Sustained-Release Enteric Formulations of Lactobacillus plantarum Based on Granulation Technology: Preparation and Therapeutic Evaluation in Acute Colitis

Sustained-Release Enteric Formulations of Lactobacillus plantarum Based on Granulation Technology: Preparation and Therapeutic Evaluation in Acute Colitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sustained-Release Enteric Formulations of Lactobacillus plantarum Based on Granulation Technology: Preparation and Therapeutic Evaluation in Acute Colitis Xiwei Zhang, Jianfeng Wan, Xile Wang, Xiaocan Zhang, Miaomiao Jiang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7469110/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Apr, 2026 Read the published version in BMC Biotechnology → Version 1 posted 9 You are reading this latest preprint version Abstract Background The clinical effectiveness of orally administered probiotics is often limited by poor survival during gastrointestinal transit and insufficient delivery to the intestinal site of action. Lactobacillus plantarum, a probiotic with anti-inflammatory potential, is highly sensitive to gastric acid and bile salts, diminishing its therapeutic efficacy for intestinal diseases. We developed a sustained-release enteric pellet formulation to enhance probiotic stability, promote targeted intestinal delivery, and evaluate its therapeutic efficacy in colitis. Results Pellets were prepared using low-temperature extrusion and coated with pH-responsive polymers (hydroxypropyl methylcellulose phthalate and Eudragit L100), creating a dual-protection barrier. Structural analysis revealed a cross-linked porous skeleton conducive to controlled release. In vitro tests confirmed gastric acid resistance and rapid release under intestinal conditions. In a dextran sulfate sodium-induced acute colitis mouse model, pellet-treated groups (both coatings) showed superior outcomes compared to uncoated probiotic powder. This included enhanced body weight recovery, reduced histopathological damage, downregulation of pro-inflammatory M1 macrophages, enhancement of anti-inflammatory M2 polarization, restoration of gut microbial diversity, and normalization of the Firmicutes to Bacteroidetes ratio. Conclusions The sustained-release enteric pellet system effectively protects Lactobacillus plantarum during gastrointestinal transit and achieves targeted intestinal release. Integrating mechanical shielding with pH-triggered dissolution significantly enhances probiotic bioavailability and therapeutic efficacy. This delivery platform represents a safe, scalable, and broadly applicable strategy for treating intestinal inflammatory diseases. Furthermore, the approach provides a versatile framework for delivering other probiotic strains or combinations. Acute colitis Lactobacillus plantarum Enteric sustained-release pellets HPMCP Eudragit L100 Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Background Probiotics, particularly Lactobacillus plantarum , have demonstrated therapeutic potential in regulating gut microbiota and promoting intestinal health[ 1 , 2 ]. However, a major challenge in probiotic-based therapy for intestinal diseases is ensuring the survival and targeted delivery of orally administered probiotics. These beneficial bacteria are exposed to harsh gastrointestinal conditions, including gastric acid, bile salts, and digestive enzymes, which drastically reduce their viability and limit clinical efficacy[ 3 ]. Conventional formulations such as fast-release powders or capsules provide insufficient protection during gastrointestinal transit and lack targeted release mechanisms, thereby failing to ensure an adequate number of viable bacteria reach the colon[ 4 ]. In recent years, multi-unit pellet systems have emerged as a promising strategy for oral drug delivery[ 5 , 6 ]. Composed of numerous small granules, these systems disperse uniformly throughout the gastrointestinal tract, reducing local irritation and minimizing the risk of dose dumping. Compared to conventional monolithic dosage forms, multi-unit systems offer the advantage of tailored release profiles, including sustained or delayed release, and can be engineered for site-specific delivery based on pH or transit time. For probiotic formulations, enteric polymer coatings have been employed to protect bacteria under acidic conditions while enabling their release in the intestine[ 7 ], thereby enhancing intestinal colonization and activity[ 8 , 9 ]. Despite these advancements, current probiotic delivery technologies exhibit limitations in optimizing viability and targeted release. Rapid-release formulations may prematurely discharge probiotics in the stomach, leading to substantial bacterial loss, while standard capsule-based systems cannot achieve the uniform dispersion and precise release control afforded by multi-unit approaches. This study proposes a novel sustained-release pelletization strategy for the intestinally targeted delivery of the probiotic Lactobacillus plantarum. The formulation employs a sucrose-based filler phase cross-linked with pH-responsive polymers to construct a release matrix capable of withstanding gastric conditions while enabling controlled release of viable bacteria in the intestinal environment. Through systematic material screening, in vitro release profiling, and in vivo evaluation using a murine model of acute colitis, the study aims to elucidate the release mechanism and therapeutic potential of the proposed delivery system. By enhancing site-specific efficacy, this strategy could reduce the per-dose probiotic requirement while maintaining or improving therapeutic outcomes, thereby alleviating patient treatment burden. Furthermore, this work provides valuable insights into the design and application of next-generation oral probiotic delivery systems and offers a viable pathway for addressing current limitations in probiotic therapy. Importantly, this technological approach offers a promising platform not only for single-strain applications but also for delivering multi-strain or composite probiotic formulations. 2. Methods 2.1 Experimental Materials Lactobacillus plantarum (viable count ≥ 1 × 10^10 CFU/g); Sodium alginate (SA), cellulose acetate phthalate (CAP) (Chemicell); hydroxypropyl methylcellulose succinate (HPMCAS), hydroxypropyl methylcellulose phthalate (HPMCP) (Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.); Eudragit L100 and S100 (Shanghai Dexiang Pharmaceutical Technology Co., Ltd.).Dextran sulfate sodium salt (DSS) (Yisheng Biotechnology, Shanghai, China).Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) (Dongguan Chuangfeng Automation Technology Co., Ltd.).Male BALB/c mice (6–8 weeks old, 25 g) were purchased from Zhengzhou University Experimental Animal Center. Electrically heated constant-temperature drying oven (Shanghai Shuli Instrument Co., Ltd.); autoclave sterilizer (Zhejiang Xinfeng Medical Devices Co., Ltd.); carbon dioxide incubator with temperature-controlled shaker; ultrapure water system (Ningbo Dansboton Environmental Technology Co., Ltd.). 2.2 Screening of Antibacterial Properties of Coating Materials The antibacterial activity of different coating materials was assessed using the inhibition zone method to identify suitable excipients for probiotic encapsulation. Thirty microliters of L. plantarum suspension (adjusted to ~ 1 × 10^8 CFU/mL after MRS liquid culture) were evenly spread onto MRS agar plates. Circular sterile filter papers (6 mm diameter) were soaked in 2% (w/v) solutions of SA, HPMCP, CAP, HPMCAS, L100, and S100, then placed onto the plates. Plates were incubated inverted at 37 ± 0.5°C for 24 ± 2 hours. Filter papers soaked in antibiotic solution and sterile water served as positive and negative controls, respectively. The diameter of inhibition zones was measured; smaller zones indicated lower antibacterial activity. All experiments were performed in triplicate, and average values were recorded. 2.3 Preparation and Optimization of Sustained-Release Granules 2.3.1 Granule Preparation and Process Optimization To avoid thermal inactivation of the probiotic during granule drying, low-temperature drying (35–40°C) was employed, while other steps (crushing, sieving, mixing, extrusion) followed conventional protocols. For HPMCP-based granules, 1% (w/v) HPMCP solution (dissolved in a 1:1 methanol-acetone mixture) was gradually added to a mortar containing freeze-dried L. plantarum powder premixed with sucrose (1:1). The mixture was ground until a non-sticky, coarse, granular material formed. The mixture was sieved sequentially through 40-mesh and then 80-mesh sieves. Particles retained on the 80-mesh sieve (particle size range: 180–425 µm) were and dried at 37–40°C for 22–26 hours. Granules were similarly prepared using SA, HPMCP, CAP, HPMCAS, L100, and S100 at concentrations of 0.5%, 1%, and 2% (w/v). Solvents were sterile water for SA, methanol-acetone for CAP and HPMCAS, and 75% ethanol for L100 and S100. 2.3.2 Dissolution Testing For in vitro dissolution studies, 100 mL of SGF and 100 mL of SIF were placed in a 40°C shaking incubator (150 rpm). After preheating to 37°C, 0.5 g of each granule formulation was added. Dissolution behavior was observed every 15 minutes for 4 hours. 2.3.3 Scanning Electron Microscopy (SEM) SEM analysis was conducted on granules formulated with HPMCP and L100, selected based on their favorable dissolution and non-antibacterial properties. Samples were analyzed by a third-party laboratory to investigate microstructural characteristics. 2.4 Induction of Acute Colitis and Treatment Protocol After a 3-day acclimation period, mice were randomly assigned into five groups: normal group (n = 6), DSS model group (n = 6), probiotic powder group (n = 6), HPMCP granule group (n = 6), and L100 granule group (n = 6). During the modeling phase, all groups except the normal group received 3% (w/v) DSS in drinking water ad libitum for 7 days, with daily replacement of freshly prepared solutions to induce acute colitis mimicking human pathophysiology(Table 1 ). Starting on Day 8, mice were treated via oral gavage every other day for a total of three doses (Days 8, 10, and 12). The administration dose was 100 mg per mouse (suspension in sterile water at 0.2 mL/10 g body weight). The HPMCP and L100 groups received suspensions of respective granules; the powder group received equivalent doses of free L. plantarum powder; the DSS and normal groups received sterile water. All gavage procedures were conducted under sterile conditions. On day 14, experimental mice were intraperitoneally administered a 1% (10 mg/mL) sodium pentobarbital solution at a dose of 100 mg/kg to induce a stable deep anesthesia state. After confirming the loss of consciousness, cervical dislocation was performed to ensure euthanasia, followed by dissection to collect colon tissues and intestinal contents. Colonic tissues were rinsed with PBS and fixed in 4% paraformaldehyde for subsequent histopathological and immunofluorescence analyses. Intestinal contents were immediately stored at − 80°C after collection for metagenomic analysis of the gut microbiota. Table 1 Experimental Design for Colitis Induction and Treatment Stage Group Intervention Modeling Phase Normal group(n = 6) Free access to drinking water (pure water) Colitis model group(n = 24) Free access to 3% DSS solution in drinking water Treatment Phase Normal group(n = 6) Sterile water DSS group(n = 6) Sterile water Lactobacillus plantarum group(n = 6) Lactobacillus plantarum powder HPMCP group(n = 6) HPMCP-L. plantarum sustained-release granules L100 group(n = 6) L100-L. plantarum sustained-release granules 2.5 Therapeutic Assessment in DSS-Induced Colitis Model The colon samples collected from dissection on day 14 (as described in section 2.4 ) were washed with PBS and fixed in 4% paraformaldehyde for hematoxylin and eosin (HE) staining to evaluate histopathological changes. The intestinal contents were stored at -80°C for subsequent microbiota analysis. 2.6 Immunofluorescence Analysis of Inflammation Resolution To evaluate anti-inflammatory effects of the sustained-release granules, immunofluorescence staining was performed. Tissue sections were incubated overnight at 4°C with primary antibodies targeting CD86, CD206, and F4/80, followed by 1-hour incubation at 37°C with appropriate secondary antibodies. Fluorescence microscopy was used for imaging and analysis of macrophage polarization. 2.7 Gut Microbiota Diversity Analysis Metagenomic analysis of microbial communities was conducted on intestinal content samples. DNA extraction and sequencing were performed by Haimujie Biomedical Technology Co., Ltd. Quality control and OTU clustering (97% similarity threshold) were performed using QIIME2. Alpha diversity (Sobs, Shannon indices), beta diversity (PCoA), and taxonomic composition at phylum and genus levels were analyzed. 3. Results 3.1 Release Profiles in Simulated Gastrointestinal Fluids Inhibition zone assays revealed that all tested coating materials exhibited negligible antibacterial activity against L. plantarum , indicating their suitability as probiotic carriers. Particles were prepared from these materials for subsequent solubility analysis. Both HPMCP and L100 exhibited prolonged dissolution times and relatively low cumulative release in simulated gastric fluid (SGF), indicating effective protection of the encapsulated probiotics under acidic conditions. At concentrations of 1% and 2%, HPMCP and L100 exhibited significantly lower release rates compared to CAP, HPMCAS, and S100, and performed better than sodium alginate (SA), indicating superior protective effects in simulated gastric fluid and effective delay in premature release. These findings demonstrate the favorable enteric properties of HPMCP and L100, which contribute to minimizing probiotic loss during gastric transit. Additionally, the disintegration times of HPMCP and L100 at 1% and 2% concentrations ranged from 60 to 100 minutes in simulated intestinal fluid—moderate compared to S100, which dissolved too rapidly, and CAP, which exhibited prolonged disintegration times nearing 180 minutes at 2%. This suggests that HPMCP and L100 provide a well-balanced release profile in the intestinal environment, achieving sustained but timely probiotic release without excessive delay. In contrast, under simulated intestinal fluid (SIF) conditions, the dissolution times were significantly shortened, and the release amounts reached complete release thresholds. By comparing the cumulative release in SGF over 4 hours (Fig. 1 A) and the time required for complete dissolution in SIF (Fig. 1 B), it is evident that L. plantarum sustained-release granules formulated with HPMCP and L100 successfully prevented premature release in the gastric phase while enabling rapid disintegration and full release in the intestinal phase. This dual-phase release behavior provides a crucial advantage for enhancing probiotic survival through the gastrointestinal tract, thereby significantly improving the bioavailability of the formulation. Based on these findings, HPMCP and Eudragit L100 were identified as optimal enteric coating materials for sustained-release probiotic delivery systems. 3.3 Morphological Characterization of Granules The HPMCP- Lactobacillus plantarum sustained-release granules (Fig. 2 A) and L100- L. plantarum sustained-release granules (Fig. 2 E) appeared as uniformly spherical particles at the macroscopic level. SEM observations revealed that at 1000× magnification, the granule surfaces appeared slightly rough. At higher magnifications (5000× and 30,000×), the granules were composed of fine subunits with uniformly distributed sucrose as filler material. These subunits were interconnected, forming a cross-linked porous matrix structure characteristic of sustained-release formulations. 3.4 Therapeutic Efficacy of Granules 3.4.1 Resolution of Inflammation Dextran sulfate sodium salt (DSS) administration induces clinical manifestations in mice that closely resemble ulcerative colitis (UC), including body weight loss, diarrhea, and hematochezia[ 10 , 11 ]. In this study, a DSS-induced colitis model was employed to evaluate the therapeutic efficacy of the orally administered sustained-release pellet formulation. Compared to the normal group, DSS-treated mice showed significant weight loss and histological signs of colitis, including epithelial damage, crypt loss, and inflammatory infiltration, confirming successful model induction. As shown in Fig. 3 A, compared with conventional Lactobacillus plantarum powder administration, both the HPMCP and L100 granule-treated groups exhibited varying degrees of epithelial structure restoration. In particular, administration of HPMCP sustained-release granules significantly reduced the level of inflammatory cell infiltration and revealed histological structures nearly indistinguishable from those of the normal control group. Compared with the DSS group and the bacterial powder group, the granule-treated groups showed markedly alleviated inflammatory cell infiltration, mucosal damage, and crypt destruction in the colonic tissue. As illustrated in Fig. 3 B and 3 C, standard markers including iNOS + , CD206, and F4/80 were used to identify M1 and M2 macrophages. DSS-induced colitis mice exhibited increased macrophage infiltration in the colon, with both M1 and M2 macrophage populations elevated compared to the control group. Oral administration of HPMCP and L100 sustained-release granules led to a reduction in M1 macrophages and a concomitant increase in M2 macrophages. These findings indicate that both HPMCP and L100 granules modulate the M1/M2 macrophage polarization balance, particularly promoting M2 polarization. This suggests that enhancing M2 polarization through L. plantarum sustained-release granules may represent a potential therapeutic strategy for inflammatory bowel disease (IBD). 3.4.2 Gut Microbiota Diversity and Composition A growing body of evidence indicates that the gut microbiota plays a direct role in the pathogenesis of inflammatory bowel disease (IBD)[ 12 , 13 ]. Distinct microbial communities within the colon form an interactive and balanced ecosystem, which becomes disrupted in the context of IBD and other colonic disorders[ 14 , 15 ]. Therefore, this study investigated whether Lactobacillus plantarum sustained-release pellets could restore gut microbial homeostasis in a murine model. Alpha diversity is an ecological metric used to assess the richness and evenness of taxa within individual samples[ 16 ]. Greater microbial species richness in an ecosystem is generally reflected by higher alpha diversity indices. As shown by the Sobs index results (Fig. 4 A), alpha diversity was significantly reduced in the probiotic powder group compared to the normal control group. This decline may be attributed to DSS-induced inflammation, which promotes the overgrowth of harmful intestinal bacteria while suppressing the proliferation of commensal microbes, ultimately leading to reduced microbial abundance. In contrast, oral administration of HPMCP- and L100-based sustained-release pellets markedly increased Sobs index values, with statistically significant differences compared to the DSS group. Notably, these results suggest that treatment with the sustained-release formulations effectively restored microbial species diversity toward levels observed in healthy controls. Principal coordinate analysis (PCoA) showed significant differences in similarity between the control group and the DSS-induced model group. PCoA (Fig. 4 C and 4 D) revealed distinct clustering of treatment groups. Microbiota profiles in the HPMCP and L100 groups were more similar to the normal group than to the DSS or powder groups, indicating restorative effects. To elucidate the compositional structure of the gut microbiota, microbial differences were analyzed at both the phylum and genus levels[ 17 ]. At the phylum level (Fig. 4 F), six dominant bacterial phyla were identified, among which Firmicutes and Bacteroidetes represented the two most abundant taxa, collectively accounting for over 90% of the total microbiota and dominating across all samples. Compared to the normal control group, the probiotic powder group exhibited an increased relative abundance of Firmicutes and a decreased abundance of Bacteroidetes. The Firmicutes-to-Bacteroidetes (F/B) ratio is widely recognized as a key indicator of microbial dysbiosis in the gut. In contrast, treatment with HPMCP- and L100-based sustained-release pellets resulted in a decreased abundance of Firmicutes and an increased abundance of Bacteroidetes relative to the powder group, indicating a regulatory effect on gut microbiota composition. Notably, the L100 group demonstrated the most pronounced modulation of the Firmicutes and Bacteroidetes populations in colitis-induced mice, with microbial profiles approaching those observed in the healthy control group. At the genus level, microbial community composition was further characterized using a heatmap analysis (Fig. 4 G). In the normal group, genera such as Bacteroides, Alistipes, and Odoribacter were present at relatively high abundances. In contrast, the probiotic powder group exhibited a marked increase in potentially pathogenic genera including Escherichia-Shigella, Enterococcus, and Staphylococcus, consistent with findings shown in Fig. 4 C, suggesting a detrimental impact on intestinal health. In the HPMCP-treated group, beneficial genera such as Candidatus_Arthromitus and Turicibacter emerged as dominant taxa with increased abundance. In the L100 group, there was a significant enrichment of Roseburia, a known short-chain fatty acid-producing genus, along with A2 and unclassified taxa within the Prevotellaceae family. These observations indicate that treatment with HPMCP- and L100-based sustained-release pellets modulated the gut microbiota by selectively enriching beneficial bacterial populations, thereby contributing to improved microbial community function. Collectively, these findings suggest that the oral administration of L. plantarum sustained-release pellets promotes microbial homeostasis and supports therapeutic efficacy in the context of inflammatory bowel disease. 4. Discussion In this study, a novel sustained-release and enteric-coated delivery system for Lactobacillus plantarum was successfully developed based on a cross-linked porous matrix constructed using pH-responsive polymers (HPMCP and Eudragit L100). This system incorporates a dual-protection mechanism of acid-resistant enteric coating and controlled-release internal matrix, which significantly improves the survival rate and therapeutic efficacy of the probiotic in a dextran sulfate sodium (DSS)-induced acute colitis mouse model, and demonstrates superior performance compared to traditional oral probiotic powders. Clinically, oral administration of Lactobacillus plantarum powder has been demonstrated to exert beneficial effects on ameliorating colitis symptoms[ 2 ]. However, its efficacy is constrained by significant degradation in gastric acid and bile salts, leading to reduced viable bacteria reaching the colon and suboptimal colonization[ 18 , 19 ]. Consequently, achieving the desired therapeutic outcomes often necessitates prolonged, frequent, and high-dose administration regimens (e.g., multiple times daily)[ 20 ]. This not only increases the patient compliance burden and treatment costs but also carries the potential for discomfort due to excessive intake. In contrast, the enteric-coated sustained-release granule strategy proposed in this study utilizes the "smart" protection afforded by HPMCP (hydroxypropyl methylcellulose phthalate) and L100 enteric coatings. This effectively shields the probiotics from gastric acid erosion, as confirmed by minimal release in simulated gastric fluid during in vitro dissolution testing, thereby ensuring the safe passage of the majority of viable bacteria through the stomach. Upon entry into the near-neutral or weakly alkaline intestinal environment (pH > 5.5-6.0), the coating rapidly dissolves. Scanning electron microscopy (SEM) analysis further revealed that the granules possess an internal sucrose-filled cross-linked porous matrix, facilitating sustained release rather than burst release. This combined mechanism of gastric protection and colon targeting plays a crucial role in ensuring the overall therapeutic efficacy of the formulation. Compared with conventional probiotic powders, the proposed delivery system combines a unique physical architecture (cross-linked porous matrix) with a chemical barrier (pH-responsive polymer coating), thereby establishing an efficient platform for dual protection and controlled release. Notably, under equivalent probiotic dosing, the HPMCP- and L100-based sustained-release granules developed in this study demonstrated significantly superior therapeutic efficacy against DSS-induced acute colitis compared to direct oral administration of freeze-dried Lactobacillus plantarum powder. This therapeutic advantage is not solely attributed to the probiotic itself, but more importantly to the optimized gastrointestinal delivery and targeted release strategy afforded by the granule system. Such a strategy effectively reduces dosing frequency and total dosage, enhances patient compliance, and achieves equal or even improved therapeutic outcomes. Therefore, this study confirms from multiple aspects that the delivery system is more effective and demonstrates superior performance. Histological evaluation (Fig. 3 A) showed that both the HPMCP and L100 granule-treated groups exhibited varying degrees of epithelial structure restoration. Notably, administration of HPMCP sustained-release granules significantly reduced inflammatory cell infiltration and presented tissue structures nearly indistinguishable from those of the normal control group. Compared with the DSS group and the probiotic powder group, the granule-treated groups showed markedly reduced inflammatory cell infiltration, mucosal damage, and crypt destruction in colonic tissue, with the HPMCP group closely resembling normal tissue. H&E staining highlights that HPMCP and L100 sustained-release granules, compared to conventional probiotic powder, effectively restore the epithelial structure of colonic tissue in DSS-induced colitis mice, significantly alleviate inflammation and tissue damage, and exhibit superior therapeutic effects. Meanwhile, immunofluorescence analysis further supported the improvement in inflammation. Immunofluorescence (Fig. 3 B, C) demonstrated that granules significantly reduced pro-inflammatory M1 macrophage (iNOS⁺/F4/80⁺) infiltration and enhanced anti-inflammatory M2 (CD206⁺/F4/80⁺) polarization, effects substantially weaker with powder. This suggests granules more effectively activate host anti-inflammatory pathways via enhanced viable probiotic delivery and sustained action[ 18 , 21 ]. Both H&E staining and immunofluorescence indicate the improvement of inflammation in colitis; moreover, the improvement of the intestinal microbiota is also an important indicator. We conducted high-throughput transcriptome sequencing to investigate changes in the intestinal microbiota. Metagenomic sequencing highlighted another key advantage: while powder minimally improved DSS-induced reductions in alpha diversity (Sobs, Shannon indices; Fig. 4 A, B), both granule types restored richness/diversity towards normal levels. PCoA analysis (Fig. 4 C, D) confirmed granule-treated microbiota (especially L100) clustered closer to healthy controls than DSS or powder groups, indicating superior restoration of ecological health. Granules also more effectively normalized the dysbiotic Bacillota /Bacteroidetes ratio elevated by powder (Fig. 4 F). At the genus level (Fig. 4 G), granule groups uniquely enriched beneficial taxa (e.g., Candidatus_Arthromitus, Turicibacter with HPMCP; Roseburia, Prevotellaceae with L100), linked to anti-inflammation and barrier function[ 22 , 23 ], contrasting with higher potential pathogens (Escherichia-Shigella, Enterococcus) in powder recipients. Collectively, the sustained-release strategy, by ensuring viable probiotic delivery and persistence, profoundly reshapes the gut microbiota towards a healthier, anti-inflammatory state, underpinning its enhanced efficacy. Crucially, at the same dosage, our delivery platform outperformed traditional powder formulations in almost all indicators, including histopathology, immune regulation, and microbiota normalization, indicating that the delivery technology enhances probiotic efficacy more effectively than simply increasing the dose. Interestingly, the therapeutic effect of this delivery system may also have considerable advantages compared to current chemical treatments for colitis. Current pharmacological treatments for intestinal inflammation, particularly inflammatory bowel disease (IBD), primarily include 5-aminosalicylic acid (5-ASA) derivatives, corticosteroids, immunosuppressants (e.g., azathioprine, methotrexate), and biologics such as anti-TNF-α monoclonal antibodies[ 24 ]. Although these agents can be effective, they are often associated with serious adverse effects, including increased risks of infection, metabolic disturbances, bone marrow suppression, and long-term malignancy[ 25 , 26 ]. In contrast, probiotics are live microorganisms that are generally regarded as safe, with side effects such as bloating or mild gastrointestinal discomfort usually being temporary and self-limiting. Beyond direct anti-inflammatory activity, the sustained-release granule system presented in this study offers a multi-targeted therapeutic approach. It not only delivers viable Lactobacillus plantarum to the intestine but also facilitates the restoration of microbial homeostasis, the reinforcement of mucosal barrier integrity, and the modulation of the immune response, specifically by enhancing M2 macrophage polarization[ 27 , 28 ]. Such mechanisms address the underlying pathophysiology of IBD rather than merely suppressing inflammation. For instance, 5-ASA acts mainly as a local anti-inflammatory agent and exhibits limited ability to regulate microbial dysbiosis. In this context, the superior epithelial repair observed in the granule-treated groups (as shown in Fig. 3 A) underscores the microbiota-mediated benefits of L. plantarum and its metabolites, such as short-chain fatty acids, in promoting epithelial regeneration and mucosal healing[ 29 ]. These findings highlight the therapeutic potential of probiotic-based sustained-release systems as safer, more holistic alternatives or adjuncts to conventional pharmacological interventions. Probiotic immunomodulation and microbiota restoration require time, potentially limiting their speed in controlling acute, severe inflammation compared to potent anti-inflammatories like corticosteroids or biologics. Consequently, probiotics alone are often insufficient for inducing remission in severe active IBD, which typically requires conventional agents[ 30 ]. Probiotic efficacy may also exhibit greater individual variation due to factors like baseline microbiota or genetics[ 31 ]. Nevertheless, in our DSS-induced acute colitis model (mimicking UC), the sustained-release granule system (particularly HPMCP granules) achieved near-normal levels of histological repair and microbiota restoration, alongside significant anti-inflammatory effects (e.g., macrophage polarization). The degree of histological repair and anti-inflammatory effects observed with the sustained-release granules, particularly HPMCP granules, in this acute DSS-colitis model appear comparable to the reported efficacy of first-line therapies like 5-ASA in managing mild-to-moderate ulcerative colitis[ 32 ], based on established clinical and preclinical knowledge. While 5-ASA remains a cornerstone for inducing and maintaining remission in this patient group, our granules demonstrated comparable efficacy in the model, coupled with a superior safety profile and the added benefit of microbiota modulation. The superior therapeutic performance of our system stems from its innovative scaffold‑based architecture, which affords excellent protection and precise release of probiotics. In dissolution data (Fig. 1 ) confirm that HPMCP and L100 coatings confer excellent acid resistance, minimizing probiotic loss in simulated gastric fluid, while the cross‑linked porous matrix (SEM, Fig. 2 ) enables controlled, sustained release in the intestinal milieu[ 33 ] Unlike traditional enteric-coated tablets or capsules where failure of a single unit results in exposure of the entire payload to gastric acid, our multi-unit granules compartmentalize the risk so that damage to individual particles causes only minor probiotic loss while the intact granules continue to deliver viable cells[ 34 ]. Moreover, embedding probiotics and sucrose fillers uniformly within a micro‑/nano‑scale polymeric network creates local protective microenvironments and controlled‑release pathways that simple powder blending cannot achieve. Compared with microencapsulation techniques, which often require high temperature, organic solvents, or shear stress that jeopardize cell viability, our Wet granulation and low‑temperature extrusion processes employ well‑established pharmaceutical excipients (HPMCP, L100, sucrose) and are readily scalable under GMP conditions. These features collectively position the scaffold‑based granule system as a robust, manufacturable platform for advanced probiotic delivery. Various probiotic strains including Bifidobacterium species and other Lactobacillus species such as Lactobacillus rhamnosus and Lactobacillus acidophilus , as well as multi-strain formulations and synbiotics which are combinations of probiotics and prebiotics, have been widely investigated for the treatment of intestinal inflammation[ 35 – 37 ]. Certain combinations have demonstrated potential synergistic effects[ 38 ]. While L. plantarum , the strain selected in this study, possesses intrinsic acid and bile resistance, adhesive capacity, and immunomodulatory properties[ 39 ], the principal contribution of this work lies not in contesting the efficacy of other strains or combinations, but in presenting a broadly applicable and efficient delivery platform. The enteric sustained-release granule system developed herein, based on a cross-linked skeleton structure and low-temperature extrusion-coating process, offers excellent compatibility and is theoretically adaptable for the delivery of other individual strains, multi-strain consortia, or even synbiotic preparations[ 40 ]. Future studies could explore optimized probiotic combinations such as Lactobacillus plantarum co-administered with selected Bifidobacterium species using this platform to further enhance therapeutic outcomes and potentially surpass the efficacy of some existing commercial formulations. Crucially, the system ensures both the stability of each strain during processing and their coordinated release within the gastrointestinal tract. Maintaining a high activity of probiotics during processing cannot be ignored either. Our granule fabrication employed meticulously maintained low temperatures (35–40°C during wet granulation and drying), minimizing heat-induced inactivation. This contrasts sharply with processes like spray drying microencapsulation, where high inlet temperatures (150–200°C) often cause significant viability loss despite lower outlet temperatures[ 41 , 42 ]. Furthermore, we proactively addressed potential excipient toxicity: all candidate coating materials (SA, HPMCP, CAP, HPMCAS, L100, S100) were pre-screened for antimicrobial activity against L. plantarum using an agar diffusion assay. No significant inhibition was observed at relevant concentrations, confirming the biocompatibility of the selected polymers (HPMCP, L100). Solvents (methanol-acetone for HPMCP, 75% ethanol for L100/S100) were also chosen for minimal antimicrobial impact. This crucial step, often overlooked when selecting materials based solely on physicochemical properties[ 43 ], ensures excipients do not harm probiotics during processing or storage. Collectively, this low-temperature processing and biocompatible excipient/solvent selection strategy effectively maintained activity during the production process, resolving the issue of a significant decline in live bacteria counts reported in other studies involving probiotic microcapsules or particles after preparation[ 41 , 44 ], and providing a basis for the formulation's efficacy. The enteric-coated sustained-release granule system developed herein represents a promising next-generation platform for oral probiotic delivery. Its gastric protection and colon-targeted sustained release are particularly well-suited for the maintenance therapy of chronic inflammatory bowel diseases (IBD, including UC and CD)[ 45 ], potentially reducing relapse frequency/severity and dependence on conventional drugs by ensuring continuous delivery of viable probiotics to the inflamed site. The inherent compatibility and scalability of the granule matrix and coating process readily allow adaptation for delivering other single probiotic strains or designing advanced formulations. This includes multi-strain probiotic consortia leveraging synergistic interactions (e.g., L. plantarum + Bifidobacterium spp. )[ 36 , 38 ] or synbiotics by incorporating prebiotics (e.g., FOS, inulin) into the matrix to selectively enhance probiotic activity in situ[ 46 ].Taken together, this modular, scalable platform offers a versatile foundation for the development of advanced probiotic. 5. Conclusions This study successfully developed and validated an enteric-coated sustained-release granule system for Lactobacillus plantarum , utilizing an innovative cross-linked porous matrix formed by HPMCP/L100. The system employs a dual protective mechanism: gastric pH-responsive coating shields against acid erosion, while the intestinal matrix enables controlled, sustained release. This revolutionizes oral probiotic delivery, significantly enhancing survival through the harsh GI tract and ensuring targeted, prolonged release within the colon. In a DSS-induced murine colitis model, at equivalent viable cell doses, the granules demonstrated profoundly superior therapeutic efficacy compared to conventional freeze-dried powder. This encompassed significantly enhanced histological repair, effective immunomodulation (e.g., M2 macrophage polarization), and comprehensive restoration of gut microbiota homeostasis – advantages directly attributable to the system's fundamental improvement of probiotic bioavailability and targeted delivery efficiency. Crucially, high viability was preserved during manufacturing via meticulously controlled low-temperature processing and stringent screening of biocompatible, non-antimicrobial excipients. The matrix-based multi-particulate system offers distinct advantages over existing technologies (e.g., single-unit enteric-coated tablets/capsules, microcapsules), including superior protection uniformity, resilience against localized failure, gentle processing, and enhanced manufacturability. Compared to conventional drugs like 5-ASA, the probiotic granules present an attractive profile: comparable efficacy (for mild-moderate colitis in the model), superior safety, and unique multi-targeted actions (anti-inflammation, barrier repair, microbiota restoration). This positions them as a promising alternative or adjunctive therapy. Beyond enabling efficient L. plantarum delivery, this versatile platform technology holds significant potential for delivering other single probiotics, multi-strain consortia, or synbiotics. Its application extends to the maintenance therapy of IBD, prevention of antibiotic-associated diarrhea (AAD), management of C. difficile infection (CDI), and other dysbiosis-related conditions. Future optimization of long-term stability, large-scale manufacturing, and pivotal clinical trials will pave the way for this next-generation delivery system to achieve transformative advances in GI health management and personalized nutritional interventions. Abbreviations Abbreviation Definition 5-ASA 5-aminosalicylic acid AAD Antibiotic-associated diarrhea CAP Cellulose acetate phthalate CD86 Cluster of differentiation 86 CD206 Cluster of differentiation 206 CDI Clostridioides difficile infection DSS Dextran sulfate sodium salt F4/80 F4/80 antigen (EMR1, mouse macrophage marker) HE Hematoxylin and eosin HPMCAS Hydroxypropyl methylcellulose succinate HPMCP Hydroxypropyl methylcellulose phthalate IBD Inflammatory bowel disease iNOS Inducible nitric oxide synthase L100 Eudragit L100 L. plantarum Lactobacillus plantarum MRS de Man, Rogosa and Sharpe (broth/agar) OTU Operational taxonomic unit PBS Phosphate-buffered saline PCoA Principal coordinate analysis S100 Eudragit S100 SA Sodium alginate SEM Scanning electron microscopy SGF Simulated gastric fluid SIF Simulated intestinal fluid Sobs Number of observed species (or OTUs) UC Ulcerative colitis Declarations Ethics approval and consent to participate The animal study protocol was approved by the Ethics Committee of the Animal Center of Zhengzhou University (protocol code ZZU-LAC20241217[01] and date of approval is Dec. 2024). Availability of data and materials Not applicable. Competing interests The authors declare that they have no competing interests Funding This research was funded by 2019 scientific research support for high-level talent project (project number: 31401225) and 2023 natural science project (project number: 51300023) from Henan University of Technology, grant number: 501100003489. Author Contribution Xile Wang and Xiaocan Zhang wrote the original draft; Shuang Liang and Miaomiao Jiang prepared all figures; Xiwei Zhang and Jianfeng Wan check all the data and figures; Hongqing Zhang acquired fundings; Guangzhou Zhou reviewed and edited the final manuscript; Guangzhou Zhou and Xiwei Zhang supervised this project. All authors have reviewed and agreed to the published version of the manuscript. References Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, et al. 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Lichtenstein GR, Loftus EV, Isaacs KL, Regueiro MD, Gerson LB, Sands BE. ACG Clinical Guideline: Management of Crohn's Disease in Adults. American Journal of Gastroenterology. 2018;113(4):481-517. Peyrin-Biroulet L, Loftus EV, Colombel J-F, Sandborn WJ. The Natural History of Adult Crohn's Disease in Population-Based Cohorts. American Journal of Gastroenterology. 2010;105(2):289-97. Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nature Reviews Immunology. 2008;8(12):958-69. Lu Y, Li X, Liu S, Zhang Y, Zhang D. Toll-like Receptors and Inflammatory Bowel Disease. Frontiers in Immunology. 2018;9. Peng L, Li Z-R, Green RS, Holzmanr IR, Lin J. Butyrate Enhances the Intestinal Barrier by Facilitating Tight Junction Assembly via Activation of AMP-Activated Protein Kinase in Caco-2 Cell Monolayers. The Journal of Nutrition. 2009;139(9):1619-25. Derwa Y, Gracie DJ, Hamlin PJ, Ford AC. Systematic review with meta‐analysis: the efficacy of probiotics in inflammatory bowel disease. Alimentary Pharmacology & Therapeutics. 2017;46(4):389-400. Zmora N, Zilberman-Schapira G, Suez J, Mor U, Dori-Bachash M, Bashiardes S, et al. Personalized Gut Mucosal Colonization Resistance to Empiric Probiotics Is Associated with Unique Host and Microbiome Features. Cell. 2018;174(6):1388-405.e21. Ford AC, Achkar J-P, Khan KJ, Kane SV, Talley NJ, Marshall JK, et al. Efficacy of 5-Aminosalicylates in Ulcerative Colitis: Systematic Review and Meta-Analysis. American Journal of Gastroenterology. 2011;106(4):601-16. Singh BN. Modified-release solid formulations for colonic delivery. Recent patents on drug delivery & formulation. 2007;1(1):53-63. Arévalo-Pérez R, Maderuelo C, Lanao JM. Recent advances in colon drug delivery systems. Journal of Controlled Release. 2020;327:703-24. Maroni A, Zema L, Del Curto MD, Foppoli A, Gazzaniga A. Oral colon delivery of insulin with the aid of functional adjuvants. Advanced Drug Delivery Reviews. 2012;64(6):540-56. Didari T, Solki S, Mozaffari S, Nikfar S, Abdollahi M. A systematic review of the safety of probiotics. Expert Opinion on Drug Safety. 2014;13(2):227-39. Chapman CMC, Gibson GR, Rowland I. Health benefits of probiotics: are mixtures more effective than single strains? European Journal of Nutrition. 2011;50(1):1-17. Xiao Y, Lu C, Liu Y, Kong L, Bai H, Mu H, et al. Encapsulation of Lactobacillus rhamnosus in Hyaluronic Acid-Based Hydrogel for Pathogen-Targeted Delivery to Ameliorate Enteritis. ACS Applied Materials & Interfaces. 2020;12(33):36967-77. Plaza-Díaz J, Ruiz-Ojeda F, Vilchez-Padial L, Gil A. Evidence of the Anti-Inflammatory Effects of Probiotics and Synbiotics in Intestinal Chronic Diseases. Nutrients. 2017;9(6). de Vries MC, Vaughan EE, Kleerebezem M, de Vos WM. Lactobacillus plantarum—survival, functional and potential probiotic properties in the human intestinal tract. International Dairy Journal. 2006;16(9):1018-28. Mehta RS, Mayers JR, Zhang Y, Bhosle A, Glasser NR, Nguyen LH, et al. Gut microbial metabolism of 5-ASA diminishes its clinical efficacy in inflammatory bowel disease. Nature Medicine. 2023;29(3):700-9. Heidebach T, Först P, Kulozik U. Influence of casein-based microencapsulation on freeze-drying and storage of probiotic cells. Journal of Food Engineering. 2010;98(3):309-16. Picot A, Lacroix C. Encapsulation of bifidobacteria in whey protein-based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt. International Dairy Journal. 2004;14(6):505-15. Gbassi GK, Vandamme T. Probiotic Encapsulation Technology: From Microencapsulation to Release into the Gut. Pharmaceutics. 2012;4(1):149-63. Cook MT, Tzortzis G, Charalampopoulos D, Khutoryanskiy VV. Microencapsulation of probiotics for gastrointestinal delivery. Journal of Controlled Release. 2012;162(1):56-67. Sartor RB, Mazmanian SK. Intestinal Microbes in Inflammatory Bowel Diseases. The American Journal of Gastroenterology Supplements. 2012;1(1):15-21. Markowiak P, Śliżewska K. Effects of Probiotics, Prebiotics, and Synbiotics on Human Health. Nutrients. 2017;9(9). Nano. 2022;16(3):4041–58. Luo H, Wu F, Wang X, Lin S, Zhang M, Cao Z, et al. Encoding bacterial colonization and therapeutic modality by wrapping with an adhesive drug-loadable nanocoating. Mater Today. 2023;62:98–110. Niess J-H, Laroui H, Ingersoll SA, Liu HC, Baker MT, Ayyadurai S et al. Dextran Sodium Sulfate (DSS) Induces Colitis in Mice by Forming Nano-Lipocomplexes with Medium-Chain-Length Fatty Acids in the Colon. PLoS ONE. 2012;7(3). Eichele DD, Kharbanda KK. Dextran sodium sulfate colitis murine model: An indispensable tool for advancing our understanding of inflammatory bowel diseases pathogenesis. World J Gastroenterol. 2017;23(33):6016–29. Dziubańska-Kusibab PJ, Berger H, Battistini F, Bouwman BAM, Iftekhar A, Katainen R, et al. Colibactin DNA-damage signature indicates mutational impact in colorectal cancer. Nat Med. 2020;26(7):1063–9. de Vos WM, Tilg H, Van Hul M, Cani PD. Gut microbiome and health: mechanistic insights. Gut. 2022;71(5):1020–32. Ni J, Wu GD, Albenberg L, Tomov VT. Gut microbiota and IBD: causation or correlation? Nat Reviews Gastroenterol Hepatol. 2017;14(10):573–84. Heeney DD, Gareau MG, Marco ML. Intestinal Lactobacillus in health and disease, a driver or just along for the ride? Curr Opin Biotechnol. 2018;49:140–7. Lozupone CA, Knight R. Species divergence and the measurement of microbial diversity. FEMS Microbiol Rev. 2008;32(4):557–78. Proctor LM, Creasy HH, Fettweis JM, Lloyd-Price J, Mahurkar A, Zhou W, et al. Integr Hum Microbiome Project Nat. 2019;569(7758):641–8. Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol. 2011;11(11):723–37. Corcoran BM, Stanton C, Fitzgerald GF, Ross RP. Survival of Probiotic Lactobacilli in Acidic Environments Is Enhanced in the Presence of Metabolizable Sugars. Appl Environ Microbiol. 2005;71(6):3060–7. Kechagia M, Basoulis D, Konstantopoulou S, Dimitriadi D, Gyftopoulou K, Skarmoutsou N, et al. Health Benefits Probiotics: Rev ISRN Nutr. 2013;2013:1–7. Platt AM, Randolph GJ. Dendritic Cell Migration Through the Lymphatic Vasculature to Lymph Nodes. Development and Function of Myeloid Subsets. Advances in Immunology2013. pp. 51–68. Tamanai-Shacoori Z, Smida I, Bousarghin L, Loreal O, Meuric V, Fong SB, et al. Roseburia spp.: a marker of health? Future Microbiol. 2017;12:157–70. Iljazovic A, Roy U, Gálvez EJC, Lesker TR, Zhao B, Gronow A, et al. Perturbation of the gut microbiome by Prevotella spp. enhances host susceptibility to mucosal inflammation. Mucosal Immunol. 2021;14(1):113–24. Lichtenstein GR, Loftus EV, Isaacs KL, Regueiro MD, Gerson LB, Sands BE. ACG Clinical Guideline: Management of Crohn's Disease in Adults. Am J Gastroenterol. 2018;113(4):481–517. Peyrin-Biroulet L, Loftus EV, Colombel J-F, Sandborn WJ. The Natural History of Adult Crohn's Disease in Population-Based Cohorts. Am J Gastroenterol. 2010;105(2):289–97. Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol. 2008;8(12):958–69. Lu Y, Li X, Liu S, Zhang Y, Zhang D. Toll-like Receptors and Inflammatory Bowel Disease. Front Immunol. 2018;9. Peng L, Li Z-R, Green RS, Holzmanr IR, Lin J. Butyrate Enhances the Intestinal Barrier by Facilitating Tight Junction Assembly via Activation of AMP-Activated Protein Kinase in Caco-2 Cell Monolayers. J Nutr. 2009;139(9):1619–25. Derwa Y, Gracie DJ, Hamlin PJ, Ford AC. Systematic review with meta-analysis: the efficacy of probiotics in inflammatory bowel disease. Aliment Pharmacol Ther. 2017;46(4):389–400. Zmora N, Zilberman-Schapira G, Suez J, Mor U, Dori-Bachash M, Bashiardes S, et al. Personalized Gut Mucosal Colonization Resistance to Empiric Probiotics Is Associated with Unique Host and Microbiome Features. Cell. 2018;174(6):1388–e40521. Ford AC, Achkar J-P, Khan KJ, Kane SV, Talley NJ, Marshall JK, et al. Efficacy of 5-Aminosalicylates in Ulcerative Colitis: Systematic Review and Meta-Analysis. Am J Gastroenterol. 2011;106(4):601–16. Singh BN. Modified-release solid formulations for colonic delivery. Recent Pat Drug Deliv Formul. 2007;1(1):53–63. Arévalo-Pérez R, Maderuelo C, Lanao JM. Recent advances in colon drug delivery systems. J Controlled Release. 2020;327:703–24. Maroni A, Zema L, Del Curto MD, Foppoli A, Gazzaniga A. Oral colon delivery of insulin with the aid of functional adjuvants. Adv Drug Deliv Rev. 2012;64(6):540–56. Didari T, Solki S, Mozaffari S, Nikfar S, Abdollahi M. A systematic review of the safety of probiotics. Exp Opin Drug Saf. 2014;13(2):227–39. Chapman CMC, Gibson GR, Rowland I. Health benefits of probiotics: are mixtures more effective than single strains? Eur J Nutr. 2011;50(1):1–17. Xiao Y, Lu C, Liu Y, Kong L, Bai H, Mu H, et al. Encapsulation of Lactobacillus rhamnosus in Hyaluronic Acid-Based Hydrogel for Pathogen-Targeted Delivery to Ameliorate Enteritis. ACS Appl Mater Interfaces. 2020;12(33):36967–77. Plaza-Díaz J, Ruiz-Ojeda F, Vilchez-Padial L, Gil A. Evidence of the Anti-Inflammatory Effects of Probiotics and Synbiotics in Intestinal Chronic Diseases. Nutrients. 2017;9(6). de Vries MC, Vaughan EE, Kleerebezem M, de Vos WM. Lactobacillus plantarum—survival, functional and potential probiotic properties in the human intestinal tract. Int Dairy J. 2006;16(9):1018–28. Mehta RS, Mayers JR, Zhang Y, Bhosle A, Glasser NR, Nguyen LH, et al. Gut microbial metabolism of 5-ASA diminishes its clinical efficacy in inflammatory bowel disease. Nat Med. 2023;29(3):700–9. Heidebach T, Först P, Kulozik U. Influence of casein-based microencapsulation on freeze-drying and storage of probiotic cells. J Food Eng. 2010;98(3):309–16. Picot A, Lacroix C. Encapsulation of bifidobacteria in whey protein-based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt. Int Dairy J. 2004;14(6):505–15. Gbassi GK, Vandamme T. Probiotic Encapsulation Technology: From Microencapsulation to Release into the Gut. Pharmaceutics. 2012;4(1):149–63. Cook MT, Tzortzis G, Charalampopoulos D, Khutoryanskiy VV. Microencapsulation of probiotics for gastrointestinal delivery. J Controlled Release. 2012;162(1):56–67. Sartor RB, Mazmanian SK. Intestinal Microbes in Inflammatory Bowel Diseases. Am J Gastroenterol Supplements. 2012;1(1):15–21. Markowiak P, Śliżewska K. Effects of Probiotics, Prebiotics, and Synbiotics on Human Health. Nutrients. 2017;9(9). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 21 Apr, 2026 Read the published version in BMC Biotechnology → Version 1 posted Editorial decision: Revision requested 18 Oct, 2025 Reviews received at journal 12 Oct, 2025 Reviews received at journal 10 Oct, 2025 Reviewers agreed at journal 02 Oct, 2025 Reviewers agreed at journal 02 Oct, 2025 Reviewers invited by journal 08 Sep, 2025 Editor assigned by journal 02 Sep, 2025 Submission checks completed at journal 02 Sep, 2025 First submitted to journal 02 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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(SIF).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7469110/v1/99e61b39b07f3563669c4d5b.png"},{"id":91401553,"identity":"87f31344-eaf5-4a50-8723-d93176414cf3","added_by":"auto","created_at":"2025-09-16 07:07:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":894031,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy (SEM) analysis of sustained-release granules.(A) Macroscopic view of HPMCP-\u003cem\u003eL. plantarum\u003c/em\u003e granules;(B–D) SEM images of HPMCP granules at 1000× (B), 5000× (C), and 30,000× (D) magnifications;(E) Macroscopic view of L100-\u003cem\u003eL. plantarum\u003c/em\u003e granules;(F–H) SEM images of L100 granules at 1000× (F), 5000× (G), and 30,000× (H) magnifications.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7469110/v1/563eff612b62a7d31424d0d8.png"},{"id":91401151,"identity":"7d5a01ab-2392-4759-b300-b68bc78f529a","added_by":"auto","created_at":"2025-09-16 06:59:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1337926,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence staining results of different treatment groups in mice after therapy completion. (A)HE staining of colon sections across treatment groups post-treatment; (B)Representative immunofluorescence images of M1⁺cells (green, iNOS⁺) and F4/80⁺macrophages (red) in colon tissue; (C) Representative immunofluorescence images of M2⁺ cells (green, CD206⁺) and F4/80⁺ macrophages (red) in colon tissue.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7469110/v1/3815d476f90873b6095f6571.png"},{"id":91401552,"identity":"ac43aab1-4453-4f9e-8913-31de0e36a23a","added_by":"auto","created_at":"2025-09-16 07:07:59","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":904939,"visible":true,"origin":"","legend":"\u003cp\u003eMetagenomic sequencing of gut microbiota in different treatment groups of mice.(A) Observed species index (Sobs) at the operational taxonomic unit (OTU) level across treatments;(B)α-Diversity analysis displayed by Shannon curves at OTU level;(C) Principal coordinate analysis (PCoA);(D) Box plots of PCoA distances between treatment groups;(E) Venn diagram of common and unique bacterial species among groups;(F) Microbial composition at phylum level;(G) Heatmap of relative abundance of microbiome at genus level.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7469110/v1/9d2990d2fb074b24d58a0acf.jpeg"},{"id":107928123,"identity":"b098b042-85ec-4438-9eac-ca389acfd294","added_by":"auto","created_at":"2026-04-27 16:08:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3932960,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7469110/v1/c9704218-2ec4-47a2-8e0c-31230a36d487.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sustained-Release Enteric Formulations of Lactobacillus plantarum Based on Granulation Technology: Preparation and Therapeutic Evaluation in Acute Colitis","fulltext":[{"header":"1. Background","content":"\u003cp\u003eProbiotics, particularly \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, have demonstrated therapeutic potential in regulating gut microbiota and promoting intestinal health[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, a major challenge in probiotic-based therapy for intestinal diseases is ensuring the survival and targeted delivery of orally administered probiotics. These beneficial bacteria are exposed to harsh gastrointestinal conditions, including gastric acid, bile salts, and digestive enzymes, which drastically reduce their viability and limit clinical efficacy[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Conventional formulations such as fast-release powders or capsules provide insufficient protection during gastrointestinal transit and lack targeted release mechanisms, thereby failing to ensure an adequate number of viable bacteria reach the colon[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn recent years, multi-unit pellet systems have emerged as a promising strategy for oral drug delivery[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Composed of numerous small granules, these systems disperse uniformly throughout the gastrointestinal tract, reducing local irritation and minimizing the risk of dose dumping. Compared to conventional monolithic dosage forms, multi-unit systems offer the advantage of tailored release profiles, including sustained or delayed release, and can be engineered for site-specific delivery based on pH or transit time. For probiotic formulations, enteric polymer coatings have been employed to protect bacteria under acidic conditions while enabling their release in the intestine[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], thereby enhancing intestinal colonization and activity[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite these advancements, current probiotic delivery technologies exhibit limitations in optimizing viability and targeted release. Rapid-release formulations may prematurely discharge probiotics in the stomach, leading to substantial bacterial loss, while standard capsule-based systems cannot achieve the uniform dispersion and precise release control afforded by multi-unit approaches.\u003c/p\u003e\u003cp\u003eThis study proposes a novel sustained-release pelletization strategy for the intestinally targeted delivery of the probiotic Lactobacillus plantarum. The formulation employs a sucrose-based filler phase cross-linked with pH-responsive polymers to construct a release matrix capable of withstanding gastric conditions while enabling controlled release of viable bacteria in the intestinal environment. Through systematic material screening, in vitro release profiling, and in vivo evaluation using a murine model of acute colitis, the study aims to elucidate the release mechanism and therapeutic potential of the proposed delivery system. By enhancing site-specific efficacy, this strategy could reduce the per-dose probiotic requirement while maintaining or improving therapeutic outcomes, thereby alleviating patient treatment burden. Furthermore, this work provides valuable insights into the design and application of next-generation oral probiotic delivery systems and offers a viable pathway for addressing current limitations in probiotic therapy. Importantly, this technological approach offers a promising platform not only for single-strain applications but also for delivering multi-strain or composite probiotic formulations.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Experimental Materials\u003c/h2\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e (viable count\u0026thinsp;\u0026ge;\u0026thinsp;1 \u0026times; 10^10 CFU/g); Sodium alginate (SA), cellulose acetate phthalate (CAP) (Chemicell); hydroxypropyl methylcellulose succinate (HPMCAS), hydroxypropyl methylcellulose phthalate (HPMCP) (Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.); Eudragit L100 and S100 (Shanghai Dexiang Pharmaceutical Technology Co., Ltd.).Dextran sulfate sodium salt (DSS) (Yisheng Biotechnology, Shanghai, China).Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) (Dongguan Chuangfeng Automation Technology Co., Ltd.).Male BALB/c mice (6\u0026ndash;8 weeks old, 25 g) were purchased from Zhengzhou University Experimental Animal Center. Electrically heated constant-temperature drying oven (Shanghai Shuli Instrument Co., Ltd.); autoclave sterilizer (Zhejiang Xinfeng Medical Devices Co., Ltd.); carbon dioxide incubator with temperature-controlled shaker; ultrapure water system (Ningbo Dansboton Environmental Technology Co., Ltd.).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Screening of Antibacterial Properties of Coating Materials\u003c/h2\u003e\u003cp\u003eThe antibacterial activity of different coating materials was assessed using the inhibition zone method to identify suitable excipients for probiotic encapsulation. Thirty microliters of \u003cem\u003eL. plantarum\u003c/em\u003e suspension (adjusted to ~\u0026thinsp;1 \u0026times; 10^8 CFU/mL after MRS liquid culture) were evenly spread onto MRS agar plates. Circular sterile filter papers (6 mm diameter) were soaked in 2% (w/v) solutions of SA, HPMCP, CAP, HPMCAS, L100, and S100, then placed onto the plates. Plates were incubated inverted at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C for 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2 hours. Filter papers soaked in antibiotic solution and sterile water served as positive and negative controls, respectively. The diameter of inhibition zones was measured; smaller zones indicated lower antibacterial activity. All experiments were performed in triplicate, and average values were recorded.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Preparation and Optimization of Sustained-Release Granules\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 Granule Preparation and Process Optimization\u003c/h2\u003e\u003cp\u003eTo avoid thermal inactivation of the probiotic during granule drying, low-temperature drying (35\u0026ndash;40\u0026deg;C) was employed, while other steps (crushing, sieving, mixing, extrusion) followed conventional protocols. For HPMCP-based granules, 1% (w/v) HPMCP solution (dissolved in a 1:1 methanol-acetone mixture) was gradually added to a mortar containing freeze-dried \u003cem\u003eL. plantarum\u003c/em\u003e powder premixed with sucrose (1:1). The mixture was ground until a non-sticky, coarse, granular material formed. The mixture was sieved sequentially through 40-mesh and then 80-mesh sieves. Particles retained on the 80-mesh sieve (particle size range: 180\u0026ndash;425 \u0026micro;m) were and dried at 37\u0026ndash;40\u0026deg;C for 22\u0026ndash;26 hours.\u003c/p\u003e\u003cp\u003eGranules were similarly prepared using SA, HPMCP, CAP, HPMCAS, L100, and S100 at concentrations of 0.5%, 1%, and 2% (w/v). Solvents were sterile water for SA, methanol-acetone for CAP and HPMCAS, and 75% ethanol for L100 and S100.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 Dissolution Testing\u003c/h2\u003e\u003cp\u003eFor in vitro dissolution studies, 100 mL of SGF and 100 mL of SIF were placed in a 40\u0026deg;C shaking incubator (150 rpm). After preheating to 37\u0026deg;C, 0.5 g of each granule formulation was added. Dissolution behavior was observed every 15 minutes for 4 hours.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3 Scanning Electron Microscopy (SEM)\u003c/h2\u003e\u003cp\u003eSEM analysis was conducted on granules formulated with HPMCP and L100, selected based on their favorable dissolution and non-antibacterial properties. Samples were analyzed by a third-party laboratory to investigate microstructural characteristics.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Induction of Acute Colitis and Treatment Protocol\u003c/h2\u003e\u003cp\u003eAfter a 3-day acclimation period, mice were randomly assigned into five groups: normal group (n\u0026thinsp;=\u0026thinsp;6), DSS model group (n\u0026thinsp;=\u0026thinsp;6), probiotic powder group (n\u0026thinsp;=\u0026thinsp;6), HPMCP granule group (n\u0026thinsp;=\u0026thinsp;6), and L100 granule group (n\u0026thinsp;=\u0026thinsp;6). During the modeling phase, all groups except the normal group received 3% (w/v) DSS in drinking water ad libitum for 7 days, with daily replacement of freshly prepared solutions to induce acute colitis mimicking human pathophysiology(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eStarting on Day 8, mice were treated via oral gavage every other day for a total of three doses (Days 8, 10, and 12). The administration dose was 100 mg per mouse (suspension in sterile water at 0.2 mL/10 g body weight). The HPMCP and L100 groups received suspensions of respective granules; the powder group received equivalent doses of free \u003cem\u003eL. plantarum\u003c/em\u003e powder; the DSS and normal groups received sterile water. All gavage procedures were conducted under sterile conditions.\u003c/p\u003e\u003cp\u003eOn day 14, experimental mice were intraperitoneally administered a 1% (10 mg/mL) sodium pentobarbital solution at a dose of 100 mg/kg to induce a stable deep anesthesia state. After confirming the loss of consciousness, cervical dislocation was performed to ensure euthanasia, followed by dissection to collect colon tissues and intestinal contents. Colonic tissues were rinsed with PBS and fixed in 4% paraformaldehyde for subsequent histopathological and immunofluorescence analyses. Intestinal contents were immediately stored at \u0026minus;\u0026thinsp;80\u0026deg;C after collection for metagenomic analysis of the gut microbiota.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExperimental Design for Colitis Induction and Treatment\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStage\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIntervention\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eModeling Phase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNormal group(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFree access to drinking water (pure water)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColitis model group(n\u0026thinsp;=\u0026thinsp;24)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFree access to 3% DSS solution in drinking water\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eTreatment Phase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNormal group(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSterile water\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDSS group(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSterile water\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e group(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e powder\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHPMCP group(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eHPMCP-L. plantarum\u003c/em\u003e sustained-release granules\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL100 group(n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eL100-L. plantarum\u003c/em\u003e sustained-release granules\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Therapeutic Assessment in DSS-Induced Colitis Model\u003c/h2\u003e\u003cp\u003eThe colon samples collected from dissection on day 14 (as described in section \u003cspan refid=\"Sec9\" class=\"InternalRef\"\u003e2.4\u003c/span\u003e) were washed with PBS and fixed in 4% paraformaldehyde for hematoxylin and eosin (HE) staining to evaluate histopathological changes. The intestinal contents were stored at -80\u0026deg;C for subsequent microbiota analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Immunofluorescence Analysis of Inflammation Resolution\u003c/h2\u003e\u003cp\u003eTo evaluate anti-inflammatory effects of the sustained-release granules, immunofluorescence staining was performed. Tissue sections were incubated overnight at 4\u0026deg;C with primary antibodies targeting CD86, CD206, and F4/80, followed by 1-hour incubation at 37\u0026deg;C with appropriate secondary antibodies. Fluorescence microscopy was used for imaging and analysis of macrophage polarization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Gut Microbiota Diversity Analysis\u003c/h2\u003e\u003cp\u003eMetagenomic analysis of microbial communities was conducted on intestinal content samples. DNA extraction and sequencing were performed by Haimujie Biomedical Technology Co., Ltd. Quality control and OTU clustering (97% similarity threshold) were performed using QIIME2. Alpha diversity (Sobs, Shannon indices), beta diversity (PCoA), and taxonomic composition at phylum and genus levels were analyzed.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Release Profiles in Simulated Gastrointestinal Fluids\u003c/h2\u003e\u003cp\u003eInhibition zone assays revealed that all tested coating materials exhibited negligible antibacterial activity against \u003cem\u003eL. plantarum\u003c/em\u003e, indicating their suitability as probiotic carriers. Particles were prepared from these materials for subsequent solubility analysis.\u003c/p\u003e\u003cp\u003eBoth HPMCP and L100 exhibited prolonged dissolution times and relatively low cumulative release in simulated gastric fluid (SGF), indicating effective protection of the encapsulated probiotics under acidic conditions. At concentrations of 1% and 2%, HPMCP and L100 exhibited significantly lower release rates compared to CAP, HPMCAS, and S100, and performed better than sodium alginate (SA), indicating superior protective effects in simulated gastric fluid and effective delay in premature release. These findings demonstrate the favorable enteric properties of HPMCP and L100, which contribute to minimizing probiotic loss during gastric transit. Additionally, the disintegration times of HPMCP and L100 at 1% and 2% concentrations ranged from 60 to 100 minutes in simulated intestinal fluid\u0026mdash;moderate compared to S100, which dissolved too rapidly, and CAP, which exhibited prolonged disintegration times nearing 180 minutes at 2%. This suggests that HPMCP and L100 provide a well-balanced release profile in the intestinal environment, achieving sustained but timely probiotic release without excessive delay. In contrast, under simulated intestinal fluid (SIF) conditions, the dissolution times were significantly shortened, and the release amounts reached complete release thresholds. By comparing the cumulative release in SGF over 4 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and the time required for complete dissolution in SIF (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), it is evident that \u003cem\u003eL. plantarum\u003c/em\u003e sustained-release granules formulated with HPMCP and L100 successfully prevented premature release in the gastric phase while enabling rapid disintegration and full release in the intestinal phase.\u003c/p\u003e\u003cp\u003eThis dual-phase release behavior provides a crucial advantage for enhancing probiotic survival through the gastrointestinal tract, thereby significantly improving the bioavailability of the formulation. Based on these findings, HPMCP and Eudragit L100 were identified as optimal enteric coating materials for sustained-release probiotic delivery systems.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Morphological Characterization of Granules\u003c/h2\u003e\u003cp\u003eThe HPMCP-\u003cem\u003eLactobacillus plantarum\u003c/em\u003e sustained-release granules (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and L100-\u003cem\u003eL. plantarum\u003c/em\u003e sustained-release granules (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) appeared as uniformly spherical particles at the macroscopic level. SEM observations revealed that at 1000\u0026times; magnification, the granule surfaces appeared slightly rough. At higher magnifications (5000\u0026times; and 30,000\u0026times;), the granules were composed of fine subunits with uniformly distributed sucrose as filler material. These subunits were interconnected, forming a cross-linked porous matrix structure characteristic of sustained-release formulations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Therapeutic Efficacy of Granules\u003c/h2\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.4.1 Resolution of Inflammation\u003c/h2\u003e\u003cp\u003eDextran sulfate sodium salt (DSS) administration induces clinical manifestations in mice that closely resemble ulcerative colitis (UC), including body weight loss, diarrhea, and hematochezia[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In this study, a DSS-induced colitis model was employed to evaluate the therapeutic efficacy of the orally administered sustained-release pellet formulation. Compared to the normal group, DSS-treated mice showed significant weight loss and histological signs of colitis, including epithelial damage, crypt loss, and inflammatory infiltration, confirming successful model induction.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, compared with conventional \u003cem\u003eLactobacillus plantarum\u003c/em\u003e powder administration, both the HPMCP and L100 granule-treated groups exhibited varying degrees of epithelial structure restoration. In particular, administration of HPMCP sustained-release granules significantly reduced the level of inflammatory cell infiltration and revealed histological structures nearly indistinguishable from those of the normal control group. Compared with the DSS group and the bacterial powder group, the granule-treated groups showed markedly alleviated inflammatory cell infiltration, mucosal damage, and crypt destruction in the colonic tissue. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, standard markers including iNOS\u003csup\u003e+\u003c/sup\u003e, CD206, and F4/80 were used to identify M1 and M2 macrophages. DSS-induced colitis mice exhibited increased macrophage infiltration in the colon, with both M1 and M2 macrophage populations elevated compared to the control group. Oral administration of HPMCP and L100 sustained-release granules led to a reduction in M1 macrophages and a concomitant increase in M2 macrophages. These findings indicate that both HPMCP and L100 granules modulate the M1/M2 macrophage polarization balance, particularly promoting M2 polarization. This suggests that enhancing M2 polarization through \u003cem\u003eL. plantarum\u003c/em\u003e sustained-release granules may represent a potential therapeutic strategy for inflammatory bowel disease (IBD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.4.2 Gut Microbiota Diversity and Composition\u003c/h2\u003e\u003cp\u003eA growing body of evidence indicates that the gut microbiota plays a direct role in the pathogenesis of inflammatory bowel disease (IBD)[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Distinct microbial communities within the colon form an interactive and balanced ecosystem, which becomes disrupted in the context of IBD and other colonic disorders[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, this study investigated whether \u003cem\u003eLactobacillus plantarum\u003c/em\u003e sustained-release pellets could restore gut microbial homeostasis in a murine model.\u003c/p\u003e\u003cp\u003eAlpha diversity is an ecological metric used to assess the richness and evenness of taxa within individual samples[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Greater microbial species richness in an ecosystem is generally reflected by higher alpha diversity indices. As shown by the Sobs index results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), alpha diversity was significantly reduced in the probiotic powder group compared to the normal control group. This decline may be attributed to DSS-induced inflammation, which promotes the overgrowth of harmful intestinal bacteria while suppressing the proliferation of commensal microbes, ultimately leading to reduced microbial abundance. In contrast, oral administration of HPMCP- and L100-based sustained-release pellets markedly increased Sobs index values, with statistically significant differences compared to the DSS group. Notably, these results suggest that treatment with the sustained-release formulations effectively restored microbial species diversity toward levels observed in healthy controls.\u003c/p\u003e\u003cp\u003ePrincipal coordinate analysis (PCoA) showed significant differences in similarity between the control group and the DSS-induced model group. PCoA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) revealed distinct clustering of treatment groups. Microbiota profiles in the HPMCP and L100 groups were more similar to the normal group than to the DSS or powder groups, indicating restorative effects.\u003c/p\u003e\u003cp\u003eTo elucidate the compositional structure of the gut microbiota, microbial differences were analyzed at both the phylum and genus levels[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. At the phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), six dominant bacterial phyla were identified, among which Firmicutes and Bacteroidetes represented the two most abundant taxa, collectively accounting for over 90% of the total microbiota and dominating across all samples. Compared to the normal control group, the probiotic powder group exhibited an increased relative abundance of Firmicutes and a decreased abundance of Bacteroidetes. The Firmicutes-to-Bacteroidetes (F/B) ratio is widely recognized as a key indicator of microbial dysbiosis in the gut. In contrast, treatment with HPMCP- and L100-based sustained-release pellets resulted in a decreased abundance of Firmicutes and an increased abundance of Bacteroidetes relative to the powder group, indicating a regulatory effect on gut microbiota composition. Notably, the L100 group demonstrated the most pronounced modulation of the Firmicutes and Bacteroidetes populations in colitis-induced mice, with microbial profiles approaching those observed in the healthy control group.\u003c/p\u003e\u003cp\u003eAt the genus level, microbial community composition was further characterized using a heatmap analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). In the normal group, genera such as Bacteroides, Alistipes, and Odoribacter were present at relatively high abundances. In contrast, the probiotic powder group exhibited a marked increase in potentially pathogenic genera including Escherichia-Shigella, Enterococcus, and Staphylococcus, consistent with findings shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, suggesting a detrimental impact on intestinal health. In the HPMCP-treated group, beneficial genera such as Candidatus_Arthromitus and Turicibacter emerged as dominant taxa with increased abundance. In the L100 group, there was a significant enrichment of Roseburia, a known short-chain fatty acid-producing genus, along with A2 and unclassified taxa within the Prevotellaceae family. These observations indicate that treatment with HPMCP- and L100-based sustained-release pellets modulated the gut microbiota by selectively enriching beneficial bacterial populations, thereby contributing to improved microbial community function. Collectively, these findings suggest that the oral administration of \u003cem\u003eL. plantarum\u003c/em\u003e sustained-release pellets promotes microbial homeostasis and supports therapeutic efficacy in the context of inflammatory bowel disease.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, a novel sustained-release and enteric-coated delivery system for \u003cem\u003eLactobacillus plantarum\u003c/em\u003e was successfully developed based on a cross-linked porous matrix constructed using pH-responsive polymers (HPMCP and Eudragit L100). This system incorporates a dual-protection mechanism of acid-resistant enteric coating and controlled-release internal matrix, which significantly improves the survival rate and therapeutic efficacy of the probiotic in a dextran sulfate sodium (DSS)-induced acute colitis mouse model, and demonstrates superior performance compared to traditional oral probiotic powders.\u003c/p\u003e\u003cp\u003eClinically, oral administration of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e powder has been demonstrated to exert beneficial effects on ameliorating colitis symptoms[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, its efficacy is constrained by significant degradation in gastric acid and bile salts, leading to reduced viable bacteria reaching the colon and suboptimal colonization[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Consequently, achieving the desired therapeutic outcomes often necessitates prolonged, frequent, and high-dose administration regimens (e.g., multiple times daily)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This not only increases the patient compliance burden and treatment costs but also carries the potential for discomfort due to excessive intake. In contrast, the enteric-coated sustained-release granule strategy proposed in this study utilizes the \"smart\" protection afforded by HPMCP (hydroxypropyl methylcellulose phthalate) and L100 enteric coatings. This effectively shields the probiotics from gastric acid erosion, as confirmed by minimal release in simulated gastric fluid during in vitro dissolution testing, thereby ensuring the safe passage of the majority of viable bacteria through the stomach. Upon entry into the near-neutral or weakly alkaline intestinal environment (pH\u0026thinsp;\u0026gt;\u0026thinsp;5.5-6.0), the coating rapidly dissolves. Scanning electron microscopy (SEM) analysis further revealed that the granules possess an internal sucrose-filled cross-linked porous matrix, facilitating sustained release rather than burst release. This combined mechanism of gastric protection and colon targeting plays a crucial role in ensuring the overall therapeutic efficacy of the formulation.\u003c/p\u003e\u003cp\u003eCompared with conventional probiotic powders, the proposed delivery system combines a unique physical architecture (cross-linked porous matrix) with a chemical barrier (pH-responsive polymer coating), thereby establishing an efficient platform for dual protection and controlled release. Notably, under equivalent probiotic dosing, the HPMCP- and L100-based sustained-release granules developed in this study demonstrated significantly superior therapeutic efficacy against DSS-induced acute colitis compared to direct oral administration of freeze-dried \u003cem\u003eLactobacillus plantarum\u003c/em\u003e powder. This therapeutic advantage is not solely attributed to the probiotic itself, but more importantly to the optimized gastrointestinal delivery and targeted release strategy afforded by the granule system. Such a strategy effectively reduces dosing frequency and total dosage, enhances patient compliance, and achieves equal or even improved therapeutic outcomes. Therefore, this study confirms from multiple aspects that the delivery system is more effective and demonstrates superior performance.\u003c/p\u003e\u003cp\u003eHistological evaluation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) showed that both the HPMCP and L100 granule-treated groups exhibited varying degrees of epithelial structure restoration. Notably, administration of HPMCP sustained-release granules significantly reduced inflammatory cell infiltration and presented tissue structures nearly indistinguishable from those of the normal control group. Compared with the DSS group and the probiotic powder group, the granule-treated groups showed markedly reduced inflammatory cell infiltration, mucosal damage, and crypt destruction in colonic tissue, with the HPMCP group closely resembling normal tissue. H\u0026amp;E staining highlights that HPMCP and L100 sustained-release granules, compared to conventional probiotic powder, effectively restore the epithelial structure of colonic tissue in DSS-induced colitis mice, significantly alleviate inflammation and tissue damage, and exhibit superior therapeutic effects. Meanwhile, immunofluorescence analysis further supported the improvement in inflammation.\u003c/p\u003e\u003cp\u003eImmunofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C) demonstrated that granules significantly reduced pro-inflammatory M1 macrophage (iNOS⁺/F4/80⁺) infiltration and enhanced anti-inflammatory M2 (CD206⁺/F4/80⁺) polarization, effects substantially weaker with powder. This suggests granules more effectively activate host anti-inflammatory pathways via enhanced viable probiotic delivery and sustained action[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Both H\u0026amp;E staining and immunofluorescence indicate the improvement of inflammation in colitis; moreover, the improvement of the intestinal microbiota is also an important indicator.\u003c/p\u003e\u003cp\u003eWe conducted high-throughput transcriptome sequencing to investigate changes in the intestinal microbiota. Metagenomic sequencing highlighted another key advantage: while powder minimally improved DSS-induced reductions in alpha diversity (Sobs, Shannon indices; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B), both granule types restored richness/diversity towards normal levels. PCoA analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D) confirmed granule-treated microbiota (especially L100) clustered closer to healthy controls than DSS or powder groups, indicating superior restoration of ecological health. Granules also more effectively normalized the dysbiotic Bacillota /Bacteroidetes ratio elevated by powder (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). At the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), granule groups uniquely enriched beneficial taxa (e.g., Candidatus_Arthromitus, Turicibacter with HPMCP; Roseburia, Prevotellaceae with L100), linked to anti-inflammation and barrier function[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], contrasting with higher potential pathogens (Escherichia-Shigella, Enterococcus) in powder recipients. Collectively, the sustained-release strategy, by ensuring viable probiotic delivery and persistence, profoundly reshapes the gut microbiota towards a healthier, anti-inflammatory state, underpinning its enhanced efficacy.\u003c/p\u003e\u003cp\u003eCrucially, at the same dosage, our delivery platform outperformed traditional powder formulations in almost all indicators, including histopathology, immune regulation, and microbiota normalization, indicating that the delivery technology enhances probiotic efficacy more effectively than simply increasing the dose. Interestingly, the therapeutic effect of this delivery system may also have considerable advantages compared to current chemical treatments for colitis.\u003c/p\u003e\u003cp\u003eCurrent pharmacological treatments for intestinal inflammation, particularly inflammatory bowel disease (IBD), primarily include 5-aminosalicylic acid (5-ASA) derivatives, corticosteroids, immunosuppressants (e.g., azathioprine, methotrexate), and biologics such as anti-TNF-α monoclonal antibodies[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Although these agents can be effective, they are often associated with serious adverse effects, including increased risks of infection, metabolic disturbances, bone marrow suppression, and long-term malignancy[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In contrast, probiotics are live microorganisms that are generally regarded as safe, with side effects such as bloating or mild gastrointestinal discomfort usually being temporary and self-limiting. Beyond direct anti-inflammatory activity, the sustained-release granule system presented in this study offers a multi-targeted therapeutic approach. It not only delivers viable \u003cem\u003eLactobacillus plantarum\u003c/em\u003e to the intestine but also facilitates the restoration of microbial homeostasis, the reinforcement of mucosal barrier integrity, and the modulation of the immune response, specifically by enhancing M2 macrophage polarization[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Such mechanisms address the underlying pathophysiology of IBD rather than merely suppressing inflammation. For instance, 5-ASA acts mainly as a local anti-inflammatory agent and exhibits limited ability to regulate microbial dysbiosis. In this context, the superior epithelial repair observed in the granule-treated groups (as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) underscores the microbiota-mediated benefits of \u003cem\u003eL. plantarum\u003c/em\u003e and its metabolites, such as short-chain fatty acids, in promoting epithelial regeneration and mucosal healing[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These findings highlight the therapeutic potential of probiotic-based sustained-release systems as safer, more holistic alternatives or adjuncts to conventional pharmacological interventions.\u003c/p\u003e\u003cp\u003eProbiotic immunomodulation and microbiota restoration require time, potentially limiting their speed in controlling acute, severe inflammation compared to potent anti-inflammatories like corticosteroids or biologics. Consequently, probiotics alone are often insufficient for inducing remission in severe active IBD, which typically requires conventional agents[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Probiotic efficacy may also exhibit greater individual variation due to factors like baseline microbiota or genetics[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Nevertheless, in our DSS-induced acute colitis model (mimicking UC), the sustained-release granule system (particularly HPMCP granules) achieved near-normal levels of histological repair and microbiota restoration, alongside significant anti-inflammatory effects (e.g., macrophage polarization). The degree of histological repair and anti-inflammatory effects observed with the sustained-release granules, particularly HPMCP granules, in this acute DSS-colitis model appear comparable to the reported efficacy of first-line therapies like 5-ASA in managing mild-to-moderate ulcerative colitis[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], based on established clinical and preclinical knowledge. While 5-ASA remains a cornerstone for inducing and maintaining remission in this patient group, our granules demonstrated comparable efficacy in the model, coupled with a superior safety profile and the added benefit of microbiota modulation.\u003c/p\u003e\u003cp\u003eThe superior therapeutic performance of our system stems from its innovative scaffold‑based architecture, which affords excellent protection and precise release of probiotics. In dissolution data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) confirm that HPMCP and L100 coatings confer excellent acid resistance, minimizing probiotic loss in simulated gastric fluid, while the cross‑linked porous matrix (SEM, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) enables controlled, sustained release in the intestinal milieu[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] Unlike traditional enteric-coated tablets or capsules where failure of a single unit results in exposure of the entire payload to gastric acid, our multi-unit granules compartmentalize the risk so that damage to individual particles causes only minor probiotic loss while the intact granules continue to deliver viable cells[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Moreover, embedding probiotics and sucrose fillers uniformly within a micro‑/nano‑scale polymeric network creates local protective microenvironments and controlled‑release pathways that simple powder blending cannot achieve. Compared with microencapsulation techniques, which often require high temperature, organic solvents, or shear stress that jeopardize cell viability, our Wet granulation and low‑temperature extrusion processes employ well‑established pharmaceutical excipients (HPMCP, L100, sucrose) and are readily scalable under GMP conditions. These features collectively position the scaffold‑based granule system as a robust, manufacturable platform for advanced probiotic delivery.\u003c/p\u003e\u003cp\u003eVarious probiotic strains including Bifidobacterium species and other \u003cem\u003eLactobacillus\u003c/em\u003e species such as \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e, as well as multi-strain formulations and synbiotics which are combinations of probiotics and prebiotics, have been widely investigated for the treatment of intestinal inflammation[\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Certain combinations have demonstrated potential synergistic effects[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. While \u003cem\u003eL. plantarum\u003c/em\u003e, the strain selected in this study, possesses intrinsic acid and bile resistance, adhesive capacity, and immunomodulatory properties[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], the principal contribution of this work lies not in contesting the efficacy of other strains or combinations, but in presenting a broadly applicable and efficient delivery platform. The enteric sustained-release granule system developed herein, based on a cross-linked skeleton structure and low-temperature extrusion-coating process, offers excellent compatibility and is theoretically adaptable for the delivery of other individual strains, multi-strain consortia, or even synbiotic preparations[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Future studies could explore optimized probiotic combinations such as \u003cem\u003eLactobacillus plantarum\u003c/em\u003e co-administered with selected Bifidobacterium species using this platform to further enhance therapeutic outcomes and potentially surpass the efficacy of some existing commercial formulations. Crucially, the system ensures both the stability of each strain during processing and their coordinated release within the gastrointestinal tract.\u003c/p\u003e\u003cp\u003eMaintaining a high activity of probiotics during processing cannot be ignored either. Our granule fabrication employed meticulously maintained low temperatures (35\u0026ndash;40\u0026deg;C during wet granulation and drying), minimizing heat-induced inactivation. This contrasts sharply with processes like spray drying microencapsulation, where high inlet temperatures (150\u0026ndash;200\u0026deg;C) often cause significant viability loss despite lower outlet temperatures[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Furthermore, we proactively addressed potential excipient toxicity: all candidate coating materials (SA, HPMCP, CAP, HPMCAS, L100, S100) were pre-screened for antimicrobial activity against \u003cem\u003eL. plantarum\u003c/em\u003e using an agar diffusion assay. No significant inhibition was observed at relevant concentrations, confirming the biocompatibility of the selected polymers (HPMCP, L100). Solvents (methanol-acetone for HPMCP, 75% ethanol for L100/S100) were also chosen for minimal antimicrobial impact. This crucial step, often overlooked when selecting materials based solely on physicochemical properties[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], ensures excipients do not harm probiotics during processing or storage. Collectively, this low-temperature processing and biocompatible excipient/solvent selection strategy effectively maintained activity during the production process, resolving the issue of a significant decline in live bacteria counts reported in other studies involving probiotic microcapsules or particles after preparation[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and providing a basis for the formulation's efficacy.\u003c/p\u003e\u003cp\u003eThe enteric-coated sustained-release granule system developed herein represents a promising next-generation platform for oral probiotic delivery. Its gastric protection and colon-targeted sustained release are particularly well-suited for the maintenance therapy of chronic inflammatory bowel diseases (IBD, including UC and CD)[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], potentially reducing relapse frequency/severity and dependence on conventional drugs by ensuring continuous delivery of viable probiotics to the inflamed site. The inherent compatibility and scalability of the granule matrix and coating process readily allow adaptation for delivering other single probiotic strains or designing advanced formulations. This includes multi-strain probiotic consortia leveraging synergistic interactions (e.g., \u003cem\u003eL. plantarum\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eBifidobacterium spp.\u003c/em\u003e)[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] or synbiotics by incorporating prebiotics (e.g., FOS, inulin) into the matrix to selectively enhance probiotic activity in situ[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].Taken together, this modular, scalable platform offers a versatile foundation for the development of advanced probiotic.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study successfully developed and validated an enteric-coated sustained-release granule system for \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, utilizing an innovative cross-linked porous matrix formed by HPMCP/L100. The system employs a dual protective mechanism: gastric pH-responsive coating shields against acid erosion, while the intestinal matrix enables controlled, sustained release. This revolutionizes oral probiotic delivery, significantly enhancing survival through the harsh GI tract and ensuring targeted, prolonged release within the colon. In a DSS-induced murine colitis model, at equivalent viable cell doses, the granules demonstrated profoundly superior therapeutic efficacy compared to conventional freeze-dried powder. This encompassed significantly enhanced histological repair, effective immunomodulation (e.g., M2 macrophage polarization), and comprehensive restoration of gut microbiota homeostasis \u0026ndash; advantages directly attributable to the system's fundamental improvement of probiotic bioavailability and targeted delivery efficiency. Crucially, high viability was preserved during manufacturing via meticulously controlled low-temperature processing and stringent screening of biocompatible, non-antimicrobial excipients.\u003c/p\u003e\u003cp\u003eThe matrix-based multi-particulate system offers distinct advantages over existing technologies (e.g., single-unit enteric-coated tablets/capsules, microcapsules), including superior protection uniformity, resilience against localized failure, gentle processing, and enhanced manufacturability. Compared to conventional drugs like 5-ASA, the probiotic granules present an attractive profile: comparable efficacy (for mild-moderate colitis in the model), superior safety, and unique multi-targeted actions (anti-inflammation, barrier repair, microbiota restoration). This positions them as a promising alternative or adjunctive therapy.\u003c/p\u003e\u003cp\u003eBeyond enabling efficient \u003cem\u003eL. plantarum\u003c/em\u003e delivery, this versatile platform technology holds significant potential for delivering other single probiotics, multi-strain consortia, or synbiotics. Its application extends to the maintenance therapy of IBD, prevention of antibiotic-associated diarrhea (AAD), management of C. difficile infection (CDI), and other dysbiosis-related conditions. Future optimization of long-term stability, large-scale manufacturing, and pivotal clinical trials will pave the way for this next-generation delivery system to achieve transformative advances in GI health management and personalized nutritional interventions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eAbbreviation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eDefinition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003e5-ASA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003e5-aminosalicylic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eAAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eAntibiotic-associated diarrhea\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eCAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eCellulose acetate phthalate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eCD86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eCluster of differentiation 86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eCD206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eCluster of differentiation 206\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eCDI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003e\u003cem\u003eClostridioides difficile\u003c/em\u003e infection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eDSS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eDextran sulfate sodium salt\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eF4/80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eF4/80 antigen (EMR1, mouse macrophage marker)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eHE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eHematoxylin and eosin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eHPMCAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eHydroxypropyl methylcellulose succinate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eHPMCP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eHydroxypropyl methylcellulose phthalate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eIBD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eInflammatory bowel disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eiNOS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eInducible nitric oxide synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eL100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eEudragit L100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eMRS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003ede Man, Rogosa and Sharpe (broth/agar)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eOTU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eOperational taxonomic unit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003ePCoA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003ePrincipal coordinate analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eS100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eEudragit S100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eSodium alginate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eScanning electron microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eSGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eSimulated gastric fluid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eSIF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eSimulated intestinal fluid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eSobs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eNumber of observed species (or OTUs)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24.6046%;\"\u003e\n \u003cp\u003eUC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75.3954%;\"\u003e\n \u003cp\u003eUlcerative colitis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study protocol was approved by the Ethics Committee of the Animal Center of Zhengzhou University (protocol code ZZU-LAC20241217[01] and date of approval is Dec. 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by 2019 scientific research support for high-level talent project (project number: 31401225) and 2023 natural science project (project number: 51300023) from Henan University of Technology, grant number: 501100003489.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXile Wang and Xiaocan Zhang wrote the original draft; Shuang Liang and Miaomiao Jiang prepared all figures; Xiwei Zhang and Jianfeng Wan check all the data and figures; Hongqing Zhang acquired fundings; Guangzhou Zhou reviewed and edited the final manuscript; Guangzhou Zhou and Xiwei Zhang supervised this project. All authors have reviewed and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, et al. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Reviews Gastroenterol Hepatol. 2014;11(8):506\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSanders ME, Guarner F, Guerrant R, Holt PR, Quigley EMM, Sartor RB, et al. An update on the use and investigation of probiotics in health and disease. Gut. 2013;62(5):787\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAli U, Saeed M, Ahmad Z, Shah F-u-H, Rehman MA, Mehmood T, et al. Stability and Survivability of Alginate Gum-Coated Lactobacillus rhamnosus GG in Simulated Gastrointestinal Conditions and Probiotic Juice Development. 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Advances in Immunology2013. pp. 51\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTamanai-Shacoori Z, Smida I, Bousarghin L, Loreal O, Meuric V, Fong SB, et al. Roseburia spp.: a marker of health? Future Microbiol. 2017;12:157\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIljazovic A, Roy U, G\u0026aacute;lvez EJC, Lesker TR, Zhao B, Gronow A, et al. Perturbation of the gut microbiome by Prevotella spp. enhances host susceptibility to mucosal inflammation. Mucosal Immunol. 2021;14(1):113\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLichtenstein GR, Loftus EV, Isaacs KL, Regueiro MD, Gerson LB, Sands BE. ACG Clinical Guideline: Management of Crohn's Disease in Adults. Am J Gastroenterol. 2018;113(4):481\u0026ndash;517.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeyrin-Biroulet L, Loftus EV, Colombel J-F, Sandborn WJ. 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Aliment Pharmacol Ther. 2017;46(4):389\u0026ndash;400.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZmora N, Zilberman-Schapira G, Suez J, Mor U, Dori-Bachash M, Bashiardes S, et al. Personalized Gut Mucosal Colonization Resistance to Empiric Probiotics Is Associated with Unique Host and Microbiome Features. Cell. 2018;174(6):1388\u0026ndash;e40521.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFord AC, Achkar J-P, Khan KJ, Kane SV, Talley NJ, Marshall JK, et al. Efficacy of 5-Aminosalicylates in Ulcerative Colitis: Systematic Review and Meta-Analysis. Am J Gastroenterol. 2011;106(4):601\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh BN. Modified-release solid formulations for colonic delivery. Recent Pat Drug Deliv Formul. 2007;1(1):53\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAr\u0026eacute;valo-P\u0026eacute;rez R, Maderuelo C, Lanao JM. Recent advances in colon drug delivery systems. J Controlled Release. 2020;327:703\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaroni A, Zema L, Del Curto MD, Foppoli A, Gazzaniga A. Oral colon delivery of insulin with the aid of functional adjuvants. Adv Drug Deliv Rev. 2012;64(6):540\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDidari T, Solki S, Mozaffari S, Nikfar S, Abdollahi M. A systematic review of the safety of probiotics. Exp Opin Drug Saf. 2014;13(2):227\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChapman CMC, Gibson GR, Rowland I. Health benefits of probiotics: are mixtures more effective than single strains? Eur J Nutr. 2011;50(1):1\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXiao Y, Lu C, Liu Y, Kong L, Bai H, Mu H, et al. Encapsulation of Lactobacillus rhamnosus in Hyaluronic Acid-Based Hydrogel for Pathogen-Targeted Delivery to Ameliorate Enteritis. ACS Appl Mater Interfaces. 2020;12(33):36967\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePlaza-D\u0026iacute;az J, Ruiz-Ojeda F, Vilchez-Padial L, Gil A. Evidence of the Anti-Inflammatory Effects of Probiotics and Synbiotics in Intestinal Chronic Diseases. Nutrients. 2017;9(6).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede Vries MC, Vaughan EE, Kleerebezem M, de Vos WM. Lactobacillus plantarum\u0026mdash;survival, functional and potential probiotic properties in the human intestinal tract. Int Dairy J. 2006;16(9):1018\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMehta RS, Mayers JR, Zhang Y, Bhosle A, Glasser NR, Nguyen LH, et al. Gut microbial metabolism of 5-ASA diminishes its clinical efficacy in inflammatory bowel disease. Nat Med. 2023;29(3):700\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHeidebach T, F\u0026ouml;rst P, Kulozik U. Influence of casein-based microencapsulation on freeze-drying and storage of probiotic cells. J Food Eng. 2010;98(3):309\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePicot A, Lacroix C. Encapsulation of bifidobacteria in whey protein-based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt. Int Dairy J. 2004;14(6):505\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGbassi GK, Vandamme T. Probiotic Encapsulation Technology: From Microencapsulation to Release into the Gut. Pharmaceutics. 2012;4(1):149\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCook MT, Tzortzis G, Charalampopoulos D, Khutoryanskiy VV. Microencapsulation of probiotics for gastrointestinal delivery. J Controlled Release. 2012;162(1):56\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSartor RB, Mazmanian SK. Intestinal Microbes in Inflammatory Bowel Diseases. Am J Gastroenterol Supplements. 2012;1(1):15\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarkowiak P, Śliżewska K. Effects of Probiotics, Prebiotics, and Synbiotics on Human Health. Nutrients. 2017;9(9).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbit","sideBox":"Learn more about [BMC Biotechnology](http://bmcbiotechnol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bbit/default.aspx","title":"BMC Biotechnology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Acute colitis, Lactobacillus plantarum, Enteric sustained-release pellets, HPMCP, Eudragit L100","lastPublishedDoi":"10.21203/rs.3.rs-7469110/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7469110/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eThe clinical effectiveness of orally administered probiotics is often limited by poor survival during gastrointestinal transit and insufficient delivery to the intestinal site of action. Lactobacillus plantarum, a probiotic with anti-inflammatory potential, is highly sensitive to gastric acid and bile salts, diminishing its therapeutic efficacy for intestinal diseases. We developed a sustained-release enteric pellet formulation to enhance probiotic stability, promote targeted intestinal delivery, and evaluate its therapeutic efficacy in colitis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003ePellets were prepared using low-temperature extrusion and coated with pH-responsive polymers (hydroxypropyl methylcellulose phthalate and Eudragit L100), creating a dual-protection barrier. Structural analysis revealed a cross-linked porous skeleton conducive to controlled release. In vitro tests confirmed gastric acid resistance and rapid release under intestinal conditions. In a dextran sulfate sodium-induced acute colitis mouse model, pellet-treated groups (both coatings) showed superior outcomes compared to uncoated probiotic powder. This included enhanced body weight recovery, reduced histopathological damage, downregulation of pro-inflammatory M1 macrophages, enhancement of anti-inflammatory M2 polarization, restoration of gut microbial diversity, and normalization of the Firmicutes to Bacteroidetes ratio.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions \u003c/strong\u003eThe sustained-release enteric pellet system effectively protects Lactobacillus plantarum during gastrointestinal transit and achieves targeted intestinal release. Integrating mechanical shielding with pH-triggered dissolution significantly enhances probiotic bioavailability and therapeutic efficacy. This delivery platform represents a safe, scalable, and broadly applicable strategy for treating intestinal inflammatory diseases. Furthermore, the approach provides a versatile framework for delivering other probiotic strains or combinations.\u003c/p\u003e","manuscriptTitle":"Sustained-Release Enteric Formulations of Lactobacillus plantarum Based on Granulation Technology: Preparation and Therapeutic Evaluation in Acute Colitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-16 06:59:55","doi":"10.21203/rs.3.rs-7469110/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-18T10:35:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-12T20:46:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-10T19:23:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72293068305984014479549616879587345744","date":"2025-10-02T12:48:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66905102137090345831400200612396686530","date":"2025-10-02T12:48:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-08T16:42:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-02T10:57:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-02T08:35:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Biotechnology","date":"2025-09-02T08:32:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbit","sideBox":"Learn more about [BMC Biotechnology](http://bmcbiotechnol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bbit/default.aspx","title":"BMC Biotechnology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"82c054d0-18f8-4f3d-b117-2bb39cca5925","owner":[],"postedDate":"September 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:06:20+00:00","versionOfRecord":{"articleIdentity":"rs-7469110","link":"https://doi.org/10.1186/s12896-026-01157-7","journal":{"identity":"bmc-biotechnology","isVorOnly":false,"title":"BMC Biotechnology"},"publishedOn":"2026-04-21 15:59:14","publishedOnDateReadable":"April 21st, 2026"},"versionCreatedAt":"2025-09-16 06:59:55","video":"","vorDoi":"10.1186/s12896-026-01157-7","vorDoiUrl":"https://doi.org/10.1186/s12896-026-01157-7","workflowStages":[]},"version":"v1","identity":"rs-7469110","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7469110","identity":"rs-7469110","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}