorbing fats. However, when present in excessive amounts, they can be toxic to the gut microbiota, disrupting microbial diversity and promoting the growth of harmful bacteria [15]. For beneficial bacteria like F. prausnitzii, exposure to high bile acid concentrations can hinder growth, impair metabolic functions, and compromise their protective roles, thereby destabilizing the gut ecosystem [16].

One promising approach to supporting F. prausnitzii under bile acid stress is the use of prebiotics. The International Scientific Association of Probiotics and Prebiotics (ISAPP) defined “dietary prebiotics” as ingredients that are selectively fermented, causing specific changes in the composition and/or activity of the gastrointestinal microbiota, which in turn offer health benefits to the host [17,18].

Fructooligosaccharides (FOSs), the well-known prebiotics, are particularly effective at enhancing butyrate production and promoting the growth of F. prausnitzii [19]. These oligosaccharides reach the colon intact, where they act as a substrate for beneficial bacteria, countering the negative effects of bile acid-induced dysbiosis [20].

Additional prebiotics, such as arabinoxylan (a hemicellulose in cereal grains) and inulin (a soluble fiber found in many fruits and vegetables) are fermented by gut microbiota and produce beneficial microbial metabolites, such as short-chain fatty acids that stimulate the growth of F. prausnitzii [21,22,23]. Golden Kiwi Fiber (GKF), derived from the fruit, supports microbial diversity and promotes the growth of beneficial gut bacteria, including F. prausnitzii [24,25]. Additionally, non-digestible polysaccharides such as pectin (found in fruits like apples and citrus) and resistant starches (present in green bananas and legumes) have been shown to modulate gut health by resisting digestion in the small intestine and reaching the colon, where they promote the growth of beneficial microbes like F. prausnitzii [26,27]. These polysaccharides also improve gut barrier function and modulate inflammatory responses. For example, citrus pectin remains largely undigested during transit through the upper gastrointestinal tract, stimulating the growth of beneficial gut bacteria and their metabolites, thereby increasing SCFA production [26].

Certain vitamins, such as riboflavin (vitamin B2), have been shown to support the growth and metabolic activity of anaerobic bacteria like F. prausnitzii [28,29]. Riboflavin and its derivatives flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) act as redox-active molecules that function as extracellular electron shuttles, facilitating electron transfer and maintaining cellular redox balance under anaerobic or stress conditions [30]. This mechanism may enhance bacterial survival in oxygen-sensitive environments and partially alleviate bile acid-induced growth inhibition [31].

The objective of this study was to identify prebiotics that effectively promoted the growth of F. prausnitzii under bile acid-induced stress. The effects of seven structurally distinct prebiotics on F. prausnitzii growth kinetics, including specific growth rate and doubling time, were quantified using continuous OD600 monitoring. Growth responses in the presence and absence of bile acids were compared to determine whether specific prebiotics mitigated bile-induced growth inhibition. In parallel, selected prebiotics were exposed to F. prausnitzii and the bacterial supernatants were then co-incubated with monocytic THP-1 cells to evaluate the potential effects of prebiotic exposure on THP-1 cell cytokine production, providing insight into their capacity to modulate immune response. Together, these analyses provided evidence for the potential of prebiotics as dietary or therapeutic interventions to improve gut health and restore microbial balance by enhancing F. prausnitzii abundance and its associated immune-modulating functions.

2. Results

2.1. Impact of Prebiotics on the Growth Dynamics of Faecalibacterium

The impact of various prebiotic fibers on bacterial growth was evaluated by measuring doubling time across increasing concentrations (0%, 0.5%, 1%, 1.5%, 2%), with a positive control (PC) included for comparison (Figure 1a–g). This concentration range was chosen to cover low-to-moderate prebiotic levels commonly used in in vitro gut fermentation studies, allowing evaluation of dose-dependent effects on bacterial growth without causing medium saturation or inhibitory effects [32,33]. Across all prebiotics tested, bacterial doubling time was consistently lower at 0–2% compared with the PC, indicating enhanced growth in the presence of prebiotics. Additionally, several prebiotics exhibited dose-dependent reductions in doubling time, with significant differences observed between concentrations.

Figure 1.

Figure 1

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Effect of different prebiotic fibers on F. prausnitzii doubling time at varying concentrations. Bacterial doubling time (in hours) was measured in the presence of increasing concentrations (0%, 0.5%, 1%, 1.5%, 2%) of seven prebiotic fibers: (a) FOSs (fructooligosaccharides), (b) inulin, (c) RS (resistant starch), (d) RF (riboflavin), (e) AX (arabinoxylan), (f) pectin, and (g) GKF (golden kiwi fiber). A positive control (PC) was included to represent maximum growth conditions. Across all panels, lower doubling times indicate faster bacterial growth. Most prebiotics significantly reduced doubling time in a concentration-dependent manner, particularly inulin, pectin, and RF, which showed the greatest growth-promoting effects. Statistical significance was determined using one-way ANOVA with correction for multiple comparisons. ns = not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Error bars represent standard deviation (SD) from biological replicates (n ≥ 3).

In Figure 1a, FOSs showed no significant difference in doubling time between 0%, 0.5%, and 1% (ns), indicating limited effect at low concentrations. However, 2% FOSs significantly reduced doubling time compared to both the positive growth control (PC) and lower concentrations (*** p < 0.0001), suggesting enhanced bacterial growth at higher doses of FOSs. In Figure 1b, inulin significantly reduced doubling time at all concentrations compared to the PC (**** p < 0.0001). Additionally, 1%, 1.5%, and 2% inulin all led to significantly decreased doubling times than 0.5% and 0% (p < 0.05 to **** p < 0.0001), indicating a clear dose-dependent growth-promoting effect. Resistant starch (RS), shown in Figure 1c, significantly decreased doubling time at 0.5%, 1.5%, and 2% compared to the PC (**** p < 0.0001). While no significant difference was observed between 0.5% and 1%, further reductions at 1.5% and 2% indicate that higher RS concentrations support more robust bacterial growth. In Figure 1d, riboflavin (RF) significantly decreased doubling time compared to PC at all concentrations (** p < 0.01 to **** p < 0.0001), with stepwise reductions across increasing doses. Notably, 1.5% and 2% RF showed significantly lower doubling times than 0.5% and 1%, supporting a strong dose-dependent effect. Arabinoxylan (AX), Figure 1e, resulted in significantly reduced doubling times at all concentrations compared to PC (*** p < 0.0001). Interestingly, there was no further reduction beyond 1%, suggesting that the stimulatory effect of AX may plateau at higher concentrations. This plateau likely reflects saturation of substrate utilization or physical limitations such as increased medium viscosity that can slow fermentation kinetics at higher polysaccharide levels, rather than a loss of prebiotic activity [34,35]. In Figure 1f, pectin markedly reduced doubling time at all concentrations compared to PC (*** p < 0.0001). The trend was clearly dose-dependent, with each increasing concentration from 0.5% to 2% associated with a statistically significant further reduction in doubling time (*** p < 0.001 to *** p < 0.0001), indicating high efficacy in promoting bacterial growth.

Finally, GKF, Figure 1g, significantly lowered doubling time at 0.5% and 1% compared to PC (** p < 0.01 to **** p < 0.0001) but further increases to 1.5% and 2% did not produce additional reductions (ns), suggesting an early saturation point. The early plateau in doubling time with GKF likely reflects the structural complexity of kiwifruit fiber and limits of microbial utilization. F. prausnitzii may preferentially consume the more accessible polysaccharides at lower concentrations, while more complex components require additional enzymatic processing. Once these pathways are saturated, further increases in GKF does not enhance growth, leading to an early saturation effect [36]. Dietary fibers with greater structural complexity (e.g., GKF) restrict the number and efficiency of microbes that can ferment them, resulting in slower or limited utilization compared with simpler carbohydrates, consistent with hierarchical fiber specificity and utilization by gut bacteria [37].

2.2. Impact of Prebiotics on the Doubling Time of Faecalibacterium Under Growth Stress

We evaluated the impact of increasing bile salt concentrations (0%, 0.1%, 0.25%, 0.5%) chosen to reflect physiologically relevant levels in the human small intestine [38,39] on F. prausnitzii doubling time as shown in Figure 2 (panels a–h) using MRS medium alone (panel a) and in the presence of various prebiotic fibers: FOSs, inulin, pectin, RF, RS, GKF, and AX (panels b–h).

Figure 2.

Figure 2

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Impact of increasing bile salt concentrations on bacterial doubling time in MRS medium and in the presence of different prebiotics. Bacterial doubling time was measured in MRS medium alone (a) and in MRS supplemented with 1% of various prebiotic fibers: (b) FOSs, (c) inulin, (d) pectin, (e) riboflavin (RF), (f) resistant starch (RS), (g) golden kiwi fiber (GKF), and (h) arabinoxylan (AX), under increasing concentrations of bile salts (0%, 0.1%, 0.25%, 0.5%). In MRS alone, doubling time of F. prausnitzii increased significantly with bile concentration, indicating bile-induced growth inhibition. The addition of certain prebiotics, particularly FOSs and AX, mitigated this effect at lower bile concentrations (0.1, 0.25 w/v%), while others (e.g., GKF) showed limited protective capacity. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post hoc test (vs. 0% bile) with multiple comparisons. ns = not significant; * p < 0.05; ** p < 0.01. Error bars represent standard deviation (SD) from biological replicates (n ≥ 3).

In Figure 2a, doubling time increased significantly with rising bile concentration in MRS medium alone, from a baseline of ~2 h at 0% bile to >6 h at 0.5% bile (p < 0.01), representing an approximate 200% increase in doubling time. This confirms the bile-induced stress and growth inhibition.

In Figure 2b, the presence of FOSs partially mitigated bile-induced growth inhibition of F. prausnitzii. Although doubling times increased significantly in 0.25% and 0.5% bile compared to 0% (* p < 0.05 to p < 0.01), the magnitude of the increase was less than observed for MRS alone. Notably, 0.1% bile had no significant impact (ns), suggesting a protective effect for FOSs at lower bile concentrations. In Figure 2c, inulin did not significantly alter doubling time across bile concentrations (ns), indicating limited bile-protective capacity. Although a trend toward increased doubling time was observed at 0.5% bile, the differences were not statistically significant. In Figure 2d, pectin significantly increased doubling time in 0.25% and 0.5% bile (* p < 0.05 to p < 0.01), consistent with a bile-induced stress response. However, the shift from 0% to 0.1% bile was not significant, suggesting pectin confers partial tolerance at low bile levels. In Figure 2e, riboflavin (RF) significantly increased doubling time in 0.25% and 0.5% bile (p < 0.01), while no significant difference was observed between 0% and 0.1% bile. This pattern resembles pectin, with a threshold effect beyond 0.1% bile. In Figure 2f, resistant starch (RS) also demonstrated a threshold response. There was no significant change from 0% to 0.1% and 0.25% bile (ns), but doubling time increased significantly at 0.5% bile (p < 0.01), indicating delayed bile sensitivity.

In Figure 2g, golden kiwi fiber (GKF) showed progressive increases in doubling time of F. prausnitzii with rising bile concentration. Each increase from 0% to 0.1%, 0.25%, and 0.5% bile was statistically significant (* p < 0.05), suggesting a linear bile-induced stress pattern with this fiber. Finally, in Figure 2h, arabinoxylan (AX) showed no significant increase in doubling time between 0% and 0.25% bile (ns), but 0.5% bile resulted in a significant increase (* p < 0.05). This again suggests a threshold-type stress response.

2.3. Cytokine Production of THP-1 Cells Induced by Prebiotic-Modified F. prausnitzii Supernatant

THP-1 cell viability was not affected by exposure to F. prausnitzii supernatants, including those generated in the presence of prebiotics, as assessed by LDH release (Supplementary Figures S1a and S2A). Supernatants from F. prausnitzii cultures exposed to prebiotics significantly modulated TNF-α expression in THP-1 cells (p = 0.043) (Figure 3) indicating context dependent immune regulation. Compared with negative control, several prebiotic conditioned supernatants stimulated marked proinflammatory TNFα upregulation upon exposure to THP-1 cells, indicating that these supernatants possess immunomodulatory activity. Among the treatments, MRS and pectin produced the strongest TNF induction, followed by AX, FOSs, and GKF. RF and RS elicited lower responses, suggesting weaker immunomodulatory potential.

Figure 3.

Figure 3

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TNF expression in THP-1 cells stimulated with F. prausnitzii culture supernatants derived from different prebiotics. The gene expression level of TNF-α was normalized with GAPDH as a control housekeeping gene and calculated using the 2−ΔΔCt method. Values of triplicate experiments are demonstrated as mean ± SE. Statistical differences among groups were determined using one-way ANOVA, with p < 0.05 considered significant.

The prominent effect of pectin was comparable to MRS, indicating that this prebiotic substrate may support F. prausnitzii metabolism in a way that enhances its proinflammatory signaling capacity. Given that TNF plays a central role in immune activation, these findings suggest that pectin could be as effective as MRS in driving immunomodulatory effects through F. prausnitzii-derived metabolites.

Exposure of THP-1 cells to F. prausnitzii supernatants derived from different prebiotics exposure showed variable IL-10 responses (Supplementary Figure S2A). AX, GKF, pectin, RF, and RS tended to enhance IL-10-fold change compared with the negative control, while FOSs and inulin produced smaller increases. However, the overall variation across replicates resulted in no significant difference among treatments (p = 0.53). This suggests that although some prebiotic substrates may favor the production of metabolites capable of promoting IL-10, none exceeded the effect observed with MRS, and the evidence for a strong anti-inflammatory effect remains inconclusive.

Similarly, stimulation with F. prausnitzii supernatants derived from different prebiotics exposure resulted in a wide range of IL-6 induction (Supplementary Figure S2B). FOSs, inulin, and MRS supernatants triggered pronounced upregulation compared with the negative control, whereas AX, RF, and RS showed little or no induction. Despite these apparent differences, the variation was not statistically significant (p = 0.30). These findings suggest that some prebiotics, particularly FOSs and inulin, may enhance the immunomodulatory activity of F. prausnitzii through IL-6 induction, though the effect did not surpass that of MRS alone.

3. Discussion

F. prausnitzii, a key butyrate-producing bacterium, plays a crucial role in gut homeostasis and anti-inflammatory processes [16]. However, its survival and growth in the presence of bile acids, which are toxic to bacterial cells, presents a significant challenge for its colonization in the gut, especially in conditions associated with bile acid-related dysbiosis, such as ileal inflammatory diseases associated with inflammatory bowel disease (IBD) [40,41]. This study demonstrates the significant role of prebiotics in supporting the growth of F. prausnitzii and shows that prebiotics may improve bacterial resilience to cell stress, such as that induced by exposure to bile acid, highlighting the potential of prebiotics to enhance microbial resilience, thereby promoting gut health under stressful conditions normally associated with IBD.

In this study, we tested seven prebiotics representing four different prebiotic classes to assess their impact on F. prausnitzii growth; Oligosaccharides (FOSs, arabinoxylan); Non-digestible Polysaccharides (Inulin, Golden Kiwi Fiber); Non-starch Polysaccharides (Pectin, Resistant Starch); and Vitamin (Riboflavin). Our study demonstrated that FOSs significantly supported F. prausnitzii growth under stressful growth conditions, consistent with previous findings [42,43] which highlighted FOSs’ ability to enhance the growth of beneficial gut bacteria, and promote butyrate production, a metabolite demonstrated to be crucial for gut health. However, some studies [44,45] reported that FOS supplementation does not uniformly improve gut microbiota composition, particularly in patients with altered health conditions or dysbiosis microbiota, such as those receiving enteral nutrition. Inulin also promoted F. prausnitzii growth in our study, aligning with previously reported findings [46,47], of moderate increases in F. prausnitzii abundance with inulin supplementation. Both FOSs and inulin supported F. prausnitzii growth under stress-induced conditions following exposure to bile acid, but their effectiveness may vary depending on the prebiotic type and the specific gut environment. Riboflavin was particularly beneficial in supporting F. prausnitzii viability under oxygen-limited conditions (bile acid exposure), as this anaerobic bacterium is sensitive to oxidative stress. Riboflavin’s role in enhancing microbial resilience aligns with studies showing its potential to promote butyrate-producing bacteria in the gut [28,48,49]. The lack of a similar effect from GKF could be due to differences in its polysaccharide structure or fermentation profile, which might require more time for microbial adaptation [50]. Although GKF supplementation has shown positive effects on F. prausnitzii abundance in constipated individuals [24], its effects on growth in our study were less pronounced. Pectin, a soluble fiber, enhanced F. prausnitzii growth under stress, which is consistent with previous studies showing pectin’s ability to support beneficial bacteria and protect against bile acid toxicity [16].

Our findings show that the capacity of F. prausnitzii supernatants (following exposure to prebiotics) to modulate immunocyte response depends on the prebiotic substrate used during culture. Pectin and MRS-derived supernatants induced TNF-α expression in monocytic THP-1 cells, indicating context-dependent immune activation.

In contrast, increases in THP-1-derived IL-10 were modest and inconsistent following supernatant exposure, and differences were not statistically significant. IL-6 responses also varied across prebiotic substrates, particularly with FOSs and inulin, but differences in cytokine response did not reach significance.

Different prebiotic structures can influence microbial fermentation products. For instance, pectin fermentation yields distinct short-chain fatty acid profiles compared with other fibers, a pattern linked to its chemical structure and degree of branching. Studies show that pectin substrates are fermented into acetate and butyrate and differentially stimulate taxa including F. prausnitzii, with structural features such as methylation and molecular weight affecting fermentation outcomes [26,51,52]. Thus, pectin treatment may result in increased active metabolites, resulting in altered immunocyte activating capacity. Overall, these findings indicate that the immunomodulatory profile of F. prausnitzii is not fixed. Instead, it appears to be shaped by the metabolic outputs generated from different prebiotic substrates. These results underscore the importance of substrate selection when evaluating synbiotic strategies targeting immune modulation. We note, however, that this study is limited to a single F. prausnitzii strain, uses only monocytic THP-1 cells as a surrogate for immune response, and does not include direct metabolite or in vivo analyses. Future work incorporating multi-strain validation, metabolomic profiling, diverse immune models, and formal safety testing will be important to fully understand the functional and translational potential of prebiotics in shaping F. prausnitzii activity.

4. Materials and Methods

4.1. Bacterial Strains and Growth Conditions

The reference strain of F. prausnitzii used in all experiments was obtained from the DSMZ-German Collection of Microorganism and Cell Cultures (DSM17677, strain designation A2-165). The strain was revived using chopped meat carbohydrate media (Anaerobe systems, Morgan Hill, CA, USA) supplemented with 30% filtered rumen fluid (Bar diamond Inc. Parma, ID, USA) under anaerobic conditions. Single colonies were purified and verified by Sanger sequencing. The pure bacterial colony was stored in glycerol (20% v/v) at −80 °C until needed.

4.2. Prebiotic Effect In Vitro and Growth Parameters

Seven prebiotics were used in this study including; Fructooligosaccharides (FOSs) (BENEO, Mannheim, Germany), arabinoxylan (Comet Bio, Schaumburg, IL, USA), Inulin from chicory, (Sigma-Aldrich, St. Louis, MO, USA) Golden Kiwi Fiber (Livs Pharma, Weston, FL, USA), citrus pectin, (Thermo Scientific, Waltham, MA, USA), Resistant Starch (ADM, Chicago, IL, USA), and Riboflavin, (Dot Scientific Inc., Burton, MI, USA). The chosen prebiotics display a degree of polymerization ≥3. Commercial prebiotics with a reported degree of polymerization >3 were selected to model resistance to host digestion and relevance to colonic microbial fermentation, and all substrates were evaluated under identical experimental conditions. Different concentrations of the prebiotics were evaluated in co-culture with F. prausnitzii, as described previously [53]. Prebiotics from filter-sterilized stock solutions were added with a reduced version of the De Man–Rogosa–Sharpe (rMRS) broth (Millipore, Sigma-Aldrich, Germany) to reach final concentrations ranging from 0.5 to 2%.

To compare the effect of prebiotics on the growth of F. prausnitzii, growth kinetics experiments in the absence (control) or presence of varying concentrations (0.5–2%) of prebiotics were performed. Overnight cultures of F. prausnitzii were grown in rMRS and washed twice using 0.85% (w/v) sterile saline solution by centrifugation (12,000× g, 5 min). Next, the inoculum of ~1.5 × 108 Colony Forming Units (CFUs)/mL was adjusted to 0.5 McFarland’s standard and added to each well of a microtiter plate (polystyrene, 96 well flat bottom, Millipore Sigma, NY, USA). The cultures were incubated anaerobically at 37 °C for 24 h in triplicate together with a negative control (un-inoculated MRS medium) and growth control (inoculated MRS containing 2% glucose). Optical density was measured every 60 min up to 24 h at a wavelength of 600 nm (OD600) using a Cerillo Alto Plate Reader (Opentrons, Long Island City, NY, USA). Each experiment was conducted in 3 biologic replicates.

4.3. Resistance to Bile Salts

To determine if the bacterial growth rates in the presence or absence of prebiotics was affected by stress, we co-incubated F. prausnitzii and prebiotics with bile, using published methodology [54]. Log-phase bacterial cultures were incubated in MRS broth containing 1% prebiotic solution and (0–0.5% w/v) porcine bile (Sigma-Aldrich, St. Louis, MO, USA). The cultures were incubated anaerobically at 37 °C for 24 h in triplicates together with negative (un-inoculated MRS+ bile medium) and positive controls (inoculated MRS containing 2% glucose). Optical density was measured every 60 min at a wavelength of 600 nm (OD600) using a Cerillo Alto Plate Reader (Opentrons, Long Island City, NY, USA). Each experiment was conducted 3 times to generate biological replicates.

4.4. Exposure of THP-1 to Prebiotic Supernatants from F. prausnitzii

To determine whether the supernatant of F. prausnitzii following culture with various prebiotics could modulate immune cell response, THP-1 (human monocytic cell line) cells were obtained from ATCC (American Type Culture Collection: TIB-202) and grown in suspension in RPMI + Glutamax supplemented with 10% (v/v) FBS in a humidified 37 °C, 5% CO2 incubator. Low passage (passage ≤ 15) cells were plated in 24-well tissue culture treated plates (Corning Incorporated Costar, Corning, NY, USA) and incubated for 72 h with 100 nM of phorbol 12-myristiate-13 acetate (PMA) (Sigma-Aldrich, St. Louis MO, USA) to induce monocytic differentiation. THP-1 cells were exposed to prepared supernatants from F. prausnitzii at a final concentration of 100 µg/mL The pH of all F. prausnitzii culture supernatants was adjusted to neutral (pH 7.0) prior to stimulation. Cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. Lipopolysaccharide (LPS) (10 ng/mL) was used as a positive monocyte stimulation for comparison to the supernatants of F. prausnitzii. Negative control supernatants were generated from F. prausnitzii incubated in De Man–Rogosa–Sharpe (MRS) broth without glucose. Positive control supernatants were generated from F. prausnitzii incubated in MRS media containing 2% glucose.

To assess potential cytotoxic effects of prebiotic-conditioned bacterial supernatants, THP-1 cell viability was evaluated using an LDH Cytotoxicity Assay Kit (#37291), (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. LDH activity was measured in cell-free culture supernatants, with untreated cells serving as spontaneous release controls and detergent-lysed cells serving as maximum release controls.

Following 24 h of stimulation, the THP-1 cells were centrifuged at 250× g and RNA extraction and RT-qPCR was performed as described [55].

5. Conclusions

In conclusion, our study shows that prebiotics, particularly FOSs, inulin, pectin, and RF, support the growth of F. prausnitzii and influence its responses to bile salt stress in vitro. These prebiotics reduced bacterial doubling times and partially mitigated bile-induced growth inhibition. Bacterial Supernatants derived from different prebiotic co-cultures modulated TNF-α expression in THP-1 cells, with pectin showing the strongest alteration, suggesting substrate-dependent bacterial response and supernatant immunomodulatory capacity. However, other cytokine responses (IL-10 and IL-6) were variable and not statistically significant, and no in vivo or multi-strain validation was performed, limiting the interpretation of immunomodulatory capacity. Overall, our findings indicate that the effects of prebiotics on F. prausnitzii are likely influenced by the chemical structure of the substrate. Further studies are needed to identify the specific metabolites responsible for the observed effects and to assess functional probiotic effects in vivo.

Acknowledgments

The authors have reviewed and edited the output and take full responsibility for the content of this publication.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27041698/s1.

ijms-27-01698-s001.zip (313.1KB, zip)

Author Contributions

Conceptualization, M.A.G.; methodology, S.A., T.S.M. and M.A.G.; software, S.A.; validation, S.A.; formal analysis, S.A. and T.S.M.; investigation, S.A.; resources, T.S.M. and M.A.G.; data curation, S.A.; writing—original draft preparation, S.A. and K.D.R., writing—review and editing, S.A., K.D.R., T.S.M. and M.A.G.; visualization, S.A.; supervision, M.A.G.; project administration, M.A.G.; funding acquisition, M.A.G. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials; further inquiries can be directed to the corresponding author.

Conflicts of Interest

This research was sponsored in part by a grant from BIOHM Health, LLC. The presentation of the results of this study does not constitute endorsement by any of the researchers or their affiliations. BIOHM Health, LLC had no role in the collection, analysis, or interpretation of the data. M.A.G. is a founding partner of BIOHM Health, LLC.

Funding Statement

The work was supported in part by a grant from BIOHM Health, LLC. BH10074. M.A.G. and T.S.M. were supported by the National Institute of Allergy and Infectious Disease RO1 AI172944.

Footnotes

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Supplementary Materials

ijms-27-01698-s001.zip (313.1KB, zip)

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials; further inquiries can be directed to the corresponding author.

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