A montmorillonite-based oral fermentation system enables long-lasting in-situ biosynthesis to restore intestinal homeostasis

Domain: neuroscience
Published: 2026-02-06
Editorial outcome: major_revision

Abstract
The disruption of intestinal homeostasis represents a hallmark of different diseases. Here, we report a montmorillonite-based oral fermentation system (MOFS) that achieves long-lasting in-situ preparation of beneficial bioproducts for intestinal homeostasis restoration. MOFS is fabricated by loading metabolic substrates into the lamellar structure of montmorillonite via cation replacement and adsorbing the substrate-related bacteria through electrostatic interactions. The water-impermeable property and mucoadhesive ability of montmorillonite endow MOFS with high oral availability and extended retention in the gut. The continuous release of loaded substrates and the formation of biofilms by carried bacteria enable long-term yet efficient synthesis of beneficial metabolites in the intestine, which comprehensively improve gut microbiota composition, suppress intestinal inflammation, and increase the integrity of the gut barrier. In murine models of imbalanced intestinal homeostasis, MOFS demonstrates superior efficacies by alleviating the associated symptoms, even in comparison to clinical therapeutics.

Introduction
Intestinal homeostasis, or the healthy and balanced state of the intestine, is established by intricate interactions between the intestinal barrier, the gut microbiota, and the host immune system1. Under normal physiological conditions, the intestinal barrier ensures the selective permeability of dietary nutrients and the physical separation of the gut microbiota from the host immune system1. Meanwhile, the gut microbiota actively shapes the host immune system and modulates intestinal barrier functions2,3. For instance, the gut symbiont Clostridium sporogenes (C. sporogenes) metabolizes tryptophan to indole and its derivatives, which have been reported to promote the expression of tight junction proteins to enhance intestinal barrier functions and stimulate the host immune system to play anti-inflammatory effects4. In addition, the host immune system reinforces intestinal barrier functions by priming protective immune responses against pathogens and maintaining tolerance to commensal microbiota and food antigens5,6. Usually, the breakdown of intestinal homeostasis may occur upon subjecting to external attacks7,8. As a hallmark of multiple diseases, this often causes the pathogenesis of inflammatory bowel diseases (IBD), acquired immunodeficiency syndrome, viral hepatitis, kidney injury, diabetes, and even cancers1,9. Therefore, strategies capable of restoring intestinal homeostasis hold great significance for disease treatment.

Conventional approaches to protect and restore intestinal homeostasis mainly focus on the intervention of the gut microbiota, the intestinal barrier, or the host immune system by small molecular therapeutics or macromolecular biologicals10,11. For example, a domain-binding small molecule divertin has been documented to reverse the intestinal barrier by blocking myosin light chain kinase recruitment12. Moreover, biopharmaceuticals, such as Janus kinase inhibitors and ustekinumab, have been applied clinically to restore intestinal immune homeostasis13,14. Unfortunately, the application of these oral therapeutics always faces a short half-life due to the instability in the intraluminal environments of the gastrointestinal tract. To overcome these challenges, a variety of advanced drug modification methods and delivery systems have been developed during the past few years15–17. For example, sodium butyrate formulated with enteric coating or encapsulation has been prepared to avoid the absorption of butyrate in the stomach, while increasing its concentration in the colon, where it plays a therapeutic effect by recovering microbial balance18. In addition, hydrophilic polysaccharide inulin has been conjugated with hydrophobic compound ursodeoxycholic acid to self-assemble into oral nanoscale devices for enhancing T-cell immunity modulation19. Despite these improvements, previous therapeutics have an overall low oral bioavailability caused by limited gut retention. More importantly, they are difficult to comprehensively restore intestinal homeostasis, as which highly depends on the multiple interactions among the gut microbiota, the intestinal barrier, and the host immune system.

Here, we present a montmorillonite-based oral fermentation system (MOFS) that provides durable in-situ synthesis of beneficial bioproducts for intestinal homeostasis restoration (Fig. 1a). MOFS is constructed by carrying metabolic substrates in the interlayer of montmorillonite (MMT) through cation replacement and then loading the substrate-associated bacteria via electrostatic interactions. As a proof-of-concept study, we fabricate MOFS using tryptophan and lactulose as the substrates and employing Enterococcus faecalis (E. faecalis), a lactic acid bacterium that metabolizes tryptophan and lactulose separately to indole and lactic acid, as the substrate-related microbe. The water-impermeable property of MMT endows MOFS with strong resistance to harsh gastrointestinal environments. Meanwhile, the mucoadhesive capacity of MMT enables the immobilization of MOFS on the mucus layer. In addition to the resulting high oral availability and prolonged retention in the intestine, the continuous release of carried tryptophan and lactulose and the formation of E. faecalis biofilms ensure long-lasting yet efficient production of lactic acid and indole in the gut (Fig. 1b). These beneficial bioproducts synergistically regulate the microbiota structure, mucosal immune responses, and barrier functions of the intestine. In two murine models of infective enteritis and IBD associated with Candida albicans (C. albicans) infection, MOFS effectively mitigates the related symptoms by restoring intestinal homeostatic balance (Fig. 1c). Given its flexibility of using cation replacement and electrostatic interactions to combine other metabolic substrates and bacterial strains, we anticipate the broad application of MOFS for in-situ biosynthesis of diverse therapeutics for treating different diseases.

Results
Design, preparation and characterization of MOFS
We chose MMT, a clinically used nanosheet-structured layered clay, as a carrier material due to its distinctive physicochemical characteristics, such as high stability, mucoadhesive ability, high adsorption capacity, water impermeability, and high cation-exchange capacity20. MMT is a 2:1 smectite with a permanent layer charge arising from isomorphic substitution21. Negative surfaces are balanced by exchangeable interlayer cations (e.g., Na⁺, Ca2+), conferring a high cation-exchange capacity21. It has been indicated that the abundant lipophilic components of the bacterial cell walls, such as lipoproteins and liposaccharides, make them easily adsorbed by MMT22. Indole, a principal intercellular signaling molecule in gut microbial ecosystems, plays dual regulatory roles in intestinal physiology by modulating the expression of tight junction proteins and orchestrating immune responses through aryl hydrocarbon receptor-mediated pathways23. Lactic acid refers to a readily accessible fuel for intestinal epithelia and an important metabolic buffer that affects the cell microenvironment, accounting for its key role in maintaining gut homeostasis24,25. Given these facts, we selected indole and lactic acid as targeted beneficial bioproducts for restoring intestinal homeostasis. Indole and lactic acid are mainly derived from the metabolism of tryptophan and lactulose by gut microorganisms, such as E. faecalis, Streptococcus rosenbergii, and C. sporogenes26,27. As a symbiotic specie, E. faecalis is widely used in dairy productions27. Thus, we chose tryptophan and lactulose as the substrates and E. faecalis as the substrate-related microbe to fabricate MOFS. The ability of E. faecalis to metabolize tryptophan and lactulose to indole and lactic acid was tested using Kovac’s reagent and methyl red, respectively. As shown in Supplementary Fig. 1a, there was a cherry red ring occurred on the top of the culture medium, suggesting the presence of indole. This could be explained by the combination of indole with p-dimethylaminobenzaldehyde in Kovac’s reagent. Similarly, after adding methyl red to the culture medium, the color changed from yellow to red, indicating the production of lactic acid that triggered an acidic environment and the change of the medium color (Supplementary Fig. 1b). We then optimized the conditions for the fabrication of MOFS by measuring the viability and proliferation of E. faecalis after incubation with different contents of MMT, tryptophan, and lactulose, respectively. As plotted in Supplementary Fig. 1c, the viability of E. faecalis increased with the amount of MMT, reaching a significant proliferation at 25 mg/ml. E. faecalis presented high viability in the medium supplemented with 0.6 mg/ml of tryptophan and 0.6 mg/ml of lactulose (Supplementary Fig. 1d–f). To ensure sufficient adsorption of bacteria by MMT, the incubation time was optimized. By measuring the number of adsorbed bacteria in MMT, a high ratio of 99% E. faecalis was obtained after a 4-hour incubation (Supplementary Fig. 1g). In comparison with free E. faecalis (Bac), scanning electron microscope (SEM) images showed that bacteria adsorbed in MMT (MMT@Bac) were covered by lamellar MMT (Fig. 2a). The adsorption of E. faecalis by MMT was further confirmed by laser scanning confocal microscopy (LSCM). We labeled MMT with Rhodamine B (RhB) by 1-(3-dimethylaminopropyl)−3-ethylcarbodiimide (EDC) and N-hydroxy succinimide (NHS). E. faecalis adsorbed in the resulting RhB-labeled MMT (RhB-MMT) was further stained with 4’,6-diamidino-2-phenylindole (DAPI). As expected, LSCM images displayed a consistent colocalization between E. faecalis and MMT (Fig. 2b and Supplementary Fig. 2). Note that there were rare free E. faecalis appeared around MMT, indicating efficient adsorption of E. faecalis by MMT.

On the basis of these results, we prepared MOFS by mixing MMT with tryptophan and lactulose at a weight content ratio of 40:1:1 and subsequently incubating with 8×108 colony forming units (CFUs)/ml of E. faecalis for 4 hours. To facilitate comparison, the obtained MMT carrying bacteria, tryptophan, and lactulose was defined as MMT@Bac@TL. Meanwhile, MMT@Bac, MMT loaded with tryptophan (MMT@T), MMT loaded with lactulose (MMT@L), MMT loaded with E. faecalis and tryptophan (MMT@Bac@T), and MMT loaded with E. faecalis and lactulose (MMT@Bac@L) were prepared as controls, respectively. MMT@Bac@TL showed limited fluctuations in the viability of carried bacteria, demonstrating both the preparation procedure and the components of MMT, tryptophan, and lactulose had a negligible impact on E. faecalis vitality (Supplementary Fig. 1h). The characteristic X-ray powder diffractometer (XRD) reflection of MMT appeared at 2θ = 6.34° (1.25 nm). In MMT@T and MMT@L, the basal space of MMT increased slightly from 1.25 to 1.31 and 1.33 nm, due to the insertion of tryptophan and lactulose. This indicated that tryptophan and lactulose were intercalated inside the interlayer space of MMT (Fig. 2c). The cation exchange capacity of MMT enabled high loading contents of tryptophan and lactulose, which were calculated to 91% and 93% (Supplementary Fig. 1i), respectively. To verify the releases of tryptophan and lactulose, MMT@Bac@TL were incubated in simulated intestinal fluid (SIF), and the accumulated release at different time points was measured using an ultraviolet-visible spectrophotometer. After incubation for 12 hours, the total releases of tryptophan and lactulose separately reached 62% and 83% (Fig. 2d and e). We further measured the ζ-potentials of MMT, MMT@Bac, MMT@Bac@T, MMT@Bac@L, and MMT@Bac@TL, respectively. In contrast to the ζ-potential of MMT (−8.0 mV), the ζ-potential of MMT@Bac largely decreased (−27.8 mV), but this reduction was mitigated as the loading of tryptophan and lactulose (Fig. 2f). Collectively, these results proved MMT@Bac@TL could be simply constructed by taking advantage of MMT to adsorb both the metabolic substrates and the substrate-related bacteria.

High oral availability and extended gut retention of MOFS
Living bacteria suffer from deactivation and disintegration following oral ingestion due to the presence of gastric acid and intestinal juice stimulations28,29. We speculated that the water-impermeable property of MMT might protect the loaded bacteria from harsh gastrointestinal environments. To verify this, we examined the protective effect of MMT during gastrointestinal transit by counting the numbers of survived bacteria at different time points after incubation in simulated gastric fluid (SGF) and SIF, respectively. As expected, the presence of MMT could increase the survival of E. faecalis in gastric acid. After a 15-minute culture in SGF, 91% of the bacteria in the Bac group were deactivated. In contrast, the influence on E. faecalis viability was negligible in the MMT@Bac@TL group (Fig. 2g). It was found that MMT@Bac also had a higher survival rate compared to the Bac group, indicating the protective effect of MMT toward E. faecalis. The ability of MMT to protect loaded bacteria was further confirmed by incubation with SIF, as both MMT@Bac and MMT@Bac@TL enabled a greatly increased survival rate compared to the Bac group (Fig. 2h). MMT is a clay containing multiple isomeric substituents, where the aluminum and silicon atoms at the crystal edges undergo partial hydrolysis in water, forming silanol and aluminum hydroxyl groups30,31. These unsaturated edge sites are more reactive than saturated base sites and can bind to mucin in the mucus layer of the intestinal wall through hydrogen bonding, van der Waals forces, and hydrophobic interactions30,31. To examine the retention on the mucus layer, an equal area of ileum and colon segments was sampled to expose the mucus layer, followed by incubation with Bac, MMT@Bac, and MMT@Bac@TL, respectively. The numbers of E. faecalis in the different segments were counted after an overnight culture. As shown in Fig. 2i, compared to free bacteria, MMT@Bac@TL exhibited ~3-fold increment in adhesion to the mucus layer of the ileum compared to Bac or MMT@Bac. Likewise, MMT@Bac@TL presented the highest number of bacteria adhered to the mucus layer of the colon, further supporting its strongest adhesive ability (Fig. 2j).

The oral availability and gut retention of MMT@Bac@TL were also evaluated in vivo. To quantify the number of survived E. faecalis, the contents of the lumen were separately collected from different intestinal segments for bacterial plate counting 4 hours after oral administration of 4×107 CFUs of Bac, MMT@Bac, and MMT@Bac@TL in mice, respectively. Interestingly, the count of survived E. faecalis in the luminal contents collected from mice orally delivered with MMT@Bac@TL was substantially higher than those in the Bac and MMT@Bac groups. As plotted in Supplementary Fig. 3a, the amounts of living E. faecalis in the mucus layer from cecum and colon segments were increased by 48 times compared to either Bac or MMT@Bac. Similarly, it was found that in the luminal contents of the cecum and colon segment, the number of survived E. faecalis in the MMT@Bac@TL group reached 2×106 CFUs/g, which was higher than those of Bac (~3-fold) and MMT@Bac (~8-fold) (Supplementary Fig. 3b). As calculated, MMT@Bac@TL exhibited the highest total survival of delivered bacteria 4-hour post administration (Supplementary Fig. 3c). We further examined the long-term adhesive ability of MMT@Bac@TL in mouse intestine and found that the amounts of living E. faecalis in the luminal content and the mucus layer of the entire intestine from mice orally delivered with MMT@Bac@TL were higher than those with Bac and MMT@Bac 72 hours after oral ingestion (Supplementary Fig. 3d, e). Correspondingly, the total number of survived bacteria in the entire intestine was the highest in the MMT@Bac@TL group (Supplementary Fig. 3f). We also counted the number of survived bacteria in the luminal content of each segment, including the ileum, cecum, and colon (Fig. 2k–r). The plotted results indicated that rare living E. faecalis was detected in each location in the Bac and MMT@Bac groups. While there were a large number of survived bacteria in these locations in the MMT@Bac@TL group. Particularly, the total survivals were over 106 CFUs/g in the ileum (Fig. 2m), the cecum (Fig. 2p), and the colon (Fig. 2r), respectively. Give MMT is known to bind mucin in the mucus layer30,31, the differential retention of MMT@Bac@TL across gut segments might be resulted from the structural and thickness variations in the mucus layer32. In addition, pH gradients along the gastrointestinal tract and gut motility could also play a role by influencing the binding33. Taken together, these results demonstrated the high oral availability and extended gut retention of MMT@Bac@TL.

MOFS-enabled long-lasting in-situ synthesis of beneficial bioproducts
To determine the metabolic efficiency of MMT@Bac@TL, the levels of indole and lactic acid were measured using corresponding test kits after incubation for the indicated time points. The levels of indole in the MMT@Bac@TL group after 72 hours of incubation were ~1.7- and ~2.3-fold higher than those of Bac+T (a simple mixture of E. faecalis and tryptophan) and MMT@Bac@T, respectively (Fig. 3a). Meanwhile, the concentration of lactic acid produced by MMT@Bac@TL reached ~0.5 mmol/L after 24 hours of incubation. Differently, only ~0.1 mmol/L lactic acid was produced by Bac+L (a simple mixture of E. faecalis and lactulose) or MMT@Bac@L (Fig. 3b). The higher productions of indole and lactic acid in MMT@Bac@TL compared to the simple mixture of bacteria with metabolic substrates (Bac+T or Bac+L) might be ascribed to the catalytic characteristic of MMT34,35. In addition, the synergistic effects of tryptophan and lactulose on bacterial growth and metabolism contributed to the MMT@Bac@TL-mediated high production of indole and lactic acid. Lactulose serves as a prebiotic, promoting the growth of lactic acid bacteria36. In turn, this facilitates the metabolism of tryptophan, resulting in increased indole production. Meanwhile, indole has been shown to modulate the growth, viability, biofilm formation, motility, and stress resistance in different bacteria37. We next evaluated the influence of metabolic substrate supplements on the metabolic profile of MMT@Bac@TL in vivo. Mice were orally delivered with Bac, MMT@Bac, or MMT@Bac@TL, and the concentrations of indole and lactic acid in different intestinal segments were measured 4 hours after gavage. As shown in Fig. 3c-e, the concentration of indole in the MMT@Bac@TL group was the highest among all the groups. The concentration of indole in the cecum was increased by ~2.7- and ~2.0 times compared to Bac or MMT@Bac (Fig. 3c). The total levels of indole in the cecum and colon segment sampled from mice treated with Bac and MMT@Bac were only ~0.7 mmol/g prot, which was increased by ~2 times in the MMT@Bac@TL group 4 hours after oral administration (Fig. 3d). Accompanied by promoted production of indole, the concentration of lactic acid in the MMT@Bac@TL group was increased by ~4- and ~2-fold compared to those in the Bac and MMT@Bac group 12 hours after oral administration, respectively (Fig. 3f). Moreover, we monitored the metabolites over a time course. The concentration of indole in the MMT@Bac@TL group was increased by ~1.4-fold compared to those in the Bac and MMT@Bac group 24 hours after oral administration, respectively (Supplementary Fig. 4a). Indole levels at 48 hours after oral administration and lactate levels at 24 hours and 48 hours showed no significant differences (Supplementary Fig. 4b–d). Notably, both the levels of indole and lactic acid in the MMT@Bac@TL group were increased by ~2-fold compared to the Bac group 72 hours after oral gavage. In contrast, the administration of MMT@Bac failed to enhance lactic acid production throughout the time course (Fig. 3g, h). These dynamic changes in indole and lactic acid production observed over time in the MMT@Bac@TL group might be associated with transitions, substrate availability, and microbial adaptation of the sample in the gut. Collectively, these data showed that MMT@Bac@TL enabled long-lasting in-situ biosynthesis in the intestine, and that the production of beneficial metabolites could be amplified by co-loading tryptophan and lactulose.

It has been reported that bacteria resided in biofilms express distinct properties compared to their free-living counterparts, including increased metabolic activities38,39. For instance, intestinal bacteria associated with biofilms can metabolize diet and mucin-related carbohydrate complexes into short-chain fatty acids, such as acetates, propionates, and butyrates38,40. As efficient surface adhesion is a prerequisite for biofilm formation, we speculated that the mucoadhesion ability of MMT@Bac@TL might promote the formation of E. faecalis biofilms, thereby enhancing bacterial metabolism. To confirm this hypothesis, we next assessed the formation of biofilms by MMT@Bac@TL using Congo red test. Typically, on Congo red plates, bacterial strains that produce rough and black colonies are recognized as a sign of biofilm formation. After 24 and 48 hours of incubation, the groups containing MMT, including MMT@Bac and MMT@Bac@TL, changed to brown or black, while Bac remained in red (Supplementary Fig. 5a). We further quantified the formation of biofilms using Congo red staining after 24 and 48 hours of incubation in SIF. In the MMT@Bac@TL group, the absorbance at 495 nm showed approximately 3-fold (24 hours) and 2-fold (48 hours) increments compared to the Bac group, respectively (Supplementary Fig. 5b). The enhanced biofilm-forming ability of MMT@Bac@TL was also confirmed by crystal violet staining. After culturing each group in a 96-well plate for 6 or 48 hours, an absorbance measurement at 540 nm was performed to quantify biofilm production. Consistent with the results of the Congo red assay, MMT@Bac@TL showed ~18 and ~34-fold increments compared to the Bac group after 6 and 48 hours of incubation, respectively (Fig. 3i, j). The enhanced biofilm formation observed in MMT@Bac could be attributed to MMT’s high surface area, which provides an adhesive scaffold that stabilized the development and maturation of biofilm matrix41. In addition, we measured the biofilm production in the intestinal environment by culturing Bac, MMT@Bac, and MMT@Bac@TL in both isolated intestinal segments and SIF. Following 48 and 72 hours of incubation, the tissues were stained with DAPI and Alexa Fluor 633-concanavalin A (ConA) to observe biofilm formation. As shown in Fig. 3k and Supplementary Fig. 6a, the mean fluorescence intensity (MFI) of ConA in the MMT@Bac@TL group was significantly higher than other groups, indicating a denser biofilm layer on the inner surface of the intestine. The ConA MFI of MMT@Bac@TL was ~40-fold higher than MMT@Bac and ~14-fold higher than Bac (Supplementary Fig. 6b). Meanwhile, the formation of biofilms was further confirmed by SEM. Unlike the smooth surface of bare bacteria, MMT@Bac displayed a rough surface due to the presence of MMT. An enhanced bacterial aggravation on a rougher surface was observed in the MMT@Bac@TL group compared to MMT@Bac as the formation of biofilms (Supplementary Fig. 6c). Moreover, we evaluated the biofilm formation by MMT@Bac@TL in vivo. Mice were orally administered with Bac, MMT@Bac, or MMT@Bac@TL, and the colon segments were collected 6 days post-treatments. As depicted in Supplementary Fig. 7, compared to either Bac- or MMT@Bac-treated mice, more biofilms were observed on the colon sections sampled from MMT@Bac@TL-treated mice. Both in vitro and in vivo experiments revealed that the superior biosynthesis efficiency of MMT@Bac@TL was benefited from the formation of biofilms, which might be associated with the upregulated energy metabolism of bacteria in biofilms. An upregulated energy metabolism characterized by enhanced glycolysis and tricarboxylic acid cycle activity has been observed in intestinal biofilms38,39. The enhanced energy metabolism supplies additional adenosine triphosphate (ATP) and central carbon intermediates to fuel anabolic pathways, such as amino acid, lipid, and exopolysaccharide biosynthesis42. Thus, we further evaluated potential metabolic pathways underlying the biofilm-mediated increase in metabolite production by comparing pyruvate and ATP levels between MMT@Bac@TL with and without biofilms. Given that biofilm formation was observed after 3 days of SIF culture, we cultured MMT@Bac@TL in PBS using the same time schedule as a biofilm-negative control. As shown in Supplementary Fig. 8, compared to MMT@Bac@TL cultured in PBS, the MMT@Bac@TL in SIF presented increases in the levels of pyruvate (~10-fold) and ATP (~2-fold).

Positive modulation of the gut microbiota by MOFS
The gut microbiota is considered as one of the key elements contributing to the regulation of intestinal homeostasis7. The imbalance of gut homeostasis caused by the changes in microbial structure, namely gut dysbiosis, is linked to a broad spectrum of diseases1. Probiotic bacteria play significant roles in maintaining a healthy microbial structure by secreting beneficial metabolites3. For example, indole and lactic acid can downregulate the expressions of virulence genes and/or movement/invasion-related genes, thereby reducing the virulence and biofilm formation of pathogens3,37,43. Given the enhanced production of metabolites, we next investigated the effects of MMT@Bac@TL on the modulation of the gut microbiota. We first cultured a classical probiotic strain of Escherichia coli Nissle 1917 (EcN) in a culture medium supplied with Bac, MMT@Bac, or MMT@Bac@TL. EcN cultured in pure culture medium was set as a control. Plate counting of EcN was conducted after 2 and 4 hours of incubation at 37°C on Luria-Bertani (LB) agar plates containing 50 μg/ml kanamycin to eliminate E. faecalis. Compared to other groups, the supplement of MMT@Bac@TL significantly promoted the growth of EcN (Fig. 4a, b). After 2 hours of culture, the growth rate of EcN in the medium group reached only ~64%. The supplement of MMT@Bac substantially increased the growth rate. The growth rate was 144-fold higher in the MMT@Bac@TL group in contrast to the MMT@Bac group. This effect of MMT@Bac@TL on EcN growth was observed with incubation extending to 4 hours. We further tested the effect of MMT@Bac@TL on the growth of pathogenic Salmonella Typhimurium (S. typhimurium). As shown in Fig. 4c, MMT@Bac@TL presented a 25-fold increment in the inhibition of S. typhimurium growth. The supplement of Bac could not inhibit the growth of S. typhimurium. Although MMT@Bac showed an inhibitory effect on S. typhimurium, with the culture time prolonged to 6 hours, only MMT@Bac@TL could effectively inhibit the growth of S. typhimurium (Fig. 4d). The different effects of MMT@Bac@TL on EcN and S. typhimurium might be relevant to the context where the interspecies signaling molecule indole can modulate the expression of crucial regulatory genes in S. typhimurium44,45. On the other hand, lactic acid can cause a drop in pH, inhibiting the growth of pathogens24. To further understand the influence of MMT@Bac@TL on the gut microbiota, we studied the variation of microbial composition in mice after suffering from antibiotic cocktails. As illustrated in Supplementary Fig. 9, following 2-week treatments with a cocktail of neomycin sulfate, vancomycin, ampicillin, and metronidazole, mice were orally administered with phosphate-buffered saline (PBS), Bac, MMT@Bac, or MMT@Bac@TL at a dose per day for 7 days. Mice without any treatment were set as a healthy control. The similarity of microbial communities among groups was confirmed using principal component analysis (PCA). As validated in Fig. 4e, the microbial structure of mice treated with MMT@Bac@TL was closer to that of healthy mice. Taxonomic analysis and linear discriminant analysis effect size (LEfSe) indicated that oral administration of MMT@Bac@TL enriched the abundances of Bacilli, Synergistota, and Staphylococcales (Fig. 4f–i and Supplementary Fig. 10). It has been identified that most species from Bacilli class show a potent effect against pathogens by producing bacteriocin46. Synergistota has been identified as a species capable of producing butyrate, which has received considerable attention for its multiple beneficial effects on intestinal homeostasis47. Although the strains from the Staphylococcales order are recognized as pathogens, a group of antimicrobial peptides and anti-inflammatory peptides produced by Staphylococcus aureus (S. aureus) have been identified48. For example, the extracellular adherence protein expressed by S. aureus has been demonstrated to be an anti-inflammatory factor that inhibits host leukocyte recruitment, T-cell proliferation, and delayed-type hypersensitivity responses49. Overall, these data verified the capability of MMT@Bac@TL to positively regulate the gut microbiome.

Suppression of intestinal inflammation by MOFS
The advances in understanding the compositions and functions of individual microbial species and complex microbial communities highlight the importance of microbial metabolism to the host immune system5. Recent studies report that lactic acid acts as an essential signaling molecule that shapes the innate and adaptive immune responses by upregulating the expressions of anti-inflammatory genes or M2-like genes25. It has also been documented that as important metabolites of lactobacillus species, indole and its derivatives enhance the M2 polarization of macrophages, subsequently reducing intestinal inflammation50. Given the critical role of macrophages in the maintenance of intestinal homeostasis, we first evaluated the effect of MMT@Bac@TL on the polarization of macrophages. J774A.1 cells treated with lipopolysaccharide (LPS) were cultured with blank medium, Bac, MMT@Bac, MMT@Bac@T, MMT@Bac@L, and MMT@Bac@TL, respectively. Cells without any treatment were set as a control. After 24 hours of culture, the percentage of M2 macrophages in the MMT@Bac@TL group was ~2-fold higher than those in other groups (Fig. 5a–d). Furthermore, the M2/M1 classification ratio also showed a significant increase, indicating a notable M2 polarization effect (Fig. 5e). The augmented M2 polarization was followed by a notable increase in the levels of cytokines and chemokines involved in regulating intestinal homeostasis (Fig. 5f–h). Although its association with inflammation responses, interleukin-1β (IL-1β) is crucial for maintaining the homeostasis of intestinal immune responses, such as immune globulin A (IgA) production51. Vascular endothelial growth factor (VEGF) plays a vital role in epithelial cell restitution by preserving mucosal homeostasis following mucosal damage52. Likewise, transforming growth factor-β (TGF-β) is a key regulator of the regenerative process of the intestine53. As evidenced, the levels of IL-1β, VEGF, and TGF-β secreted by macrophages cultured with MMT@Bac@TL were the highest. In addition, interleukin-4 (IL-4) was 30% increased by MMT@Bac@TL compared to the medium group, contributing to an increased ratio of IL-4 to IL-6 (Supplementary Fig. 11a, b). Meanwhile, the ratio of interleukin-10 (IL-10) to nitric oxide (NO) in the MMT@Bac@TL group was 17% higher than in the medium group (Supplementary Fig. 11c). IL-4 and IL-10 are key mediators of mucosal immune regulation to control inflammation54,55. In contrast, increases in IL-6 and NO are commonly involved in the aggravation of immunopathology during gut inflammation56,57. Dendritic cells (DCs) are extensively involved in maintaining intestinal homeostasis by mediating the tolerance to food antigens, limiting the reactivity to the gut microbiota, and responding to intestinal pathogens58. In addition to macrophage polarization, MMT@Bac@TL facilitated the maturity of DCs (Supplementary Fig. 12). We further examined the effects of MMT@Bac@TL on the intestinal immune system in vivo. Consist with the effects of MMT@Bac@TL on macrophages and DCs, MMT@Bac@TL promoted the M2 polarization of macrophages and the maturation of DCs in the mesenteric lymph nodes (MLNs) (Fig. 5i, j and Supplementary Fig. 13a, b). DCs are know to regulate the production of IgA by plasma cells59. Indeed, accompanied by increased DCs, there were more IgA+ B cell populations in the Peyer’s patches (PPs) sampled from MMT@Bac@TL-treated mice compared to other treatments (Fig. 5k and Supplementary Fig. 13c). Note that IgA plays a pivotal role in gut homeostasis through orchestrating the mucosal responses, namely protecting the mucosal surface from pathogen invasion while tolerating commensal bacteria6,60 Briefly, MMT@Bac@TL presented a favorable ability to regulate gut immune responses for maintaining intestinal homeostasis.

Protection of intestinal barrier integrity by MOFS
The composition of the gut microbiome influences the intestinal immune system, which in turn affects the integrity of the gut barrier2. In light of this, we then evaluated the capability of MMT@Bac@TL to protect the intestinal barrier from external insults. The integrity of the intestinal barrier is typically related to the mucus layer and tight junctions1. Under normal physiological conditions, goblet cells continually produce mucin to replenish and maintain the mucus layer. Recent studies disclose that the gut microbiota and its metabolites modulate the functions of goblet cells61. For instance, indole has been demonstrated to enhance mucus secretion by upregulating IL-10 and MUC gene expression62. We hence cultured HT29-MTX-E12 cells, a mucus-producing intestinal cell line, with 50 μg/ml LPS for 6 hours and subsequently treated them with 1×105 CFUs of Bac, MMT@Bac, MMT@Bac@T, MMT@Bac@L, and MMT@Bac@TL for an additional 6 hours, respectively. The level of mucus was then assessed using Alcian Blue/Periodic acid-Schiff (AB/PAS) staining, which can distinguish neutral mucin from acid mucin, thereby providing a comprehensive analysis of the mucus composition. As illustrated in Fig. 6a, the proportion of mucus was significantly reduced due to LPS stimulation. As expected, incubation with MMT@Bac@TL showed the highest accumulation of mucus compared to other treatments. It was worth noting that MMT@Bac@TL exhibited an ~1.1-fold increase in acidic mucin production compared to the medium group (Fig. 6b). Meanwhile, the level of neutral mucin was decreased under the treatment of MMT@Bac@TL in contrast to the medium group (Fig. 6c). This resulted in an increased ratio of acidic mucin to neutral mucin, which was close to the normal condition (Fig. 6d). It has been identified that secreted mucin becomes more acidic and develop higher N- and O-linked glycosylation levels during maturation, which are implicated in the protection of the mucus layer from pathogen invasion63,64. In addition to the mucus layer, numerous studies have found that indole and lactic acid can enhance endothelial cell barrier functions by inducing the expression of genes involved with endothelial cell structures, including tight junctions, adherent junctions, and actin cytoskeleton65. As a widely used in vitro model of the intestinal barrier, Caco-2 cell monolayer was treated with 50 μg/ml LPS for 12 hours and then incubated with 1×104 CFUs of Bac, MMT@Bac, MMT@Bac@T, MMT@Bac@L, and MMT@Bac@TL for 7 hours, respectively. We first measured the effect of MMT@Bac@TL on cell permeability by quantifying the fluorescence intensity of FITC-dextran (4 kDa) that permeated from the upper to the lower chamber of the transwell device. As shown in Supplementary Fig. 14, compared to the cell culture medium-treated group, the fluorescence intensity of FITC-dextran was lower in the MMT@Bac@TL group, indicating decreased cell permeability. Given that tight junction barrier loss is an important contributor to increased barrier permeability, we next measured the expressions of tight junction proteins zona occludens 1 (ZO-1) and occludin under different treatments. The cells were stained with specific antibodies against the tight junction protein ZO-1 and occludin, which are critical components of the intestinal barrier. Expectedly, LPS stimulation reduced the expression of ZO-1 and occludin, which were notably recovered after incubation with MMT@Bac@TL. In contrast, other treatments presented negligible effects in the protection of tight junctions, particularly the expression of occludin (Fig. 6e and Supplementary Fig. 15a). Furthermore, in the antibiotic cocktail-induced mouse model of gut dysbiosis, we verified the potential of MMT@Bac@TL to protect the intestinal barrier. Compared to healthy mice, the antibiotic cocktail led to a marked depletion of goblet cells. In stark contrast to the sparse mucus observed in other treatment groups, the reduced expression of mucus was reversed by the MMT@Bac@TL treatment, which resulted in a level closely aligning with patterns found in healthy mice (Fig. 6f, h). We also validated the capability of MMT@Bac@TL to repair tight junctions in the colon of mice treated with antibiotics. Compared to the healthy control, antibiotic cocktail-induced gut dysbiosis had a more pronounced effect on the tight junction protein ZO-1 than on occludin. It is indicated that pro-inflammatory and contractile cues associated with dysbiosis rapidly remodel ZO-1 continuity and localization at cell-cell-cell borders66,67. By contrast, occludin homeostasis is more strongly influenced by context-dependent endocytosis/turnover pathways68. Notably, both decreased occludin and ZO-1 expressions triggered by antibiotic cocktail-induced gut dysbiosis could be greatly reversed by MMT@Bac@TL (Fig. 6g, i, j and Supplementary Fig. 15b). Together, MMT@Bac@TL could protect the intestinal barrier from damage under external attacks.

Potential of MOFS in treating S. typhimurium-induced enteritis
Encouraged by the beneficial effects of MMT@Bac@TL on intestinal homeostasis, we then evaluated the potential of MMT@Bac@TL in treating enteritis caused by S. typhimurium infection, which is a frequently occurred clinical scenario. A typical syndrome associated with infectious enteritis is the presence of intestinal homeostatic imbalance. However, currently available therapeutics are difficult to comprehensively restore intestinal homeostasis. To develop a murine model of enteritis, mice were infected with 5×107 CFUs of S. typhimurium via oral gavage 24 hours after pretreatment with streptomycin (Fig. 7a). The mice were orally administered with MMT, Bac, MMT@Bac, MMT@Bac@T, MMT@Bac@L, and MMT@Bac@TL one dose per day for 6 days, respectively. In each treatment group, equivalent MMT (0.05 mg), E. faecalis (1×107 CFUs), tryptophan (0.0013 mg), or lactulose (0.0013 mg) was applied. Infected mice treated with PBS were used as a negative control. The untreated mice served as a healthy control. Meanwhile, MMT, a type of medical clay used to eliminate pathogens69, was used as a positive control. As shown in Fig. 7b and c, S. typhimurium infection-induced decrease of colon length was effectively restored by MMT@Bac@TL. In contrast, other treatments failed to control this symptom. Specifically, the colon length of MMT@Bac@TL-treated mice (9.4 ± 0.1 cm) was longer than those of mice treated with PBS (7.7 ± 1.1 cm), MMT (8.2 ± 0.6 cm), Bac (7.4 ± 1.2 cm), MMT@Bac (8.0 ± 0.9 cm), MMT@Bac@T (8.3 ± 0.8 cm), and MMT@Bac@L (8.3 ± 0.9 cm), highlighting its superior treatment efficacy. To confirm that the improved effects were attributed to the enhanced production of beneficial metabolites, we measured the concentrations of indole and lactic acid in the gut. As plotted in Fig. 7d, compared to PBS, the treatments of Bac, MMT@Bac, and MMT@Bac@T could only induce a 3.0-fold increase in indole production. The indole level in the ileum sampled from mice treated with MMT@Bac@TL group was ~7.3 times higher than that in the PBS group. We also noted that the concentration of lactic acid in the MMT@Bac@TL group reached approximately 63 mmol/g in the intestine (Fig. 7e), which was increased by ~1.4 times compared to the PBS, MMT, Bac, or MMT@Bac group. After confirming its metabolic efficiency, we next tested the effect of MMT@Bac@TL on the inhibition of S. typhimurium colonization and translocation. As shown in Fig. 7f, the number of S. typhimurium in the intestine declined markedly by MMT@Bac@TL. Accompanied by a decreased colonization, rare S. typhimurium was detected in the kidney and the spleen (Fig. 7g, h). Differently, the rest treatments could not effectively inhibit the colonization of S. typhimurium in the gut, and a large number of S. typhimurium (~1010 CFUs) were translocated to the kidney and the spleen. The clearance of S. typhimurium by MMT@Bac@TL further alleviated intestinal inflammation and eventually protected the gut from damage. As illustrated in Fig. 7i and Supplementary Fig. 16a, myeloperoxidase (MPO) staining indicated that treatment with MMT@Bac@TL significantly reduced neutrophil granulocyte infiltration in the colon compared to other groups. Meanwhile, severe pathological injury on the muscular layer caused by S. typhimurium infection was mitigated by MMT@Bac@TL (Fig. 7j and Supplementary Fig. 16b). The above results verified the therapeutic potential of MMT@Bac@TL in intervening enteritis, which was ascribed to the modulation of intestinal homeostasis mediated by higher levels of beneficial metabolites.

Value of MOFS in treating colitis associated with C. albicans infection
C. albicans represented the most abundant genera in IBD patients70. It has been reported that candidalysin produced by C. albicans promotes the pathological process of IBD by inducing intestinal inflammation71. In addition to its role against pathogen infection, indole offers a viable alternative to eliminate C. albicans and its biofilms72. Lactic acid-producing bacteria are known to block C. albicans filamentation, an important virulence trait, by affecting the intestinal microenvironment43. In addition, E. faecalis was identified to secrete Fsr-dependent proteases, which inhibit hyphal morphogenesis and biofilm formation of the fungus, thereby reducing the virulence72. We hence speculated that MMT@Bac@TL could also remove pathogenic fungi in the gut during the treatment of enteritis. To clarify this, we first verified the bactericidal effect of MMT@Bac@TL on C. albicans in a rose bengal medium, the selective medium of C. albicans. After incubating C. albicans with MMT@Bac@TL at a 1:10 ratio (C. albicans to E. faecalis) for 3 hours, the CFUs of C. albicans in the MMT@Bac@TL group were reduced by 2 times compared to the medium group (Supplementary Fig. 17). The bactericidal effect of MMT@Bac@TL was increased up to 21-fold with incubation time prolonging to 24 hours. For greater relevance, we further compared the bactericidal effect of MMT@Bac@TL on pathogens with that of clinically available probiotics, including EcN and Bacillus subtilis (Bs). C. albicans were co-cultured with EcN, Bs or MMT@Bac@TL in yeast extract peptone dextrose (YPD) medium at a ratio of 1:10. The fungi cultured with medium were set as a control. After 12 hours of co-culture, samples were serially diluted, plated on YPD plates and incubated at 37°C for colony enumeration of C. albicans. As shown in Supplementary Fig. 18a, MMT@Bac@TL exhibited comparable effects in inhibiting C. albicans to those of clinically available probiotics. Encouraged by the bactericidal effect of MMT@Bac@TL on fungi, we further evaluated the bactericidal effect of MMT@Bac@TL on S. typhimurium SL1344, one of the representative pathogenic bacteria. As shown in Supplementary Fig. 18b, Bs has a negligible effect on inhibiting the growth of S. typhimurium. The counts of S. typhimurium were ~3-fold and ~2-fold lower in EcN and MMT@Bac@TL, respectively, compared to the medium group. Considering that the anti-inflammatory efficacy of living bacteria is another factor in the therapy of colitis, in addition to antimicrobial efficacy, we next compared MMT@Bac@TL with clinically available probiotics. J774A.1 cells pre-treated with LPS (1 µg/ml) for 24 hours were subsequently incubated with 102 CFUs EcN, Bs, or MMT@Bac@TL for 24 hours. The cells treated with culture medium served as the control. As shown in Supplementary Fig. 18c, the ratio of M2 to M1 macrophages was over 2-fold increase in MMT@Bac@TL compared to other treatments, indicating a potent anti-inflammatory effect of MMT@Bac@TL.

Having confirmed the antimicrobial and anti-inflammatory effects of MMT@Bac@TL, we next explored the therapeutic value of MMT@Bac@TL in a colitis model with C. albicans infection. As shown in Fig. 8a, mice were orally administered with streptomycin (20 mg/kg) and penicillin (20000 U/kg) for 2 days to clear the intestinal microbiota and then given 3% DSS water (w/v) freely over 23 days. During this period, mice were gavaged with C. albican (1×108 CFUs) every other day and simultaneously treated with MMT@Bac@TL containing 1×107 CFUs E. faecalis, 0.05 mg MMT, 0.0013 mg tryptophan, and 0.0013 mg lactulose every other day. Healthy mice and infected mice treated with PBS, 5-aminosalicylic acid (5-ASA, 6 mg/ml, 0.1 ml per day), and equivalent MMT, Bac, MMT@Bac, MMT@Bac@T, and MMT@Bac@L were applied as controls. After 12 doses of treatment, mice were euthanized, and samples were collected for further analyses. As presented in Fig. 8b, c, the average length of the colon sampled from MMT@Bac@TL-treated mice (8.6 ± 1.1 cm) was longer than those of mice treated with PBS (7.2 ± 0.7 cm), MMT (7.1 ± 1.0 cm), Bac (7.7 ± 0.9 cm), and MMT@Bac (7.5 ± 1.1 cm). The homogenates of the cecum were prepared for plate counting of C. albicans. The CFUs of C. albicans in the PBS group were as high as 2×104 CFUs/g (Fig. 8d). Notably, C. albicans was substantially lowered in the entire gut after treatment with MMT@Bac@TL (~2.0×103 CFUs). In contrast, there were ~2×104, 3×104, and 2×104 CFUs of C. albicans detected in the MMT, Bac, and MMT@Bac groups, respectively. We further used fluorescent in situ hybridization (FISH) to visualize the localization of C. albicans in the colon. Different from other treatments, a rare fluorescent signal of C. albicans was observed in the MMT@Bac@TL group (Fig. 8e and f). Meanwhile, MMT@Bac@TL induced effective defense responses against C. albicans infection as the percentage of IgA+ B cells in the MMT@Bac@TL group was 2-fold higher than that in the PBS group (Fig. 8g). IgA+ B cells have been demonstrated to play a nonredundant role against fungal infections. In addition, treatment with MMT@Bac@TL avoided the occurrence of intestinal damage and reduced the inflammation responses. While the disruption of the muscular layer and the elicitation of inflammation were obvious in the groups of 5-ASA, MMT, and Bac compared to the PBS group (Fig. 8h–j and Supplementary Fig. 19). In brief, these results elucidated the value of MMT@Bac@TL to treat fungal infection-associated IBD.

In summary, we report the design and preparation of MMT@Bac@TL that enables long-lasting in-situ synthesis of beneficial bioproducts for restoring intestinal homeostasis. MMT@Bac@TL is constructed by adsorption of E. faecalis onto the surface of MMT, which is preloaded with metabolic substrates of tryptophan and lactulose in the interlayer space through cation exchange. The water-impermeable and mucoadhesive property of MMT endows MMT@Bac@TL with high resistance and durable retention, achieving an efficient in-situ biosynthesis of beneficial metabolites of indole and lactic acid in the gut. These metabolites contribute to gut microbiota regulation, immune response balance, and intestinal barrier protection. Specifically, in an antibiotic-induced mouse model of gut dysbiosis, MMT@Bac@TL significantly inhibits the invasion of pathogenic bacteria and enhances the abundance of beneficial bacteria, recovering a normal composition and function of the gut microbiome. Meanwhile, it increases the percentages of M2 macrophages, IgA+ B cells, and mature DCs, which are conducive to intestinal homeostasis. Moreover, MMT@Bac@TL improves intestinal barrier integrity by increasing tight junction expression and mucus production. We also demonstrate the protective effects of MMT@Bac@TL in maintaining intestinal homeostasis, enabling satisfactory efficacies in treating infective enteritis and IBD with C. albicans infection. This work provides a unique platform for oral drug delivery, with an appealing characteristic of long-lasting in-situ synthesis of bioproducts. Before considering for future translation, the safety, dose, and administration frequency of MMT@Bac@TL need to be systemically examined and optimized. In our future work, we will pursue the versatility of this platform to carry other metabolic substrates and microbial species, with the aim of generating diverse therapeutic agents and exploring advanced approaches for treating different diseases.

Methods
Materials and strains
L(-)-Tryptophan (TP2893390) and 5-ASA (01122621) were purchased from Shanghai Titan Scientific Co., Ltd. MMT (M916670), lactulose (L812332), ampicillin (A830931), neomycin sulfate (N799581), metronidazole (M813525), and vancomycin (V871983) were purchased from Macklin Biochemical Co., Ltd. RhB (A5102) was purchased from Tokyo Chemical Industry Co., Ltd. Antibodies including anti-mouse CD11c-FITC (117305), CD11b-PerCPCy5.5 (101227), F4/80-phycoerythrin (PE, 123110), CD86-allophycocyanin (APC, 105011), CD86-PE/Cyanine7 (PE Cy7, 105014) CD206-PE-Cy7 (141719), CD206-Alexa Fluor® 647 (AF647, 141712), CD3-APC Cy7 (100221), CD45R/B220-PE-Cy7 (103221), and CD80-PE (104707) were purchased from Biolegend. Con A (C21402) and IgA-PE (12-5994-81) were purchased from Thermo Fisher Scientific. LPS (ST1470), DAPI (C1002), and NO detection kit (S0021S) were purchased from Beyotime Biological Technology Co. LB broth powder (A507002) was obtained from Sangon Biotech (Shanghai) Co., Ltd. E. faecalis agar (HB0268-2) and trypticase soy broth (TSB, HB4114) were purchased from Qingdao Hi-Tech Industrial Park Hope Bio-Technology Co., Ltd. E. faecalis was obtained from the American Type Culture Collections. EcN was purchased from Ningbo Bionice (China). S. typhimurium was purchased from the China General Microbiological Culture Collection Center. C. albicans was purchased from Shanghai Bioresource Collection Center. Mouse macrophage cell line J774A.1, human colon carcinoma cell line Caco-2, and DC 2.4 dendritic cell line were purchased from iCell Bioscience Inc. Mucus-producing intestinal cell line HT29-MTX-E12 was kindly provided by Wei Wu (Fudan University, Shanghai, China). Fetal bovine serum (FBS, 10099-141), penicillin-streptomycin solution (15140122), and Dulbecco’s Modified Eagle Medium (DMEM, 11965092) were purchased from Gibco. Nonessential amino acid solution (N1250) and crystal violet (G1062) were obtained from Beijing Solarbio Science & Technology Co., Ltd. HT-29-MTX-E12 cells were cultured with DMEM containing 10% FBS, 1% penicillin-streptomycin solution, and 1% nonessential amino acid. Caco-2, J774A.1, and DC 2.4 cells were cultured with DMEM containing 10% FBS and 1% penicillin-streptomycin solution. IL-4 (AF02165-A), IL-6 (AF02163-A), and IL-10 (AF02176-A) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Hunan Aifang Biotechnology Co., Ltd. VEGF (EK283), IL-1β (EK201B), and TGF-β1 (EK981) ELISA kits were obtained from Multi Sciences (Lianke) Biotech Co., Ltd.

Preparation of MMT@Bac@TL
To prepare MMT loaded with tryptophan and lactulose, MMT (0.125 g), tryptophan (0.0032 g), and lactulose (0.0032 g) were suspended in 5 ml ddH2O and then incubated at 37°C with a shaking speed of 200 rpm for 24 h. An E. faecalis colony was taken from an LB agar plate and cultured in LB liquid medium overnight (200 rpm, 37°C). Then, E. faecalis were washed with ddH2O twice and resuspended in 980 µl PBS. Afterward, MMT loaded with tryptophan and lactulose (900 μl) and E. faecalis (100 μl) were mixed and cultured at room temperature for 4 hours with shaking at 700 rpm. Similarly, MMT@Bac, MMT@Bac@T, and MMT@Bac@L are prepared in the same manner.

Characterization of MMT@Bac@TL
The morphologies of bacteria, MMT, and MMT@Bac were measured by SEM (HITACHI Regulus 8230). For SEM measurement, samples were dropped on a silicon wafer separately after serial dehydration in 30, 50, 70, 80, 90, and 100% ethanol for 10 minutes. XRD measures were performed by Bruker X-ray diffractometer (D8 ADVANCE Da Vinci). MMT, MMT@T, MMT@L, and MMT@Bac@TL were collected by centrifugation (7000 rpm, 10 minutes) and washed 3 times with ddH2O. The samples were then frozen at −80°C for 24 hours, followed by freeze-drying in a lyophilizer. The dry samples were analyzed for the changes in the d value by XRD. Bacterial numbers were determined by making serious dilutions of E. faecalis, MMT@Bac, MMT@Bac@T, MMT@Bac@L, or MMT@Bac@TL suspensions, culturing them on selective EF agar plates overnight (200 rpm, 37°C), and bacterial counts were measured using an automated colony counting system (Interscience Scan 500). The ζ-potential of MMT, MMT@Bac, MMT@Bac@T, MMT@Bac@L, or MMT@Bac@TL was determined by dynamic light scattering (DLS).

Fluorescence labeling of MMT@Bac
EDC (0.20 g, D1601), NHS (0.12 g, B0249), and RhB (0.01 g) were activated in 10 ml ddH2O for 1 hour. Then, MMT (0.1 g) was added into the reaction solution. The mixture was stirred at room temperature for 24 hours. Finally, RhB-MMT was collected after washing with ddH2O. E. faecalis was mixed with DAPI (5 µg/ml) and incubated for 30 minutes at room temperature. Afterward, RhB-MMT (900 μl) and DAPI-labeled E. faecalis (100 μl) were mixed and cultured at room temperature for 4 hours with shaking at 700 rpm. E. faecalis and MMT were applied as controls for the above characterization. E. faecalis, MMT, and MMT@Bac were characterized by LSCM (Leica TCS SP8).

Resistance assessment in vitro
A 300 µl suspension of E. faecalis, MMT@Bac, or MMT@Bac@TL (4×10⁵ CFUs) was mixed with 700 µl of SGF (pH 1.2) or SIF (pH 6.8). For SGF, 1 g of pepsin (88918F) and 384 µl of hydrochloric acid (10011061) were added to 100 ml of ddH2O, and the pH was adjusted to 1.2. For SIF, 1.36 g of potassium dihydrogen phosphate (82821B) and 2 g of trypsin (88927B) were dissolved in 100 ml of ddH2O. The pH was adjusted to 6.8 with 0.2 mol sodium hydroxide solution (G19852A). The mixtures were then further incubated at 37°C with gentle shaking. At predetermined time points, 50 µl solution was withdrawn from the medium and washed with PBS. After spreading on solid LB plates, the bacteria were incubated overnight at 37°C for counting.

Ex vivo adhesion of bacteria on the ileum
Freshly murine ileum or colon tissue was divided into ~2 cm segments and everted in order to expose the intestinal mucus layer. These sectioned ileum and colon segments were dripped with 10 µl of PBS containing an equal number of E. faecalis, MMT@Bac, or MMT@Bac@TL (1×108 CFUs/ml) for 30 minutes. At the indicated time points, the intestinal segments were flushed gently with PBS. After spreading on solid LB plates, the bacteria were incubated overnight at 37°C for counting.

Crystal violet staining
To quantify biofilms using crystal violet, the wells were washed 3 times with 250 µl of PBS to remove the planktonic cells. Then, the samples were mixed with 300 µl of methanol for 20 minutes to fix biofilm. Subsequently, a 60 min staining process was performed using 0.1% crystal violet. The nonspecific stain was removed by washing the samples with 250 µl of ddH2O. The plates were left to dry and then subjected to an elution process with 30% acetic acid for 1 hour at room temperature. Absorbance was measured at 540 nm using a microplate reader (Tecan Infinite M200 PRO).

Congo red staining
The Congo red broth and agar assays were prepared as described elsewhere73. Briefly, TSB medium was prepared with an additional 4% sucrose (G66841D) and 0.08% Congo red dye (A600324). 20 µl of Bac, MMT@Bac, or MMT@Bac@TL (1×108 CFUs) was streaked onto a Congo red-containing TSB plate for the agar test. One colony of bacteria on a TSB plate was inoculated onto the Congo red-containing TSB for the broth test. The colors of colonies were observed after 24- or 48-hour incubation. Brown to black colors were considered a positive sign of biofilm formation.

Effects of MMT@Bac@TL on the growth of bacteria
To evaluate the regulatory effects of MMT@Bac@TL on microorganisms, a series of in vitro experiments was conducted. To examine the effects of E. faecalis, MMT@Bac, or MMT@Bac@TL on the growth of bacteria and fungi, EcN, C. albicans, or S. typhimurium was cultured overnight at 37°C, and bacterial quantities were determined by plate counting. EcN (2 × 105 CFUs), C. albicans (1×106 CFUs), or S. typhimurium (2×106 CFUs) was then mixed with E. faecalis, MMT@Bac or MMT@Bac@TL in a 1:1, 1:10, or 10:1 ratio (CFUs) and incubated at 37°C with shaking. Samples were collected at 2, 4, and 6 hours or 3, 6, 12, and 24 hours, serially diluted, and spread onto selective plates.

Mucus level assay
HT29-MTX-E12 cells seeded into the 96-well plate were treated with 50 µg/ml LPS for 6 hours. Then, the cells were treated with 1×105 CFUs of Bac, MMT@Bac, MMT@Bac@T, MMT@Bac@L, and MMT@Bac@TL for 6 hours, respectively. For the acidic mucus assay, cells were fixed with 4% paraformaldehyde for 30 minutes and then incubated with 150 µl of AB solution (pH 2.5) for 30 minutes in the dark. Then, 300 µl of supernatant was collected and transferred into a 96-well plate and assayed at 630 nm using a microplate reader. Likewise, for the neutral mucus measurement, 100 µl of 10% Triton-100 was added into the plate and incubated at room temperature for 30 minutes. Subsequently, supernatant (15 µl) was collected and transferred into a 96-well plate. After adding 60 µl of periodic acid, the plate was incubated in the dark for 90 minutes, followed by incubation with 50 µl of PAS solution in the dark for 40 minutes. The resultant samples were analyzed at 550 nm using a microplate reader (BioTek).

Tight junction protein expression level
Expression levels of tight junction proteins were measured both in vitro and in vivo. For the cell sample, Caco-2 cells were pretreated with 50 µg/ml of LPS for 12 hours and then incubated with 1×104 CFUs of Bac, MMT@Bac, MMT@Bac@T, MMT@Bac@L, and MMT@Bac@TL for 7 hours, respectively. To evaluate the intestinal physical barrier, tissue sections were fixed and permeabilized prior to immunofluorescence staining. The cells and tissue sections were sequentially incubated with primary antibodies against occludin (sc-133256; Santa Cruz Biotechnology) and ZO-1 (AF5145; Affinity Biosciences), followed by species-matched secondary antibodies conjugated with fluorescent probes. Imaging was performed using LSCM (Leica TCS SP8).

In vitro immunomodulatory ability of MMT@Bac@TL
In this experiment, J774A.1 cells were pretreated with 1 µg/ml LPS for 24 hours, followed by co-incubation with 102 CFUs of E. faecalis, MMT@Bac, MMT@Bac@T, MMT@Bac@L, or MMT@Bac@TL for an additional 24 hours. The supernatants were then collected to measure the cytokine levels. The cytokines were detected using ELISA kits. Subsequently, the cells were harvested and washed twice with pre-cooled PBS for flow cytometry.

Immune response analysis
Flow cytometry was then employed to evaluate the polarization of the macrophages. The J774A.1 cells were washed and incubated with anti-CD11b, anti-F4/80, and anti-CD86 antibodies for 30 minutes at room temperature. Afterward, the cells were then stained with anti-CD206 antibodies after fixation and permeabilization with Fixation/Permeabilization Concentrate and Diluent kit (Thermo Fisher Scientific, 00-5521-00). Data were acquired using flow cytometry (BD LSRFortessa). DC 2.4 cells were treated with 1×104 CFUs of Bac, MMT@Bac, MMT@Bac@T, MMT@Bac@L, and MMT@Bac@TL for 18 hours, respectively. The samples were then incubated with extracellular antibodies (CD86). The samples were then washed twice with PBS and analyzed by flow cytometry (BD LSRFortessa). Data were analyzed using FlowJo (TreeStar).

In vivo survival assessment after oral ingestion
Male Balb/c mice at 6 to 8 weeks were fed 4×107 CFUs of Bac, MMT@Bac, or MMT@Bac@TL by oral gavage. At 4, 24, 48 or 72 hours after administration, the mice were euthanized. To evaluate the survival of the bacteria, tissues including the ileum, colon, and cecum, as well as contents, were separately collected. The samples were grounded and diluted with 1 ml of PBS. The suspensions (50 µl) were withdrawn and spread on solid E. faecalis agar plates. The bacteria were incubated overnight at 37°C before counting.

Levels of indole and lactic acid in the gut
To analyze the content of intraluminal indole, the suspensions of the ileum, colon, and cecum were processed for the indole assay kit (MIK7664, MesGen Biotech) in accordance with the manufacturer’s instructions. Specifically, after homogenizing and centrifuging the intestinal segments, the supernatant (25 µl) was mixed with 25 µl of the reaction reagent and 50 µl of PBS. The samples were then incubated at 37°C for 25 minutes. The resultant samples were analyzed at 565 nm using a microplate reader. To analyze intestinal lactic acid levels, the samples were processed using a lactic acid assay kit (A019-2-1, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions. The tissue supernatants (1 µl) were mixed with 50 µl of the enzyme working solutions and 10 µl of color reagents. The samples were then incubated at 37°C for 10 minutes. After the reaction, the stop solution (100 µl) was added and mixed well. The absorbance was measured at 530 nm. ddH2O or 3 mmol/l standard solution was used as the negative or positive control, respectively.

Recovery of the microbiota following antibiotics
Mice were given oral gavage of a broad-spectrum antibiotic solution every 24 hours for 14 days. The broad-spectrum antibiotic solution was comprised of ampicillin (1 mg/kg), vancomycin (0.5 mg/kg), metronidazole (1 mg/kg), and neomycin sulfate (1 mg/kg). The solutions were prepared freshly every 24 hours. Bac, MMT@Bac, MMT@Bac@T, MMT@Bac@L, or MMT@Bac@TL was orally gavaged with an equivalent of 4×107 CFUs of E. faecalis. Colon and cecum contents of all mice were collected and stored at −80°C for the following experiments. The colon tissue was fixed with 4% paraformaldehyde and then used for subsequent analysis.

Analysis of the biofilm in vivo
To observe the production of E. faecalis biofilms within the intestinal tract, intestinal segments were fixed with Carnoy’s fixative, embedded in paraffin, and sectioned. The extracellular matrix of the bacteria was stained with Con A. After dewaxing and rehydration, colon tissue sections (5 µm thickness) underwent antigen retrieval treatment using 1× All-purpose Powerful Antigen Retrieval Solution (Beyotime, P0088) under 98°C for 20 minutes. After PBS washes, the sections were incubated with Alexa Fluor 633-conjugated Con A (20 µg/ml) for 1 hour at room temperature, then rinsed 3 times with PBS. Finally, the specimens were stained with DAPI (5 µg/ml) and sealed with an antifade mounting medium. The slides were imaged with LSCM (TCS SP8, Leica).

Biofilm formation in isolated intestinal segments
Healthy Balb/c mice were used to harvest standardized ileal segments (2 cm) from the same anatomical location. The ileal segments were then opened longitudinally and placed mucosal side up on grooved slides. We then applied 1 ml of SIF containing 1×107 CFUs of Bac, MMT@Bac, or MMT@Bac@TL to measure biofilm formation under an intestinal simulation environment. After 72 hours of incubation at 37°C, the SIF was removed, and the mucosal surface was gently rinsed with PBS in 48-well plates. Tissues were then fixed in 4% paraformaldehyde for 30 minutes at room temperature and stained with Con A (20 µg/ml) and DAPI (5 µg/ml) for imaging.

Treatment of S. typhimurium-induced enteritis
Streptomycin (20 mg) was administered after the mice were deprived of water and food for 6 hours. After 7 days of adaptive feeding, mice were treated with streptomycin for 24 hours and orally inoculated with 5×107 CFUs of S. typhimurium. S. typhimurium was grown for 8 hours in LB supplemented with kanamycin. Bac, MMT@Bac, MMT@Bac@T, MMT@Bac@L, or MMT@Bac@TL with equivalent CFUs (1.0×107 CFUs) was administered intragastrically for treatment on the following 6 days. Mice treated with MMT (0.05 mg) were used as a benchmark. Then, the major organs, including the intestine, kidney, and spleen, were sampled for further analysis.

Treatment of colitis with fungal infection
Balb/c mice were gavaged with 0.2 ml of water containing 20 mg/kg of streptomycin, 20000 U/kg of penicillin for 2 doses. Then, mice were given drinking water containing 3% (w/v) DSS ad libitum to induce colitis. On the same day, mice in the DSS groups were orally administered with 1×108 CFUs of C. albicans every other day and simultaneously treated with Bac, MMT@Bac, MMT@Bac@T, MMT@Bac@L, and MMT@Bac@TL containing 1×107 CFUs of E. faecalis and 0.05 mg MMT every other day from day 3 to 25. Healthy mice and infected mice treated with PBS, 5-ASA (30 mg/kg), and equivalent MMT, Bac, MMT@Bac, MMT@Bac@T, and MMT@Bac@L were applied as controls. After 12 doses of treatment, mice were euthanized, and samples were collected for further analysis. Homogenates of the cecum were prepared for C. albicans plate counting. The colon was then sampled for further study. Flow cytometric analysis was performed to quantify the frequencies of IgA+ B cells in the PPs.

FISH detection for C. albicans
To observe C. albicans colonization in the intestine, intestinal segments were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned. FISH was performed in a hybridization buffer with a fungal 28S rRNA DNA oligonucleotide that is coupled to 5 µmol Cy3 labeled probe (5’-CTCTGGCTTCACCCTATTC-3’). The tissue sections were incubated in the hybridization solution (G3045, Servicebio) at 37°C for 1 hour for pre-hybridization. Slides were washed for 10 minutes at 37°C in 2×, 1×, 0.5× SSC (G3015, Servicebio). Subsequently, samples were counterstained with DAPI for 1 hour at 37°C. C. albicans was quantified manually using the cell counter plug-in for Fiji.

Histopathological analysis
The collected intestinal tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned. The obtained sections were deparaffinized and stained with H&E (C0105S, Beyotime Biotechnology) and AB-PAS (MST-8050, Fuzhou Maixin Biotechnology) according to the manufacturer’s instructions to evaluate pathological damage in the intestinal regions. To assess inflammatory infiltration in the intestine, the deparaffinized sections of intestinal samples underwent antigen retrieval using 1× All-purpose Powerful Antigen Retrieval Solution, followed by blocking endogenous peroxidase activity with 3% H2O2. The sections were then incubated with the primary anti-MPO antibody (GB11224, Servicebio) and the corresponding secondary HRP antibody (G1215, Servicebio). Visualization was performed using a 3,3’-diaminobenzidine (DAB) substrate kit according to the manufacturer’s instructions.

Microbiota 16S sequencing and analysis
The 16S rRNA gene sequencing and analysis were conducted at Biomarker Technologies. Total DNA was extracted from the contents of the cecum. Sequencing was performed on an Illumina HiSeq platform by targeting hypervariable V3-V4 regions of the bacterial 16S rRNA gene. Data analysis was performed on BMKCloud. Beta diversity was analyzed using PCA. Microbial composition was analyzed at the class level. LEfSe was analyzed at the phylum and species levels.

Statistical analysis
All statistical analyses were performed with GraphPad Prism 9 and IBM SPSS Statistics 27 software. Data points were excluded only when identified as statistical outliers by the standardized boxplot-based analysis using Tukey’s method. Experimental data were analyzed using one-way analysis of variance (ANOVA) with Fisher’s LSD post-test for multiple comparisons. Student’s t-test was used for the two-group comparisons. Data were expressed as means ± standard deviation (SD). p-value < 0.05 was considered statistically significant.

Ethical statement
Male Balb/c mice aged 6 to 8 weeks were purchased from Jiesijie Laboratory Animal Technology. Mice were housed under standard conditions (12-hour light/dark cycle, 22 ± 2°C, 55 ± 10% humidity) with ad libitum access to water and standard diet. All animal experiments were carried out according to the institutional guidelines and approved by the Animal Care and Use Committee in Shanghai Yishang Biotechnology Company (IACUC-2022-Mi-203).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Zhang Y et al present a montmorillonite-based oral fermentation system designed for restoring intestinal\nhomeostasis. This system is fabricated by loading metabolic substrates in the lamellar structure of montmorillonite via cation\nreplacement and adsorbing the substrate-related bacteria through electrostatic interactions. After oral delivery, the\ncontinuous release of loaded substrates and the formation of biofilms by carried bacteria enable long-term efficient synthesis\nof beneficial metabolites in the intestine. Importantly, it demonstrates its superior therapeutic efficacy in murine models.\nOverall, this work presents significant novelty. The data are well-organized and compelling. Thus, this work is recommended\nfor publication in Nature Communications. While, some minor issues need to be addressed to improve the manuscript.\n1.In figure 1B, the continuous release of carried tryptophan and lactulose and the formation of E. faecalis biofilms ensure\nlong-lasting efficient production of lactic acid and indole in the gut. To facilitate broad accessibility, it would be better to label\nlactic acid and indole with their corresponding structural formulas in the figure.\n2.The authors describe that the cation exchange capacity of MMT enabled high loading contents of tryptophan and\nlactulose. How does MMT load tryptophan and lactulose via cation exchange? An explanation is helpful for understanding\nthe mechanism involved in the preparation of MMT@Bac@TL.\n3.Figures 3i and j show the enhanced biofilm-forming ability of MMT@Bac compared to the Bac group. It would be beneficial\nif the authors could discuss why did MMT@Bac promote biofilm formation.\n4.On page 9, the statement “We further quantified the formation of biofilms using Congo red staining after 24 and 48 hours of\nincubation in SIF. In the MMT@Bac@TL group, the absorbance at 495 nm showed approximately 3- and 2-fold increments\ncompared to the Bac and MMT@Bac groups, respectively” is unclear. Please clarify at which time point (24 or 48 hours)\nthese comparisons were made. In addition, on page 11, the description of “The growth rate was 3-fold higher in the\nMMT@Bac@TL group in contrast to the MMT@Bac group” might need further validation.\n5.The method for detecting ex vivo formation of biofilms by Bac, MMT@Bac, and MMT@Bac@TL on ileum sections is\ninteresting. A comprehensive description of the method would be beneficial for reference by other researchers.\n6.The antibiotic cocktail-induced gut dysbiosis has a more pronounced effect on the tight junction protein ZO-1 than on\noccludin. Elucidating the underlying mechanisms for this difference may provide insights into the enhanced therapeutic\neffects of MMT@Bac@TL.\n7.It is recommended to adjust the y-axes of figures 7c and 8c to make the changes in intestinal length more visible.", "reviewer_2": "In this manuscript, the authors constructed an oral delivery system using montmorillonite loaded with Enterococcus faecalis\nand metabolic substrates, aiming to restore intestinal homeostasis through in-situ synthesis of metabolites in the intestine.\nWhile the research is clearly presented and the data are detailed, the manuscript lacks sufficient innovation and notable\nhighlights. Its core focus, constructing an oral delivery system by loading bacteria and substrates onto montmorillonite,\nhas already been explored in similar studies within the fields of clay carrier applications and probiotic delivery. It fails to\ndemonstrate breakthrough technological or mechanistic innovations, thus offering limited supplementary value to the\nexisting research framework.\nSecondly, there are several detailed issues to address: The description of the interaction mechanism between\nmontmorillonite, Enterococcus faecalis, and metabolic substrates is superficial, failing to clarify how ion exchange facilitates\nadsorption. In terms of structural characterization, the state of Enterococcus faecalis on the montmorillonite surface in the\nSEM images is insufficiently clear. Regarding animal experiments, there is inconsistency in group design: in the evaluation\nof therapeutic efficacy against Salmonella typhimurium-induced enteritis, no control group or positive drug group was\nincluded (with 6 mice per group). In contrast, for the study on fungal infection-related colitis, although healthy control and\npositive drug groups were established, only 4 colon samples were photographed per group in Figure 8b, which may easily\nlead to misunderstanding.\nAdditionally, the discussion of experimental results in this paper is not sufficiently in-depth, remaining largely at the level of\nresult description. For instance, key issues such as why the simultaneous loading of tryptophan and lactulose leads to\nsignificantly higher production of indole and lactic acid compared to the control group, and the mechanism by which biofilms\npromote metabolite production, have not been analyzed in depth.\nIn summary, I believe that this manuscript has not met the publication standards of Nature Communications.", "reviewer_3": "This manuscript reports the development of a montmorillonite-based oral fermentation system (MOFS) that enables\nsustained in-situ biosynthesis of beneficial metabolites in the gut, thereby restoring intestinal homeostasis. The work is\noriginal and well-conceived, with a rational materials design, comprehensive experimental validation, and promising\ntranslational potential. The results convincingly support the conclusions, and the study aligns well with the scope of Nature\nCommunications. However, several experimental details require clarification, and some data presentations and language\nusage need refinement.\n1. While Congo red and crystal violet staining were used to evaluate biofilm formation, additional quantitative analyses via\nSEM or CLSM analysis would strengthen the findings.\n2. The authors show MOFS retention in ileum, cecum, and colon, but it remains unclear whether this distribution relates to\nlocal pH, mucus viscosity, or motility. A brief mechanistic discussion or supporting experiment is suggested.\n3. Please include more detailed time-course measurements of tryptophan and lactulose levels in intestinal contents to\nsupport claims of sustained release in vivo.\n4. While IL-1β, VEGF, and TGF-β were assessed, additional pro-inflammatory cytokines and epithelial integrity metrics\nshould be considered to strengthen the anti-inflammatory claims.\n5. Although MOFS reduces Candida albicans, the mechanistic basis is only partially explained.\n6. In PCA and LEfSe analyses, please provide sample size per group, clearly indicate the statistical methods used, and add\np-value annotations to confirm the significance of the microbiota shifts.\n7. For greater relevance, especially in antimicrobial or anti-inflammatory efficacy studies, a benchmark group using a\nclinically available probioticshould be included to contextualize the advantage of MOFS.\n8. Some related references such as (Nat. Commun.,2022,13, 3432) are suggested to be cited."}, "author_response": "Response: We are grateful to the reviewer for providing constructive comments and\nuseful suggestions on how to further refine the conclusions of our work. The raised\nconcerns have been addressed accordingly in the revised manuscript.\n1. In figure 1B, the continuous release of carried tryptophan and lactulose and the\nformation of E. faecalis biofilms ensure long-lasting efficient production of lactic acid\nand indole in the gut. To facilitate broad accessibility, it would be better to label lactic\nacid and indole with their corresponding structural formulas in the figure.\nResponse: Thank you for the valuable suggestion to improve the scheme. We have\nadded the structural formulas for the orange ball (lactic acid) and the blue ball (indole)\nin the revised Figure 1b (Page 39, Line 1). Meanwhile, we have increased the\nsaturations of tryptophan and lactulose structural formulas so that the references of the\norange and blue balls are more distinct.\nFig. 1 Schematic illustration. a, Preparation of MOFS by cation replacement and\nabsorption. b, High resistance of MOFS and its long-lasting in situ synthesis of\nbeneficial bioproducts in the gut. c, MOFS-mediated protection of intestinal hemostasis\nby improving gut microbiota composition, suppressing intestinal inflammation, and\nincreasing the integrity of the gut barrier. Graphic created with BioRender.com.\n2. The authors describe that the cation exchange capacity of MMT enabled high loading\ncontents of tryptophan and lactulose. How does MMT load tryptophan and lactulose\nvia cation exchange? An explanation is helpful for understanding the mechanism\ninvolved in the preparation of MMT@Bac@TL.\nResponse: We thank the reviewer for this insightful question. Corresponding\nexplanations have been added in the revised manuscript (Page 5, Line 6-11): We chose\nMMT, a clinically used nanosheet-structured layered clay, as a carrier material due to\nits distinctive physicochemical characteristics, such as high stability, mucoadhesive\nability, high adsorption capacity, water impermeability, and high cation-exchange\ncapacity (Anal Chim Acta 2014, 814, 69-78). MMT is a 2:1 smectite with permanent\nlayer charge arising from isomorphic substitution (Geochim Cosmochim Acta 2024, 372,\n181-195). Negative surfaces are balanced by exchangeable interlayer cations (e.g., Na⁺,\nCa2+), conferring a high cation-exchange capacity.\n3. Figures 3i and j show the enhanced biofilm-forming ability of MMT@Bac compared\nto the Bac group. It would be beneficial if the authors could discuss why did\nMMT@Bac promote biofilm formation.\nResponse: We thank the reviewer for this constructive suggestion. In the revised\nmanuscript, we have added a brief discussion on the enhanced biofilm formation\nobserved in the MMT@Bac group (Page 11, Line 22-24): The enhanced biofilm\nformation observed in MMT@Bac could be attributed to MMT’s high surface area,\nwhich provides an adhesive scaffold that stabilized the development and maturation of\nbiofilm matrix (npj Biofilms Microbiomes 2017, 3, 4). Moreover, MMT markedly\npromoted E. faecalis proliferation (Supplementary Fig. 1c), thereby accelerating\nbiofilm formation.\n4. On page 9, the statement “We further quantified the formation of biofilms using\nCongo red staining after 24 and 48 hours of incubation in SIF. In the MMT@Bac@TL\ngroup, the absorbance at 495 nm showed approximately 3- and 2-fold increments\ncompared to the Bac and MMT@Bac groups, respectively” is unclear. Please clarify at\nwhich time point (24 or 48 hours) these comparisons were made. In addition, on page\n11, the description of “The growth rate was 3-fold higher in the MMT@Bac@TL group\nin contrast to the MMT@Bac group” might need further validation.\nResponse: We thank the reviewer for pointing this out. We have made a further\nclarification in the description. Corresponding modifications have been made in the\nrevised manuscript (Page 11, Line 13-17; Page 13, Line 15-16): We further quantified\nthe formation of biofilms using Congo red staining after 24 and 48 hours of incubation\nin SIF. In the MMT@Bac@TL group, the absorbance at 495 nm showed approximately\n3-fold (24 hours) and 2-fold (48 hours) increments compared to the Bac group,\nrespectively (Supplementary Fig. 5b). The growth rate was 144-fold higher in the\nMMT@Bac@TL group in contrast to the MMT@Bac group (Fig. 4a).\n5. The method for detecting ex vivo formation of biofilms by Bac, MMT@Bac, and\nMMT@Bac@TL on ileum sections is interesting. A comprehensive description of the\nmethod would be beneficial for reference by other researchers.\nResponse: We thank the reviewer for this helpful suggestion. We have described an ex-\nvivo mucus-biofilm production assay in detail in Methods. Corresponding descriptions\nhave been added in the revised manuscript (Page 29, Lin 24-30 and Page 30, Line 1-2):\nBiofilm formation in isolated intestinal segments\nHealthy Balb/c mice were used to harvest standardized ileal segments (2 cm) from the\nsame anatomical location. The ileal segments were then opened longitudinally and\nplaced mucosal side up on grooved slides. We then applied 200 µL of SIF containing 1\n× 107 CFUs of Bac, MMT@Bac, or MMT@Bac@TL to measure biofilm formation\nunder an intestinal simulation environment. After 72 hours of incubation at 37 °C, the\nSIF was removed and the mucosal surface was gently rinsed with PBS in 48-well plates.\nTissues were then fixed in 4% paraformaldehyde for 30 minutes at room temperature\nand stained with Con A and DAPI for imaging.\n6. The antibiotic cocktail-induced gut dysbiosis has a more pronounced effect on the\ntight junction protein ZO-1 than on occludin. Elucidating the underlying mechanisms\nfor this difference may provide insights into the enhanced therapeutic effects of\nMMT@Bac@TL.\nResponse: Thank you for this constructive suggestion. The potential mechanisms\nunderlying the distinct effects of antibiotic cocktail-induced gut dysbiosis on the tight\njunction proteins ZO-1 and occludin have been discussed in the revised manuscript\n(Page 18, Line 4-11): Compared to the healthy control, antibiotic cocktail-induced gut\ndysbiosis has a more pronounced effect on the tight junction protein ZO-1 than on\noccludin. It is indicated that pro-inflammatory and contractile cues associated with\ndysbiosis rapidly remodel ZO-1 continuity and localization at cell-cell borders (Proc\nNatl Acad Sci U S A 2010, 107, 8237-8241; Int J Mol Sci 2020, 21, 993). By contrast,\noccludin homeostasis is more strongly influenced by context-dependent\nendocytosis/turnover pathways (J Cell Biol 2010, 189, 111-126). Notably, both\ndecreased occludin and ZO-1 expressions triggered by antibiotic cocktail-induced gut\ndysbiosis could be greatly reversed by MMT@Bac@TL (Fig. 6g, i, j, and\nSupplementary Fig. 14b).\n7. It is recommended to adjust the y-axes of figures 7c and 8c to make the changes in\nintestinal length more visible.\nResponse: Thank you for the helpful suggestion. In the revised manuscript, we have\nrescaled the y-axes of Figures 7c and 8c to better emphasize changes in intestinal length.\nThese adjustments make the difference in the intestinal length among groups more\nvisible (Figure R1). The revised figures have been presented in Page 50, Line 1 and\nPage 52, Line 1.\nFigure R1 Revised Figure 7c (left) and Figure 8c (right).\nReviewer #2\nIn this manuscript, the authors constructed an oral delivery system using\nmontmorillonite loaded with Enterococcus faecalis and metabolic substrates, aiming to\nrestore intestinal homeostasis through in-situ synthesis of metabolites in the intestine.\nWhile the research is clearly presented and the data are detailed, the manuscript lacks\nsufficient innovation and notable highlights. Its core focus, constructing an oral\ndelivery system by loading bacteria and substrates onto montmorillonite, has already\nbeen explored in similar studies within the fields of clay carrier applications and\nprobiotic delivery. It fails to demonstrate breakthrough technological or mechanistic\ninnovations, thus offering limited supplementary value to the existing research\nframework.\nResponse: We thank the reviewer very much for her/his detailed overall comment on\nour work and for providing valuable suggestions on how to improve the quality of our\nmanuscript. Regarding concerns about the novelty of our work, we would like to clarify\nthat the key innovation of this work is the development of a fermentation system for\nlong-lasting in-situ biosynthesis of beneficial bioproducts by loading bacteria and\nmetabolic substrates into montmorillonite. This is conceptually different from previous\nstudies that use montmorillonite to directly deliver probiotics and small molecules,\nwhich lack the ability to sustain in-situ biosynthesis over time. Additionally, the other\nraised concerns have been addressed accordingly in the revised manuscript. Below are\nthe point-by-point responses.\nSecondly, there are several detailed issues to address: The description of the interaction\nmechanism between montmorillonite, Enterococcus faecalis, and metabolic substrates\nis superficial, failing to clarify how ion exchange facilitates adsorption.\nResponse: Thank you for pointing this out. In the revised manuscript, we have expanded\nthe discussion to clarify the mechanisms of interactions among montmorillonite, E.\nfaecalis, and metabolic substrates. Corresponding discussion has been added in the\nrevised manuscript (Page 5, Line 6-13): We chose MMT, a clinically used nanosheet-\nstructured layered clay, as a carrier material due to its distinctive physicochemical\ncharacteristics, such as high stability, mucoadhesive ability, high adsorption capacity,\nwater impermeability, and high cation-exchange capacity (Anal Chim Acta 2014, 814,\n69-78). MMT is a 2:1 smectite with permanent layer charge arising from isomorphic\nsubstitution (Geochim Cosmochim Acta 2024, 372, 181-195). Negative surfaces are\nbalanced by exchangeable interlayer cations (e.g., Na⁺, Ca2+), conferring a high cation-\nexchange capacity. It has been indicated that the abundant lipophilic components of the\nbacterial cell walls, such as lipoproteins and liposaccharides, make them easily\nadsorbed by MMT (ACS Omega 2023, 8, 3385-3395).\nIn terms of structural characterization, the state of Enterococcus faecalis on the\nmontmorillonite surface in the SEM images is insufficiently clear.\nResponse: According to the reviewer’s suggestion, we have supplemented new SEM\nimages that clearly display spherical bacterial cells attached to the surface of\nmontmorillonite (Page 40, Line 1).\nFig. 2 Characterization and stability of MMT@Bac@TL in gastrointestinal\nenvironments. a, SEM images of Bac, MMT, and MMT@Bac. Scale bar, 1 μm.\nRegarding animal experiments, there is inconsistency in group design: in the evaluation\nof therapeutic efficacy against Salmonella typhimurium-induced enteritis, no control\ngroup or positive drug group was included (with 6 mice per group). In contrast, for the\nstudy on fungal infection-related colitis, although healthy control and positive drug\ngroups were established, only 4 colon samples were photographed per group in Figure\n8b, which may easily lead to misunderstanding.\nResponse: Thank you for pointing this out. According to the suggestion, in the revised\nmanuscript, we have included a healthy control to evaluate the therapeutic efficacy\nagainst Salmonella typhimurium-induced enteritis. Meanwhile, MMT, as the medical\nclay for the pathogen elimination (Ann Pharm Fr 2025, 83, 588-604), has been applied\nas a positive control. For the study on fungal infection-related colitis, photographs of\nall samples have been presented in the revised Fig. 8b (Page 52, Line 1). Corresponding\ndiscussion has been added in the revised manuscript (Page 18, Line 22-30 and Page 19,\nLine 1-4): The mice were orally administered with MMT, Bac, MMT@Bac,\nMMT@Bac@T, MMT@Bac@L, and MMT@Bac@TL one dose per day for 6 days,\nrespectively. In each treatment group, equivalent MMT (1.3 mg), E. faecalis (1 × 107\nCFUs), tryptophan (0.3 mg), or lactulose (0.3 mg) was applied. Infected mice treated\nwith PBS were used as a negative control. The untreated mice served as a healthy\ncontrol. Meanwhile, MMT, a type of medical clay used to eliminate pathogens (Ann\nPharm Fr 2025, 83, 588-604), was used as a positive control. As shown in Fig. 7b and\nc, S. typhimurium infection-induced decrease of colon length was effectively restored\nby MMT@Bac@TL. In contrast, other treatments failed to control this symptom.\nSpecifically, the colon length of MMT@Bac@TL-treated mice (9.4 ± 0.1 cm) was\nlonger than those of mice treated with PBS (7.7 ± 1.1 cm), MMT (8.2 ± 0.6 cm),\nBac (7.4 ± 1.2 cm), MMT@Bac (8.0 ± 0.9 cm), MMT@Bac@T (8.3 ± 0.8 cm), and\nMMT@Bac@L (8.7 ± 0.4 cm), highlighting its superior treatment efficacy.\nFig. 7 Therapeutic value of MMT@Bac@TL in treating S. typhimurium-induced\nenteritis. a, Schedule of MMT@Bac@TL-mediated treatment of S. typhimurium-\ninduced enteritis. At 24 hours after infection with 5 × 107 CFUs of S. typhimurium, the\nmice were oral administered with 0.2 ml of PBS, MMT, Bac, MMT@Bac,\nMMT@Bac@T, MMT@Bac@L, and MMT@Bac@TL one dose per day for 6 days,\nrespectively. Healthy mice without any treatments were set as a control. b,c,\nPhotographs (b) and average lengths (c) of the colon sampled from treated mice. d,e,\nLevel of indole (d) and lactic acid (e) in the gut. f-h, Counts of S. typhimurium in the\nintestine (f), kidney (g), and spleen (h), respectively. i, MPO staining of the colon\nsections. Blue arrows indicate MPO-positive cells. Scale bar, 130 μm. j, H&E staining\nof the colon sections. Black arrows indicate the pathological changes of the muscular\nlayer. Scale bar, 130 μm. Data are represented as means ± SD (n = 5-6). p values were\ndetermined using one-way ANOVA. p values < 0.05 are shown.\nSupplementary Fig. 15 Quantification of pathological changes in the gut. a, Inhibition\nof intestinal inflammation by MMT@Bac@TL in enteritis mice. Quantification of\nMPO-positive cells in intestinal tissues. b, Pathological score of the colon measured by\nH&E staining. Data are represented as means ± SD (n = 3). p values were determined\nusing one-way ANOVA. p values < 0.05 are shown.\nFig. 8 Potential of MMT@Bac@TL in treating colitis with fungal infection. a,\nExperimental design for evaluating the therapeutic potential of MMT@Bac@TL in\ntreating colitis with fungal infection. Mice were orally delivered with 0.2 ml of\nstreptomycin (20 mg/kg) and penicillin (20 U/kg) one dose per day for 2 days to\neliminate the intestinal microbiota. Then, the mice were given 3% DSS water freely for\n23 days. During this time frame, the mice were gavaged with 1 × 108 CFUs of C. albican\nin combination with PBS, 5-ASA (30 mg), MMT (1.3 mg), 1 × 107 CFUs of Bac,\nMMT@Bac@T, MMT@Bac@L, or MMT@Bac@TL every other day (a dose per day).\nAfter 12 doses, the mice were euthanized and samples were collected for subsequent\nmeasurements (n = 4-7). b,c, Photographs (b) and average lengths (c) of the colon\nsampled from treated mice. d, Count of C. albicans in the cecum. e,f,\nConfocal images\n(e) and MFI (f) of C. albicans in colon sections stained with FISH (Red) and DAPI\n(Blue). Scale bar, 30 μm. g, Percentage of IgA+ B cells in the PPs. h, Percentage of\nMPO-positive area measured by Image-J (n = 3). i,j, Pathological score (i) of H&E\nstaining (j) of the colon (n = 3). Scale bar, 75 μm. Pathological changes on the mucosa\nand muscular layer were indicted by black arrows. Data are represented as means ± SD.\np values were determined using one-way ANOVA. p values < 0.05 are shown.\nAdditionally, the discussion of experimental results in this paper is not sufficiently in-\ndepth, remaining largely at the level of result description. For instance, key issues\nsuch as why the simultaneous loading of tryptophan and lactulose leads to\nsignificantly higher production of indole and lactic acid compared to the control group,\nand the mechanism by which biofilms promote metabolite production, have not been\nanalyzed in depth.\nResponse: Thank you for bringing this to our attention. To address this, we have\nexpanded relevant discussion in depth in the revised manuscript. Meanwhile, we have\nconducted new experiments to analyze the potential mechanisms underlying the\nbiofilm-mediated increase in metabolite production. The corresponding discussion has\nbeen incorporated into the revised version (Page 9, Line 26-30 and Page 10, Line 1-4):\nThe higher production of indole and lactic acid in MMT@Bac@TL compared to the\nsimple mixture of bacteria with metabolic substrates (Bac + T or Bac + L) might be\nascribed to the catalytic characteristic of MMT (Proc Natl Acad Sci U S A 2001, 98,\n8997-9001; J Chem Pharm Res 2012, 4, 991-1015). Additionally, the synergistic effects\nof tryptophan and lactulose on bacterial growth and metabolism contributed to the\nMMT@Bac@TL-mediated high productions of indole and lactic acid. Lactulose serves\nas a prebiotic, promoting the growth of lactic acid bacteria (Nat Commun 2022, 13,\n1244). In turn, this facilitates the metabolism of tryptophan, resulting in increased\nindole production. Meanwhile, indole has been shown to modulate the growth, viability,\nbiofilm formation, motility, and stress resistance in different bacteria (Trends Microbiol\n2020, 28, 566-577).\nThe corresponding discussion on the mechanisms by which biofilms promote\nmetabolite production has been incorporated into the revised manuscript (Page 12, Line\n12-26): Both in vitro and in vivo experiments revealed that the superior biosynthesis\nefficiency of MMT@Bac@TL was benefited from the formation of biofilms, which\nmight be associated with the upregulated energy metabolism of bacteria in biofilms.\nAn\nupregulated energy metabolism characterized by enhanced glycolysis and tricarboxylic\nacid cycle activity has been observed in biofilms (World J Microbiol Biotechnol 2023,\n39, 212; Nat Commun 2024, 15, 7575). The enhanced energy metabolism supplies\nadditional adenosine triphosphate (ATP) and central carbon intermediates to fuel\nanabolic pathways, such as amino acid, lipid, and exopolysaccharide biosynthesis (Sci\nRep 2019, 9, 13160). Thus, we further evaluated the potential metabolic pathways\nunderlying the biofilm-mediated increase in metabolite production by comparing\npyruvate and ATP levels between MMT@Bac@TL with and without biofilms. Given\nthat biofilm formation was observed after 3 days of SIF culture, we cultured\nMMT@Bac@TL in PBS using the same schedule as a biofilm-negative control. As\nshown in Supplementary Fig. 8, compared to MMT@Bac@TL cultured in PBS,\nMMT@Bac@TL in SIF presented increases in the levels of pyruvate (~10-fold) and\nATP (~2-fold).\nSupplementary Fig. 8 Energy metabolism of MMT@Bac@TL with or without\nbiofilms. a,b Quantification of pyruvate (a) and ATP (b), respectively. Data are\nrepresented as means ± SD (n = 3 or 6). p values were determined using Student’s t test.\np values < 0.05 are shown.\nReviewer #3\nThis manuscript reports the development of a montmorillonite-based oral fermentation\nsystem (MOFS) that enables sustained in-situ biosynthesis of beneficial metabolites in\nthe gut, thereby restoring intestinal homeostasis. The work is original and well-\nconceived, with a rational materials design, comprehensive experimental validation,\nand promising translational potential. The results convincingly support the conclusions,\nand the study aligns well with the scope of Nature Communications. However, several\nexperimental details require clarification, and some data presentations and language\nusage need refinement.\nResponse: We sincerely appreciate the reviewer’s kind and valuable suggestions for\nfurther improving our manuscript. The raised concerns have been fully addressed in the\nrevised manuscript.\n1. While Congo red and crystal violet staining were used to evaluate biofilm formation,\nadditional quantitative analyses via SEM or CLSM analysis would strengthen the\nfindings.\nResponse: Thank you for this valuable suggestion. To further validate the biofilm\nformation, we have conducted supplementary analyses using both scanning electron\nmicroscopy (SEM) and confocal laser scanning microscopy (CLSM) in the revised\nmanuscript. Corresponding results and relevant discussion have been added in the\nrevised manuscript (Page 11, Line 24-30 and Page 12, Line 1-7): Additionally, we\nmeasured the biofilm production in the intestinal environment by culturing Bac,\nMMT@Bac, and MMT@Bac@TL in both isolated intestinal segments and SIF.\nFollowing 48 and 72 hours of incubation, the tissues were stained with DAPI and Alexa\nFluor 633-concanavalin A (ConA) to observe biofilm formation. As shown in Fig. 3k\nand Supplementary Fig. 6a, the mean fluorescence intensity (MFI) of ConA in the\nMMT@Bac@TL group was significantly higher than other groups, indicating a denser\nbiofilm layer on the inner surface of the intestine. The ConA MFI of MMT@Bac@TL\nwas ~40-fold higher than MMT@Bac and ~15-fold higher than Bac (Supplementary\nFig. 6b). Meanwhile, the formation of biofilms was further confirmed by SEM. Unlike\nthe smooth surface of bare bacteria, MMT@Bac displayed a rough surface due to the\npresence of MMT. An enhanced bacterial aggravation on a rougher surface was\nobserved in the MMT@Bac@TL group compared to MMT@Bac as the formation of\nbiofilms (Supplementary Fig. 6c).\nSupplementary Fig. 6 Characterization of the biofilm formation of MMT@Bac@TL.\na, LSCM images of the biofilms in SIF. Bacteria and biofilms were shown in blue and\nred, respectively. Scale bar, 25 μm. b, MFI of ConA in different groups. Biofilms were\nidentified by Con A. c, SEM images of Bac, MMT@Bac, and MMT@Bac@TL after 3\ndays of culture in SIF. Scale bar, 75 μm. Data are represented as means ± SD (n = 3-5).\np values were determined using one-way ANOVA. p values < 0.05 are shown.\n2. The authors show MOFS retention in ileum, cecum, and colon, but it remains unclear\nwhether this distribution relates to local pH, mucus viscosity, or motility. A brief\nmechanistic discussion or supporting experiment is suggested.\nResponse: Thank you for the insightful comment. In the revised manuscript, we have\nmade a further discussion on the potential influences of local pH, mucus viscosity, and\nmotility on MOFS retention. Corresponding discussion has been added in the revised\nmanuscript (Page 7, Line 30 and Page 8, Line 1-5; Page 9, Line 10-13): MMT is a clay\ncontaining multiple isomeric substituents, where the aluminum and silicon atoms at the\ncrystal edges undergo partial hydrolysis in water, forming silanol and aluminum\nhydroxyl groups (Proc Natl Acad Sci USA 2024, 121, e2320482121; Nat Chem 2023,\n15, 119-128). These unsaturated edge sites are more reactive than saturated base sites\nand can bind to mucin in the mucus layer of the intestinal wall through hydrogen\nbonding, van der Waals forces, and hydrophobic interactions. Give MMT is known to\nbind mucin in the mucus layer, the differential retention of MMT@Bac@TL across gut\nsegments might be resulted from the structural and thickness variations in the mucus\nlayer (Immunol Rev 2014, 260, 8-20). Additionally, pH gradients along the\ngastrointestinal tract and gut motility could also play a role by influencing the binding\n(Small 2025, 21, 2410258).\n3. Please include more detailed time-course measurements of tryptophan and lactulose\nlevels in intestinal contents to support claims of sustained release in vivo.\nResponse: Thank you for the suggestion. Accordingly, we have further measured\ntryptophan and lactulose levels in intestinal contents after 24 and 48 hours of oral\nadministration. Corresponding results have been added in the revised manuscript (Page\n10, Line 17-30): Moreover, we monitored the metabolites over a time course. The\nconcentration of indole in the MMT@Bac@TL group was increased by ~1.4-fold\ncompared to those in the Bac and MMT@Bac group 24 hours after oral administration,\nrespectively (Supplementary Fig. 4a). Indole level at 48 hours after oral administration\nand lactate levels at 24 hours and 48 hours showed no significant differences\n(Supplementary Fig. 4b-d). Notably, both the levels of indole and lactic acid in the\nMMT@Bac@TL group were increased by ~2-fold compared to the Bac group 72 hours\nafter oral gavage. In contrast, the administration of MMT@Bac failed to enhance lactic\nacid production throughout the time course (Fig. 3g and h). These dynamic changes in\nindole and lactic acid productions observed over time in the MMT@Bac@TL group\nmight be associated with transitions, substrate availability, and microbial adaptation of\nthe sample in the gut. Collectively, these data showed that MMT@Bac@TL enabled\nlong-lasting in situ biosynthesis in the intestine, and that the production of beneficial\nmetabolites could be amplified by co-loading tryptophan and lactulose.\nSupplementary Fig. 4 Levels of indole and lactic acid in the gut after oral\nadministration. a,b, Concentrations of indole in the entire intestine after 24 (a) and 48\n(b) hours of oral administration. c,d, Lactic acid concentrations in the colon after 24 (c)\nand 48 (d) hours of oral administration. Data are represented as means ± SD (n = 5). p\nvalues were determined using one-way ANOVA. p values < 0.05 are shown.\n4. While IL-1β, VEGF, and TGF-β were assessed, additional pro-inflammatory\ncytokines and epithelial integrity metrics should be considered to strengthen the anti-\ninflammatory claims.\nResponse: Thank you for your valuable suggestion. Based on this suggestion, we have\nsupplemented additional cytokines and epithelial integrity metrics. Corresponding\nresults have been added in the revised manuscript (Page 15, Line 17-24; Page 17, Line\n9-17): Additionally, interleukin-4 (IL-4) was 30% increased by MMT@Bac@TL\ncompared to the medium group, contributing to an increased ratio of IL-4 to IL-6\n(Supplementary Fig. 11a and b). Meanwhile, the ratio of interleukin-10 (IL-10) to nitric\noxide (NO) in the MMT@Bac@TL group was 17% higher than in the PBS group\n(Supplementary Fig. 11c). IL-4 and IL-10 are key mediators of mucosal immune\nregulation, which control over inflammation (Curr Opin Immunol 2024, 91, 102495;\nNat Rev Gastroenterol Hepatol 2023, 20, 538-553). In contrast, increases in IL-6 and\nNO are commonly involved in the aggravation of immunopathology during gut\ninflammation (Inflamm Bowel Dis 2007, 13, 1016-1023; Front Endocrinol 2024, 15,\n1390351).\nAs a widely used in vitro model of the intestinal barrier, Caco-2 cell monolayer\nwas treated with 50 μg/ml LPS for 24 hours and then incubated with 1 × 104 CFUs of\nBac, MMT@Bac, MMT@Bac@T, MMT@Bac@L, and MMT@Bac@TL for 24 hours,\nrespectively. We first measured the effect of MMT@Bac@TL on cell permeability by\nquantifying the fluorescence intensity of FITC-dextran (4 kDa) that permeated from the\nupper to the lower chamber of the transwell device. As shown in Supplementary Fig.\n13, compared to the cell culture medium-treated group, the fluorescence intensity of\nFITC-dextran was lower in the MMT@Bac@TL group, indicating decreased cell\npermeability.\nSupplementary Fig. 11 Cytokine levels in the culture supernatant of macrophages\nunder different treatments. After 24 hours of LPS (1 µg/ml) stimulation, J774A.1 cells\nwere incubated with culture medium, Bac, MMT@Bac, MMT@Bac@T,\nMMT@Bac@L, or MMT@Bac@TL for 24 hours. a-c, Concentrations of IL-4 (a), ratio\nof IL-6 to IL-4 (b), and ratio of IL-10 to NO (c) in the supernatant. Data are represented\nas means ± SD (n = 6). p values were determined using one-way ANOVA. p values <\n0.05 are shown.\nSupplementary Fig. 13 Cell permeability measured with the FITC-dextran assay.\nFluorescence intensity in the down chamber of the transwell after 24-hour incubation\nof FITC-dextran (4kDa) with Caco-2 monolayers on the upper chamber of transwell\ninserts. Data are represented as means ± SD (n = 5). p values were determined using\none-way ANOVA. p values < 0.05 are shown.\n5. Although MOFS reduces Candida albicans, the mechanistic basis is only partially\nexplained.\nResponse: We thank the reviewer for offering the constructive suggestions. The relevant\ndiscussion has been supplemented in the revised manuscript (Page 20, Line 4-9). In\naddition to its role against pathogen infection, indole offers a viable alternative to\neliminate C. albicans and its biofilms (J Appl Microbiol 2012, 113, 622-628). Lactic\nacid-producing bacteria are known to block C. albicans filamentation, an important\nvirulence trait, by affecting the intestinal microenvironment (Gut Pathog 2023, 15, 30).\nAdditionally, E. faecalis was identified as secreting Fsr-dependent proteases, which\ninhibit hyphal morphogenesis and biofilm formation in the fungus, thereby reducing\nthe virulence (J Appl Microbiol 2012, 113, 622-628).\n6. In PCA and LEfSe analyses, please provide sample size per group, clearly indicate\nthe statistical methods used, and add p-value annotations to confirm the significance of\nthe microbiota shifts.\nResponse: We appreciate your kind suggestions. In PCA and LEfSe analyses, five\nsamples per group were used for the measurement. The p value determined by a non-\nparametric statistical test Analysis of Similarities (ANOSIM) has been presented in the\nmodified Fig. 4e. LEfSe analyses were performed with the Kruskal-Wallis rank sum\ntest. The threshold of LDA score is 3.5. Corresponding descriptions have been added\nin the revised manuscript (Page 44, Line 1 and Page 45, Line 3-6).\nFig. 4 Modulation of the gut microbiome by MMT@Bac@TL. e, Results of the PCA\nof the gut microbiota at class level (n = 5). p value was analyzed by non-parametric\nstatistical test ANOSIM. f, Cladogram of LEfSe from phylum to species levels (n = 5).\nLEfSe analyses were performed with the Kruskal-Wallis rank sum test. The threshold\nof LDA score is 3.5.\n7. For greater relevance, especially in antimicrobial or anti-inflammatory efficacy\nstudies, a benchmark group using a clinically available probiotic should be included to\ncontextualize the advantage of MOFS.\nResponse: Thank you for this valuable suggestion. To better evaluate the therapeutic\nadvantages of MOFS, we have added a benchmark comparison with the clinically\navailable probiotics EcN and Bacillus subtilis (Bs) in the revised manuscript.\nCorresponding results have been added to the revised manuscript (Page 20, Line 17-30\nand Page 21, Line 1-8): For greater relevance, we further compared the bactericidal\neffect of MMT@Bac@TL on pathogens with that of clinically available probiotics,\nincluding EcN and Bacillus subtilis (Bs). C. albicans were co-cultured with EcN, Bs or\nMMT@Bac@TL in yeast extract peptone dextrose (YPD) medium at a ratio of 1:10.\nThe fungi cultured with medium were set as a control. After 12 hours of co-culture,\nsamples were serially diluted, plated on YPD plates and incubated at 37 °C for colony\nenumeration of C. albicans. As shown in Supplementary Fig. 17a, MMT@Bac@TL\nexhibited comparable effects in inhibiting C. albicans to those of clinically available\nprobiotics. Encouraged by the bactericidal effect of MMT@Bac@TL on fungi, we\nfurther evaluated the bactericidal effect of MMT@Bac@TL on S. typhimurium SL1344,\none of the representative pathogenic bacteria. As shown in Supplementary Fig. 17b, Bs\nhas a negligible effect on inhibiting the growth of S. typhimurium. The counts of S.\ntyphimurium were ~3-fold and ~2-fold lower in EcN and MMT@Bac@TL,\nrespectively, compared to the medium group. Considering that the anti-inflammatory\nefficacy of living bacteria is another factor in the therapy of colitis, in addition to\nantimicrobial efficacy, we next compared MMT@Bac@TL with clinically available\nprobiotics. J774A.1 cells pre-treated with LPS (1 µg/ml) for 24 hours were\nsubsequently incubated with 102 CFUs EcN, Bs, or MMT@Bac@TL for 24 hours. The\ncells treated with culture medium served as the control. As shown in Supplementary\nFig. 17c, the ratio of M2 to M1 macrophages was over 4-fold increase in\nMMT@Bac@TL compared to other treatments, indicating a potent anti-inflammatory\neffect of MMT@Bac@TL.\nSupplementary Fig. 17 Comparative anti-inflammatory and antimicrobial activities of\nMOSF to clinically available probiotics. a,b, Counts of C. albicans (a) and S.\ntyphimurium (b) after co-culture with Bs, EcN or MMT@Bac@TL for 24 hours. The\nfungi or bacteria cultured in the medium served as the control. c, Flow cytometric\nanalysis of the ratio of M2 to M1 macrophages after 24 hours incubation with Bs, EcN,\nor MMT@Bac@TL. Data are represented as means ± SD (n = 5-6). p values were\ndetermined using one-way ANOVA. p values < 0.05 are shown.\n8. Some related references such as (Nat. Commun.,2022,13, 3432) are suggested to be\ncited.\nResponse: We sincerely apologize for overlooking this vital work, which is important\nto understand the development of engineered bacteria for treating gut disorders. It has\nbeen cited in the revised manuscript (Ref.17, Page 33, Line 17-19).\nWe thank all the reviewers again for taking their valuable time to review our\nmanuscript. Their kind comments and useful suggestions are the greatest support\nto our work."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This manuscript presents a highly innovative design for restoring intestinal homeostasis using a montmorillonite-based oral fermentation system. This system is fabricated by loading metabolic substrates in the lamellar structure of montmorillonite via cation replacement and adsorbing the substrate-related bacteria through electrostatic interactions. After oral delivery, the continuous release of loaded substrates and the formation of biofilms by carried bacteria enable long-term efficient synthesis of beneficial metabolites in the intestine. Importantly, the superiority over clinical therapeutics in murine models demonstrates its potential for therapeutic application. Overall, this work presents significant novelty and the in vivo fermentation system is a remarkable advancement over conventional oral delivery systems. The data are well-organized and compelling. The revision has answered all my concerns. Thus, I recommend this work for publication in Nature Communications.", "reviewer_2": "I appreciate the amount of job the authors have done in revising the manuscript, which certainly has substantiated the value of this work. However, I have to say that this work is an incremental piece to the current state of oral delivery of probiotics, and it lacks the broad interest to the wide readership that Nature Communications aims to have.", "reviewer_3": "I think that the authors have satisfactorily addressed the comments provided with the original version, and I believe the paper is suitable for publication."}, "author_response": "Response: We thank the reviewer very much for her/his positive feedback and for\nproviding helpful suggestions on how to refine the conclusions of our work.\nResponse: We are grateful that the reviewer has provided valuable feedback and has\nrecognized the efforts we have made to improve our study. We believe that the study is\nconceptually different from previous studies.\nResponse: The authors are grateful to the reviewer for offering insightful comments and\nvaluable suggestions for improving our study.\nWe thank all the reviewers again for taking their valuable time to review our\nmanuscript. Their kind comments and useful suggestions are the greatest support\nto our work."}]

Decision letter