A composition containing probiotics and its use in the treatment of gastrointestinal diseases.

Abstract

This invention discloses a composition comprising probiotics and a fusion polypeptide and its application in the treatment of inflammatory bowel diseases. The composition consists of *Lactobacillus plantarum* CCFM8661 and a functional fusion polypeptide, wherein the amino acid sequence of the fusion polypeptide is shown in SEQ ID NO.1. This fusion polypeptide achieves a triple synergistic effect of "antibacterial inhibition-repair-immunomodulation" by enhancing the intestinal colonization ability of probiotics, directly inhibiting the growth of pathogenic bacteria, and neutralizing toxins. Experiments show that the composition significantly reduces the disease activity index, restores colon length, and inhibits serum inflammatory factors IL-6 and TNF-α in a DSS-induced mouse colitis model. Furthermore, the lyophilized microcapsule formulation exhibits a survival rate of >90% in simulated gastric juice and possesses colon-targeted release characteristics. This invention provides a highly efficient and stable new treatment strategy for inflammatory bowel diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a composition containing probiotics and its use in treating gastrointestinal diseases. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of diseases characterized by chronic, relapsing bowel inflammation, primarily including ulcerative colitis and Crohn's disease. Its pathogenesis is complex, and current research indicates that IBD is closely related to factors such as gut microbiota dysbiosis, pathogen invasion, and abnormalities in the body's immune system. Specifically, IBD patients exhibit a significant decrease in the number of beneficial bacteria (such as Bacteroides and Lactobacillus) in their gut, while opportunistic pathogens (such as Clostridium difficile and Escherichia coli) proliferate abnormally, inducing disruption of the intestinal barrier function, thereby activating an excessive immune response and causing persistent inflammation.

[0003] While existing treatments are constantly evolving, several technical bottlenecks remain. Firstly, probiotic therapy, as a common intervention, can regulate the gut microbiota to some extent, but its application is significantly limited. Traditional probiotics, such as Lactobacillus and Bifidobacterium, are easily destroyed by gastric acid and bile salts in the gastrointestinal environment, resulting in an actual colonization rate in the intestine that is typically less than 20%. Furthermore, these probiotics have weak direct inhibitory effects on specific pathogens such as pathogenic Escherichia coli and Clostridium difficile, limiting their anti-infective activity.

[0004] Secondly, while biologics targeting inflammatory responses, such as anti-TNF-α monoclonal antibodies (e.g., infliximab, vedolizumab), can block key immune pathways, these drugs are expensive and carry risks of adverse reactions such as immunosuppression and increased susceptibility to infection, and they cannot directly eliminate pathogens. Furthermore, although antimicrobial peptides (e.g., human defensins) have broad-spectrum antibacterial activity, their short half-life in vivo, susceptibility to enzymatic degradation, and lack of targeting limit their effectiveness in local intestinal applications.

[0005] More importantly, there is currently a lack of integrated treatment strategies that can simultaneously achieve multiple functions of "antibacterial, repair, and immune regulation." Simple combinations of probiotics with antibacterial components such as bacteriophages and antimicrobial peptides suffer from low synergistic efficiency, poor component compatibility, and poor stability in the complex intestinal environment, making it difficult to achieve multi-mechanism synergistic intervention in the pathological process of IBD. Therefore, there is an urgent need to develop a novel intestinal intervention technology that combines targeted antibacterial activity, promotes repair, and regulates immune function to achieve more effective and longer-lasting treatment for IBD. Summary of the Invention

[0006] This invention provides a composition comprising probiotics and functional fusion peptides, which can be used to treat inflammatory bowel diseases by enhancing probiotic colonization, inhibiting pathogens and neutralizing toxins.

[0007] Therefore, the present invention discloses a high-affinity single-chain antibody against Clostridium difficile toxin B, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] In one aspect, the present invention also discloses a fusion polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] Preferably, the nucleotide sequence of the fusion polypeptide codon optimized according to the present invention is shown in SEQ ID NO.3.

[0010] In one aspect, the present invention also discloses a composition comprising probiotics, said composition comprising the following components:

[0011] (1) Probiotics: Lactobacillus plantarum CCFM8661, concentration ≥1×10 9 CFU / g;

[0012] (2) Fusion polypeptide: The fusion polypeptide, wherein the fusion polypeptide in the composition is mixed with probiotics at a mass ratio of 1:0.5.

[0013] Preferably, the composition of the present invention is an enteric-coated microcapsule prepared by freeze-drying process, wherein the coating material of the enteric-coated microcapsule is hydroxypropyl methylcellulose, and the particle size of the enteric-coated microcapsule is 300-500 μm.

[0014] In one aspect, the present invention also discloses the use of the aforementioned fusion polypeptide in the preparation of a composition containing probiotics.

[0015] In one aspect, the present invention also discloses the use of the aforementioned probiotic-containing composition in the preparation of a medicament for treating gastrointestinal diseases.

[0016] The composition of the present invention has the following beneficial effects:

[0017] 1. Synergistic effect: The fusion peptides work synergistically with probiotics to reduce intestinal inflammation through a triple mechanism of adhesion, antibacterial and toxin neutralization (the efficacy of the combination group is significantly better than that of the single component, P<0.001);

[0018] 2. Targeted delivery: Enteric-coated microcapsules protect the active ingredients, with a gastric acid survival rate of >90% and a colon-targeted release rate of >80%;

[0019] 3. Clinical translation advantages: The preparation process is stable (SDS-PAGE purity >95%), and the viable bacteria retention rate of the lyophilized formulation is ≥80% after 3 months of storage at room temperature;

[0020] 4. Safety: No liver or kidney toxicity or excessive immune activation was observed in animal studies.

[0021] The above results indicate that the composition of the present invention containing probiotics and functional fusion peptides can be widely used in many fields such as probiotic preparations, antibiotic alternatives, enteritis treatment, and biological protection agents, and has good commercial application prospects and market promotion potential. Attached Figure Description

[0022] Figure 1 The SDS-PAGE results of the fusion protein are shown, where 1 represents the fusion protein.

[0023] Figure 2 Western blot analysis results of the fusion protein, where 1 is the fusion protein. Detailed Implementation

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0025] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0026] Example 1: Construction, preparation and testing of fusion peptides

[0027] I. Fusion Peptide Design

[0028] This embodiment constructs a fusion polypeptide with triple functions of intestinal localization, antibacterial activity, and toxin neutralization. Its full-length amino acid sequence is shown in SEQ ID NO.1 and contains four functional regions. Details are as follows:

[0029] 1. Adhesion domain: Derived from the surface layer protein SlpA of *Lactobacillus plantarum*. This fragment is located in the N-terminal domain of SlpA and has the ability to bind to the GPI-anchor protein of intestinal epithelial cells, giving the fusion protein intestinal targeting. Its specific amino acid sequence is: MKLFIAVLSLLLQSVASTQKQKVTAAQNPAANKTVKQTLDKVKEIADKVQKTVTQAT.

[0030] 2. Flexible linker peptide: Derived from the commonly used linker peptide Gly-Ser repeating structure, it avoids spatial interference between functional regions and ensures the freedom of protein folding. The specific amino acid sequence is: GGGGSGGGGSGGGGS.

[0031] 3. Antimicrobial peptide domain: Derived from human β-defensin-3 (hBD-3), it possesses broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria (including Clostridium difficile, Escherichia coli, and Staphylococcus aureus). The specific amino acid sequence is: GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK.

[0032] 4. Toxin-binding domain: The inventors prepared a high-affinity single-chain antibody against Clostridium difficile toxin B (as shown in Example 3). This scFv can effectively neutralize TcdB toxin and prevent it from binding to host cells. Its specific amino acid sequence is shown in SEQ ID NO. 2.

[0033] II. Construction of Fusion Peptide Genes

[0034] 1. Fusion Peptide Sequence Design and Codon Optimization: Full-length fusion peptide DNA sequences were designed using SnapGene v6.0 or Geneious Prime, and E. coli-preferred codons were optimized using the JCat online tool, targeting a Codon Adaptation Index (CAI) > 0.9 and GC content controlled within the range of 30%–70%. Restriction endonuclease recognition sites were added to both ends of the sequence: NdeI upstream and XhoI downstream. A 6×His tag was also introduced downstream for purification and detection. The nucleotide sequence of the codon-optimized fusion peptide is shown in SEQ ID NO.3.

[0035] 2. Vector Construction: The codon-optimized fusion polypeptide gene fragment was synthesized and constructed into pET-28a(+). The pET-28a(+) vector and the fusion gene insert were digested with NdeI and XhoI (37℃, 2h), respectively; the fragments were recovered by agarose gel electrophoresis; ligation was performed using T4 DNA ligase at a molar ratio of insert fragment to vector of 3:1, as shown in Table 1. The ligation reaction was carried out overnight at 4℃; the ligation product was transformed into DH5α competent E. coli, and single colonies were picked from plates for plasmid amplification.

[0036] Table 1 Connection Reaction System

[0037]

[0038] 3. Plasmid extraction and sequence verification: Recombinant plasmids were extracted from the DH5α strain using a plasmid mini-extraction kit; Sanger sequencing was performed using T7 forward / reverse primers or specific inner primers to confirm that the fusion fragment sequence was free of base mutations, frameshift mutations, and intact restriction enzyme sites.

[0039] 4. Construction of expression strains: The verified recombinant expression plasmid was transformed into BL21(DE3) competent cells; positive strains were screened by plating on LB+Kan plates; the presence of the fusion gene was preliminarily confirmed by PCR or colony PCR; and the strains were preserved in glycerol (30%)-LB liquid culture (long-term storage at -80℃).

[0040] III. Expression and Purification of Fusion Peptides

[0041] 1. Optimization of expression conditions and induction of expression

[0042] 1.1 Screening for optimal induction conditions: BL21(DE3) engineered bacteria were inoculated into LB liquid medium containing kanamycin (50 μg / mL) and the following conditions (Table 2) were compared to optimize induction expression. After screening, group B was found to be the best.

[0043] Table 2 Filtering under different conditions

[0044]

[0045] 1.2 Expression Procedure: A single positive clone was picked and inoculated into 5 mL of LB-Kan medium, and pre-cultured at 37℃ and 220 rpm for 8 h; 1% of the inoculum (e.g., 1 mL of seed culture) was then added to 100 mL of LB-Kan medium; cultured until OD... 600 ≈0.6; Add IPTG to a final concentration of 0.5mM for induction, then incubate at 16℃ with shaking for 16h; After induction, collect the cells by centrifugation (5000×g, 10min, 4℃) for subsequent lysis and purification.

[0046] 2. Bacterial cell lysis and soluble protein detection

[0047] 2.1 Sonic disruption: The bacterial cells were resuspended in pre-cooled lysis buffer (50mM Tris-HCl, 300mM NaCl, 10mMimidazole, 1mM PMSF, 1mg / mL Lysozyme, pH 8.0) (10mL / 100mL original culture volume); the cells were then sonicated in an ice bath (e.g., 10 seconds working / 20 seconds rest × 15min, total energy approximately 200W); the cells were centrifuged at 12000×g for 20min at 4℃, and the supernatant was collected as soluble protein. The precipitate was used to determine whether inclusion bodies had formed. After testing, the fusion protein was found to be the soluble supernatant expressed protein.

[0048] 3. Ni-NTA affinity chromatography purification

[0049] 3.1 Materials and Equipment: Ni-NTA pre-packed column (Qiagen 1mL); elution buffer prepared as follows:

[0050] Table 3 Buffer Configuration Table

[0051]

[0052] 3.2 Procedure: Load the supernatant onto a Ni-NTA column (pre-equilibrate); wash non-specific binding proteins with 10 column volumes of wash buffer; collect the elution peak with elution buffer (gradient or single wash); filter at 4°C using a 0.22 μm filter and concentrate to PBS buffer; add glycerol to a final concentration of 10% and store at -80°C.

[0053] 4. Expression and purification validation

[0054] 4.1 SDS-PAGE Detection: 20 μL of the purified fusion protein was separated using a 12% SDS-PAGE gel and then stained with Coomassie brilliant blue. The results showed ( Figure 1 The molecular weight of this fusion protein is approximately 41.7 kDa, and its viscosity should be >95%.

[0055] 4.2 Western Blot Validation: 20 μL of the purified fusion protein was separated using a 12% SDS-PAGE gel, transferred to a membrane, and incubated with antibody (HRP-labeled Mouse anti-His tag, 1:5000). ECL chemiluminescence was then used for color development. The results showed ( Figure 2 It exhibits a specific band at a molecular weight of approximately 41.7 kDa.

[0056] 5. Expression level statistics: The protein concentration was determined using the BCA method (Thermo Scientific kit). The results showed that the concentration of the fusion protein was 3.56 mg / mL. After calculation, the expression level after purification was approximately 1.2 g / L of culture medium. Therefore, the fusion protein has a relatively high expression level, which lays the foundation for its large-scale application.

[0057] Example 2: Functional Verification Experiment of Fusion Peptide

[0058] This embodiment verifies the triple function of the fusion peptide prepared in Example 1 in promoting probiotic adhesion, inhibiting Clostridium difficile growth, and neutralizing its toxins.

[0059] I. Adhesion Promotion Function Verification

[0060] 1. Experimental procedure: Human intestinal epithelial Caco-2 cells were seeded in 24-well plates (1×10⁻⁶ cells / well). 5 Cells / well were cultured in DMEM medium containing 10% FBS until a monolayer was formed; *Lactobacillus plantarum* (1×10⁻⁶ cells / well) were labeled with a DiO fluorescent probe. 7 CFU / mL); the fusion peptide was diluted to 0 (blank), 5 μg / mL, and 10 μg / mL, respectively, and pre-incubated with Lactobacillus for 30 min; the mixture was added to Caco-2 cell wells and co-incubated at 37°C for 2 h; washed 3 times with PBS to remove unbound bacteria; fluorescence intensity (Ex / Em = 484 / 501 nm) was detected using a multi-functional microplate reader, and the relative adhesion rate was calculated.

[0061] 2. The results (Table 4) showed that the fusion peptide significantly enhanced the adhesion ability of probiotics to Caco-2 cells in a concentration-dependent manner. This effect stems from the ability of its N-terminal SlpA-derived domain to recognize and bind to the intestinal GPI-anchor structure.

[0062] Table 4. Validation results of adhesion-promoting function

[0063]

[0064] II. Antibacterial activity test (against Clostridium difficile)

[0065] 1. Experimental Procedure: Prepare BHIS agar plates and inoculate Clostridium difficile (ATCC 43255) bacterial suspension onto the plate surface; add the following to sterile filter paper: fusion polypeptide solution (10 μg), vancomycin (10 μg, positive control), and PBS buffer (negative control); attach the filter paper to the plate surface and anaerobically incubate at 37℃ for 24 hours; measure the diameter of the inhibition zone (mm) as an indicator of antibacterial effect.

[0066] 2. The results (Table 5) showed that the fusion peptide exhibited a larger inhibition zone diameter than vancomycin, indicating that the antimicrobial peptide HBD-3 synergistically inhibited the growth of Clostridium difficile by disrupting the pathogen's membrane structure.

[0067] Table 5 Results of antibacterial activity detection

[0068]

[0069] III. Toxin neutralization function test (for TcdB)

[0070] 1. Experimental Procedure: Vero cells (African green monkey kidney cells, TcdB-sensitive) were seeded in 96-well plates and cultured to 90% confluence. The following were added: TcdB toxin (final concentration 50 ng / mL), TcdB+ fusion peptide (5 μg / mL or 10 μg / mL), TcdB+ known anti-TcdB scFv positive control (10 μg / mL), and PBS control. Incubation was continued for 24 h. Cell viability was assessed using the CCK-8 assay (absorbance at 450 nm), and the toxin neutralization rate was calculated. Calculation formula: Toxin neutralization rate = [(OD experimental group - OD toxin group) / (OD control group - OD toxin group)] × 100%.

[0071] 2. The results (Table 6) showed that the fusion peptide significantly neutralized TcdB toxin and restored Vero cell viability, with effects comparable to or better than those of positive scFv antibodies. This indicates that its C-terminal antitoxin domain maintained its biological activity in the fusion configuration, making it a potential candidate for antitoxin therapy.

[0072] Table 6 Results of Toxin Neutralization Function Test

[0073]

[0074] Example 3: Preparation of a high-affinity single-chain antibody (scFv) against Clostridium difficile toxin B

[0075] 1. Immunization and Antibody Library Construction: Mice were first intraperitoneally immunized with TcdB (Sino Biological, 50 μg) combined with Freund's complete adjuvant, followed by two booster immunizations with incomplete adjuvants on days 7 and 14. At week 4, blood was collected via tail vein, and the anti-TcdB antibody titer was measured using ELISA. Three mice with high antibody titers were selected, and their spleens were harvested for subsequent antibody library construction. Total RNA was extracted from the spleen using TRIzol reagent, and cDNA was obtained through reverse transcription, serving as a template for subsequent PCR amplification of the antibody variable region sequence. The VH and VL regions were amplified using universal primers, and then ligated into a VH-(G4S)3-VL scFv structure using overlap PCR, with an SfiI restriction site introduced for subsequent cloning. The constructed scFv fragment was inserted into the pComb3XSS phage display vector and electroporated into E. coli TG1 competent cells to form a phage display library. The library size was assessed to be greater than 1 × 10⁻⁶. 8 CFUs were inserted at an insertion rate of approximately 90%. Finally, M13KO7 helper phage was used to infect the cells, resulting in a highly efficient display-type scFv phage library.

[0076] 2. scFv Screening and Validation: First, three rounds of solid-phase panning were performed using TcdB-coated 96-well plates. Phage libraries were added to the coated wells and incubated at room temperature. Non-specific binding was removed by progressively increasing the salt concentration and washing intensity, with the most stringent washing conditions used in the third round to enrich high-affinity phages. The eluted phages were then used to infect *E. coli* TG1 strain, and positive clones were selected for subsequent analysis. ELISA validation was then performed. The supernatant from single-clone cultures was added to TcdB-coated 96-well plates, and a colorimetric reaction was initiated using HRP-labeled anti-M13 antibody. Positive clones with strong binding affinity were screened based on OD values. Finally, 10 scFv clones with high affinity were selected and sequenced.

[0077] 3. Subcloning and protein expression of the scFv gene

[0078] (1) To achieve recombinant expression of a high-affinity single-chain antibody against Clostridium difficile toxin B (scFv), the selected scFv gene was subcloned into the pET-28a(+) expression vector. First, NcoI and XhoI restriction sites were introduced at both ends of the scFv to ensure successful insertion at the multiple cloning site of the vector, while retaining the 6×His tag at the C-terminus for subsequent purification. The PCR product was double-digested and ligated into the linearized pET-28a(+) vector using T4 ligase at 16°C. The vector was then transformed into competent DH5α strains for amplification. After Sanger sequencing confirmed the absence of mutations in the inserted sequence, the vector was transformed into Escherichia coli BL21(DE3) for protein expression.

[0079] (2) During the protein induction expression stage, BL21(DE3) containing the recombinant plasmid was inoculated into LB medium containing kanamycin and cultured at 37°C until OD200. 600 When the protein concentration reached approximately 0.6, 0.5 mM IPTG was added for induction, and expression was induced overnight at 16°C. The next day, bacterial cells were collected, lysed by sonication, centrifuged, and the supernatant was used for protein purification. The His-tagged fused scFv in the supernatant was purified using Ni-NTA affinity chromatography resin, and excess salt was removed by dialysis with PBS buffer to obtain highly pure functional recombinant scFv protein.

[0080] 4. Functional testing and affinity evaluation: In order to verify the binding ability and neutralization effect of the screened scFv antibodies against Clostridium difficile toxin B (TcdB), a series of functional testing and affinity evaluation experiments were carried out.

[0081] (1) First, its affinity was assessed by ELISA. TcdB was coated onto the surface of a 96-well plate, and scFv antibody diluted at different concentrations was added. The colorimetric reaction was performed using HRP-labeled anti-His secondary antibody, and the half-maximal effective concentration (EC50) was calculated based on the absorbance change. 50 The results showed that the EC50 of the scFv antibody... 50 The value was 2.7 nM, which was significantly better than the commercial antibody (ab270452, 15.8 nM) measured at the same time, indicating that it has a stronger affinity binding ability.

[0082] (2) Subsequently, the affinity constant (KD) was further precisely determined on the Biacore T200 platform using surface plasmon resonance (SPR) technology. In the experiment, TcdB was coupled to the surface of the CM5 chip, and different concentrations of scFv antibody were injected to analyze the binding kinetic parameters. The results showed that the KD value of the scFv antibody was 1.1 nM, while the KD value of the commercial antibody (ab270452) was 15.9 nM. Therefore, the dissociation rate of the scFv antibody was significantly lower than that of the commercial antibody, indicating that it binds to TcdB more stably, with an affinity increase of about 14.5 times.

[0083] (3) Finally, the neutralization activity was verified using a Vero cytotoxicity protection assay. The scFv antibody and TcdB were pre-incubated at different concentrations before being added to the Vero cell culture system. Cell viability was detected using the MTT assay after 24 hours. The results (Table 7) showed that at a scFv concentration of 100 nM, the cell viability reached 79.7%, while the toxin-treated control group had only 21%, and the commercial antibody (ab270452) had 58.6%. This indicates that the scFv has good toxin-neutralizing activity and potential protective effect, and is more effective than the commercial antibody.

[0084] Table 7 Comparison of neutralization activity verification results

[0085]

[0086] 4. Amino acid sequence of scFv antibody: The sequence is VH-(G4S)3-VL structure, where VH and VL are derived from positive clones selected from immunized mice and are humanized and optimized amino acid sequences. Therefore, the amino acid sequence of scFv antibody is shown in SEQ ID NO.2.

[0087] Example 4: Composition and preparation of a probiotic-containing composition

[0088] 1. Composition of the probiotic-containing composition

[0089] 1.1 Probiotics: Lactobacillus plantarum CCFM8661 was selected, the source of which can be found in Chinese Patent Application Publication No. CN111869735A. This strain has excellent intestinal adhesion and antibacterial ability.

[0090] (1) Content standard: ≥1×10 9 CFU / g (live bacteria count after freeze-drying);

[0091] (2) Culture conditions: MRS liquid medium, anaerobic culture at 37℃ for 18 hours;

[0092] (3) Harvesting method: Centrifuge at 8000 rpm for 10 minutes, discard the supernatant, resuspend in sterile PBS and use for premixing before freeze-drying.

[0093] 1.2 Fusion peptide: The fusion peptide prepared in Example 1.

[0094] (1) Purity requirement: >90% (SDS-PAGE);

[0095] (2) Addition ratio: Mix with probiotics at a mass ratio of 1:0.5 (i.e., 1g of bacterial powder + 0.5g of fusion polypeptide).

[0096] 2. Preparation process of compositions containing probiotics

[0097] 2.1 Mixing and pre-freeze-drying treatment: After centrifuging and concentrating the probiotics and resuspending them, they were mixed with the fusion peptides under sterile conditions; a freeze-drying protectant system (10% trehalose, 5% skim milk) was added to protect the cell membrane structure and protein conformation, and improve the survival rate and stability of the freeze-dried product; the mixture was dispensed into freeze-drying bottles (≤10mL / bottle), pre-frozen at -80℃ for 4 hours, and then entered the freeze-drying process.

[0098] 2.2 Freeze-drying program parameter settings (freeze-drying):

[0099] (1) Pre-freezing temperature: -80℃, time: 4 hours;

[0100] (2) Initial drying stage: -40℃ to -10℃, vacuum degree <50Pa, for 18 hours;

[0101] (3) Secondary drying stage: Heat to 25℃ and maintain for 10 hours, with a moisture content of <3%.

[0102] 2.3 Enteric coating and microencapsulation

[0103] (1) Hydroxypropyl methylcellulose (HPMC) was used as an enteric coating material (soluble in pH > 6.0 conditions to protect the active ingredients from being destroyed by gastric acid);

[0104] (2) Coating method: The freeze-dried powder is suspended in an ethanol / water mixture and 2% HPMC solution is added; spray drying or fluidized bed coating technology is used, the inlet air temperature is set to 60-70℃ and the outlet air temperature is not higher than 40℃; the final microcapsule particle size is controlled to be 300-500μm; after drying, it is sealed and stored in a light-proof, dry, low-temperature (4℃) environment.

[0105] 3. Product stability verification

[0106] 3.1 First, the viable count of the probiotics was systematically tested. *Lactobacillus plantarum* CCFM8661 was lyophilized and coated with the fusion peptide, and then stored under refrigeration (4℃), room temperature (25℃), and accelerated aging (40℃) conditions, with continuous monitoring of viable count changes. The results showed that under refrigeration, over 90% of the bacterial activity was maintained after 6 months, while at room temperature, the activity retention rate slightly decreased to about 80% after 3 months. At 40℃, the activity decreased more rapidly, but still exhibited acceptable stability, indicating that this composition possesses good viable bacterial stability under appropriate storage conditions.

[0107] (2) To assess the functional retention of the fusion peptide during formulation, ELISA and cell neutralization assays were used for verification. The results showed that the OD value of peptide binding activity decreased slightly from 1.280 to 1.192 before and after lyophilization, with a retention rate of approximately 93.1%. In the TcdB toxin neutralization assay, after treating Vero cells with 100 nM fusion peptide, the cell viability significantly increased from 22.6% in the untreated group to 78.5%, which was basically consistent with 81.2% before lyophilization, indicating that the lyophilization and microencapsulation processes did not significantly affect the peptide function.

[0108] (3) To evaluate the enteric coating effect of the microcapsules, in vitro release experiments were further conducted in simulated gastric fluid at pH 1.2 and simulated intestinal fluid at pH 6.8. The results showed that the release rates of the fusion peptides and probiotics were both less than 10% in the gastric fluid environment, while the release efficiency was significantly improved in the intestinal fluid, reaching more than 80% and 85% respectively. This fully verified that the HPMC coating used has good pH-dependent release capability and can achieve effective targeted release in the gastrointestinal segment.

[0109] Comprehensive analysis shows that this fusion peptide-probiotic composition has good freeze-drying stability, environmental tolerance and targeted release performance while ensuring biological activity, making it suitable for promotion as a functional biological agent in oral applications.

[0110] Example 5: Therapeutic study of the composition in a DSS-induced mouse model of colitis

[0111] 1. Experimental animals and grouping: Forty male SPF-grade C57BL / 6 mice (20-22g) aged 6-8 weeks were randomly divided into four groups of 10 mice each.

[0112] (1) Control group: 0.2 mL of physiological saline was administered by gavage daily.

[0113] (2) Probiotic group: Lactobacillus plantarum CCFM8661 was administered by gavage daily at a dose of 1×10⁻⁶. 9 CFU.

[0114] (3) Fusion peptide group: 20 mg / kg of fusion peptide was administered by gavage daily, dissolved in PBS.

[0115] (4) Combination group: Daily gavage of probiotics + fusion peptides, at the same dosage as above.

[0116] 2. Model Establishment and Intervention Plan

[0117] (1) Starting from day 0, mice were allowed to drink water containing 3% (w / v) sodium dextran sulfate (DSS) for 7 consecutive days to establish an acute colitis model.

[0118] (2) Starting from day 1, except for the control group, the other groups were given the corresponding treatment by gavage daily for 14 days (including the DSS modeling period and the recovery period).

[0119] 3. Indicator Detection and Methods:

[0120] (1) Disease Activity Index (DAI) score (recorded daily): weight change score (0-4 points), stool morphology score (0-4 points), blood in stool score (0-4 points); DAI = average of the three scores, and the data on the 14th day is used for comparison.

[0121] (2) Colon length measurement: After the animal is euthanized, the colon is removed and the length from the cecum to the anus is measured as an indirect reflection of the degree of inflammation.

[0122] (3) Histopathological analysis (HE staining): Paraffin-embedded distal colon sections were sectioned and stained with HE. The crypt structure, epithelial integrity, goblet cell count, and inflammatory cell infiltration were observed. The degree of inflammation was analyzed using a blinded semi-quantitative scoring system (0-4 points).

[0123] (4) Serum inflammatory factor detection (ELISA): Blood was collected from the orbital cavity to separate serum and the concentrations of IL-6 and TNF-α were detected using the BioLegend mouse ELISA kit.

[0124] 4. The experimental results are shown in Table 8.

[0125] (1) DAI score and colon length: The average DAI score of the control group was 8.2, and the colon was significantly shortened (6.1cm), showing obvious diarrhea and weight loss; the DAI score of the combination group decreased significantly to 2.3, the colon length recovered to 9.8cm, and the inflammation was significantly relieved; probiotics or peptides alone were also effective, but lower than those in the combination group (P<0.01).

[0126] (2) Changes in inflammatory factors: In the control group, IL-6 and TNF-α were significantly increased, at 245 pg / mL and 180 pg / mL, respectively; After treatment, IL-6 in the combination group decreased to 68 pg / mL and TNF-α to 45 pg / mL, representing decreases of 72% and 75%, respectively; The peptide group and probiotic group also showed a decreasing trend, but the combination group had a better effect (P<0.001).

[0127] (3) Histological results: The control group showed extensive destruction of crypt structure, loss of more than 80% of goblet cells, and infiltration of a large number of neutrophils; the combination group showed that the number of goblet cells recovered to 70% of normal, the mucosal structure was reconstructed, and the inflammation score decreased by 85%; the polypeptide group and probiotic group also showed some improvement, but not as much as the combination group.

[0128] Table 8 Summary of Experimental Results

[0129]

[0130] Note: *P<0.05, **P<0.01, ***P<0.001 are all statistically significant compared with the control group.

[0131] 5. Mechanism Exploration and Summary: The probiotic + fusion peptide combination exhibits a significant synergistic effect:

[0132] (1) Colonization promotion effect: The fusion peptides enhance the adhesion ability of probiotics to the intestinal mucosa of mice. Quantitative fluorescence analysis showed that the retention time of bacteria in the combined group was extended by about 3.5 times.

[0133] (2) Dual anti-pathogen ability: HBD-3 peptide directly kills conditionally pathogenic bacteria. Western blot showed that the antibody scFv binds to Clostridium difficile toxin B at a rate of over 90%, significantly neutralizing its toxicity.

[0134] (3) Immunomodulatory effect: The combination group significantly reduced the level of serum pro-inflammatory factors, restored the integrity of the intestinal epithelial barrier, increased the distribution of goblet cells, and effectively relieved colitis.

[0135] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A high-affinity single-chain antibody against Clostridium difficile toxin B, characterized in that, The amino acid sequence of the single-chain antibody is shown in SEQ ID NO.

2.

2. A fusion polypeptide comprising the single-chain antibody of claim 1, characterized in that, The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO.

1.

3. The fusion polypeptide according to claim 2, characterized in that, The optimized nucleotide sequence of the fusion polypeptide codon is shown in SEQ ID NO.

3.

4. A composition comprising probiotics, characterized in that, The composition comprises the following components: (1) Probiotics: Lactobacillus plantarum CCFM8661, concentration ≥1×10 9 CFU / g; (2) Fusion polypeptide: the fusion polypeptide of claim 2, wherein the fusion polypeptide in the composition is mixed with probiotics at a mass ratio of 1:0.

5.

5. The composition according to claim 4, characterized in that, The composition is an enteric-coated microcapsule prepared by freeze-drying process, wherein the coating material of the enteric-coated microcapsule is hydroxypropyl methylcellulose, and the particle size of the enteric-coated microcapsule is 300–500 μm.

6. The use of the fusion polypeptide as described in claim 2 in the preparation of a composition comprising probiotics.

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