A recombinant lactococcus microcapsule, its preparation method and application

Abstract

This invention relates to a recombinant *Lactococcus lactis* microcapsule, its preparation method, and its applications, belonging to the field of genetic engineering. To address the technical problems of low safety, high side effects, unstable efficacy, and insufficient targeting in existing methods for treating or alleviating inflammatory bowel disease (IBD), this invention provides a recombinant *Lactococcus lactis* microcapsule. By integrating a fusion gene encoding catalase and superoxide dismutase, a recombinant *Lactococcus lactis* with antioxidant function is constructed. The recombinant *Lactococcus lactis* is then coated with a mucosa-adhesive chitosan and sodium alginate composite nanomaterial to obtain a microcapsule with antioxidant enzyme expression capabilities and a nano-protective coating. The recombinant *Lactococcus lactis* microcapsule provided by this invention can enhance the activity and bioavailability of orally delivered substances under the harsh acidic environment and complex physiological barriers of the gastrointestinal tract, providing a new strategy and experimental evidence for the clinical prevention and treatment of IBD.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and in particular relates to a recombinant lactococcus microcapsule, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a nonspecific inflammatory disease of the gastrointestinal tract characterized by intestinal inflammation, tissue damage, abdominal pain, frequent or persistent diarrhea, and rectal bleeding. IBD is characterized by low mortality, high morbidity, high disability rates, and a trend towards affecting younger individuals. It is primarily caused by factors such as genetic susceptibility, environment, pathogen infection, persistent inflammatory response, gut microbiota dysbiosis, and abnormal activation of the immune response; however, its etiology remains to be fully elucidated, and currently there is no radical cure for IBD. Existing treatments for IBD suffer from several drawbacks, including: primary unresponsiveness or secondary loss of response, high cost, need for injection administration, insufficient targeting, high safety risks, low bioavailability, unstable efficacy, and potential drug resistance.

[0003] Meanwhile, the harsh acidic environment and complex physiological barriers of the gastrointestinal tract make it difficult for orally delivered active substances to maintain their activity after reaching the lesion site. Due to the chronic nature of IBD, conventional clinical medications often require long-term, scheduled use, which increases treatment costs and the incidence of adverse reactions, and even poses a potential threat to patients' lives. Therefore, those skilled in the art are eager to develop a safe, effective method for treating or alleviating inflammatory bowel disease while minimizing risks. Summary of the Invention

[0004] This invention addresses the technical problems of low safety, high side effects, unstable efficacy, and insufficient targeting in existing methods for treating or alleviating inflammatory bowel disease. It provides a recombinant lactococcus microcapsule, its preparation method, and its application.

[0005] One objective of this invention is to provide a method for preparing recombinant Lactococcus lactis microcapsules, the method comprising the following steps:

[0006] S1: By seamless cloning, the fusion gene encoding catalase and superoxide dismutase was ligated to plasmid pNZ8149 to obtain a recombinant plasmid;

[0007] S2: The recombinant plasmid in S1 was introduced into the competent cells of Lactococcus lactis NZ3900 by electroporation to obtain recombinant Lactococcus lactis expressing antioxidant enzymes;

[0008] S3: Resuspend the pretreated recombinant lactococcus in chitosan solution for 30 min to obtain mixture 1; resuspend the pretreated mixture 1 in sodium alginate solution for 30 min to obtain mixture 2; resuspend the pretreated mixture 2 in chitosan solution for 30 min to obtain mixture 3; resuspend the pretreated mixture 3 in sodium alginate solution for 30 min to obtain mixture 4, which is the recombinant lactococcus microcapsule.

[0009] In a preferred embodiment of the present invention, the nucleotide sequence of the fusion gene in S1 is shown in SEQ ID NO.27.

[0010] In a preferred embodiment of the present invention, the electroporation step in S2 is as follows: the electroporation cup and the recovery medium are pre-cooled on ice for 15 min, Lactococcus lactis NZ3900 competent cells are taken out and thawed on ice. After thawing, the recombinant plasmid is added and mixed well, and the mixture is placed in an ice bath for 5 min to obtain a mixture. The mixed solution is transferred to the electroporation cup and placed in an electroporator for electroporation treatment. After the treatment, the mixed solution in the electroporation cup is transferred to an EP tube containing recovery medium and cultured at 30°C for 3 h to obtain a bacterial culture. The bacterial culture is centrifuged and spread onto a selective medium plate and cultured at 30°C.

[0011] In a preferred embodiment of the present invention, the electric shock device is configured with the following settings: voltage of 2000 V, resistance of 200 Ω, pulse of 25 μF, and electric shock time of 4.5-5 s.

[0012] In a preferred embodiment of the present invention, the recovery culture medium is composed of: GM17 liquid culture medium with 20 mmol·L⁻¹ added. -1 MgCl2 and 2 mmol·L -1 CaCl2, 121℃, high pressure for 30 min.

[0013] In a preferred embodiment of the present invention, the GM17 liquid culture medium is obtained by autoclaving M7 broth at 121°C for 15 minutes, sterilizing and cooling it, and then adding 0.5% glucose.

[0014] In a preferred embodiment of the present invention, the selective culture medium comprises: 20 g casein peptone, 5 g yeast extract, 2.5 g gelatin, 5 g D-glucose, 5 g lactose, 5 g sucrose, 4 g sodium chloride, 1.5 g sodium acetate, and 0.5 g ascorbic acid vitamin C. The medium is brought to a final volume of 1000 mL with distilled water, adjusted to pH 6.8, autoclaved at 121°C for 15 min, sterilized, and cooled. Then, 0.5% lactose and 0.004% bromocresol purple are added.

[0015] In a preferred embodiment of the present invention, the pretreatment step in S3 is: washing 2-3 times with 0.5 M NaCl solution.

[0016] A second objective of this invention is to provide a recombinant lactococcus microcapsule, which is obtained by the above-described preparation method.

[0017] A third objective of this invention is to provide an application of recombinant lactococcus microcapsules, wherein the application is to use the above-mentioned recombinant lactococcus microcapsules in the preparation of drugs for treating or alleviating inflammatory bowel disease.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a recombinant lactococcus microcapsule, using food-grade (GRAS) lactococcus as a genetic engineering host, and constructing a recombinant lactococcus with antioxidant function by integrating a fusion gene encoding catalase (CAT) and superoxide dismutase (SOD) (nucleotide sequence shown in SEQ ID NO.27); the recombinant lactococcus is coated with a mucoadhesive chitosan (CS) and sodium alginate (SA) composite nanomaterial to obtain a recombinant lactococcus microcapsule with antioxidant enzyme expression ability and a nano-protective coating.

[0019] This invention utilizes auxotrophic complementary genes lacF As a selection marker, only *Lactococcus lactis* with the successfully inserted target gene nucleotide sequence (as shown in SEQ ID NO. 27) can grow in a selective medium containing only lactose. This invention selects a complementary gene as a selection marker to replace antibiotic resistance genes, offering advantages such as high safety, low cost, and strong specificity. Simultaneously, this invention adds bromocresol purple to the selective medium. When the recombinant *Lactococcus lactis* utilizes lactose, it produces acidic metabolites, lowering the local pH, causing bromocresol purple to turn yellow, thus improving the accuracy of the selection results.

[0020] This invention involves coating recombinant *Lactococcus lactis* with composite nanomaterials at pH 6. Transmission electron microscopy (TEM) characterization confirmed that the four-layer coating treatment did not inhibit the growth of *Lactococcus lactis*, thus improving its survival rate under harsh conditions. The biofilm-coated recombinant *Lactococcus lactis* microcapsules (LL-SC-C2A2) provided by this invention can grow normally and secrete CAT and SOD proteins extracellularly. In vivo experiments demonstrated that LL-SC-C2A2 has good biocompatibility and safety. Through the synergistic effect of recombinant *Lactococcus lactis* carrying antioxidant genes and the polysaccharide nanocoating, it improved symptoms of colitis induced by sodium dextran sulfate (DSS), protected the integrity of the intestinal barrier, restored the expression of tight junctions in colonic tissue, improved intestinal permeability and intestinal flora species diversity, and inhibited the production of pro-inflammatory factors in the colon while enhancing the expression of anti-inflammatory cytokines.

[0021] The present invention provides a recombinant lactococcus microcapsule, which offers a new strategy and experimental basis for the clinical prevention and treatment of inflammatory bowel disease. Attached Figure Description

[0022] Figure 1 This is an electrophoresis image of the fusion gene PCR amplification in Example 1;

[0023] Figure 2 This is a back-amplification electrophoresis image of the pNZ8149 plasmid in Example 1;

[0024] Figure 3 This is a PCR identification diagram of LL-SC colonies in Example 1; lanes 1-10 are the numbers of randomly selected clones.

[0025] Figure 4 This is a growth curve diagram of different strains in Example 1;

[0026] Figure 5 The diagram shows the protein expression in the ultrasonic supernatant of bacterial cultures under different conditions in Example 1; lane 1 is the pNZ8149-1 empty vector group with 1 ng / mL inducer, lane 2 is the pNZ8149-5 empty vector group with 5 ng / mL inducer, lane 3 is the pNZ8149 empty vector group without inducer; lane 4 is the SODCAT recombinant bacteria without inducer; lane 5 is the SODCAT-1 recombinant bacteria with 1 ng / mL inducer, and lane 6 is the SODCAT-5 recombinant bacteria with 5 ng / mL inducer;

[0027] Figure 6 This is a potential diagram of the recombinant lactococcus before and after encapsulation in Example 1;

[0028] Figure 7The effect of different coatings on the growth of the strain in Example 1;

[0029] Figure 8 This is a TEM (Transmission Electron Microscopy) morphology identification image from Example 1;

[0030] Figure 9 The graphs for gastrointestinal fluid resistance detection in Example 1 are shown; a is a survival rate statistical graph, and b is a colony count statistical graph.

[0031] Figure 10 The graphs shown in Example 1 depict SOD activity under different conditions; a represents different culture times, and b represents different ultrasound conditions.

[0032] Figure 11 The antioxidant capacity of the strains under different treatments in Example 1 is shown in Figure 1; a represents SOD activity and b represents CAT activity; in the x-axis, 1 represents LL-SC-C2A2 with inducer added; 2 represents LL-SC with inducer added; 3 represents LL-SC without inducer added; 4 represents LL-p8149 with inducer added; and 5 represents LL-p8149 without inducer added.

[0033] Figure 12 The image shows the safety evaluation of LL-SC-C2A2 in Example 1; A is an HE-stained image; in B, ag represents the relative expression levels of mouse blood cell parameters WBC, RBC, HGB, HCT, MCH, MCHC, and PLT, respectively; in B, hk represents the relative expression levels of mouse liver and kidney function parameters ALT, AST, BUN, and ALB, respectively; and in B, l is a statistical graph of mouse condition scores during the feeding process.

[0034] Figure 13 This is a schematic diagram of the DSS-induced colitis treatment experiment in mice in Example 2;

[0035] Figure 14 This is a graph showing the changes in mouse body weight and the DAI index in Example 2; a represents mouse body weight, and b represents the DAI index.

[0036] Figure 15 The images show the detection of colon tissue length and intestinal permeability in mice in Example 2; a represents colon tissue length, and b represents intestinal permeability.

[0037] Figure 16 This is a graph showing the detection of intracellular reactive oxygen species in Example 2;

[0038] Figure 17 Image showing H&E, AB-PAS, and TUNEL staining of colon tissue in Example 2;

[0039] Figure 18The image shows the detection of inflammatory factors in the colon tissue of mice in Example 2; a is IL-6, b is IL-1β, c is TNF-α, d is TGF-β1, e is IL-10, and f is MPO.

[0040] Figure 19 Figure 1 shows the RT-PCR analysis of colon tissue in Example 2; Figure 2 shows the representative immunohistochemical staining of MUC-2, Claudin-1, Occludin-1, and ZO-1 in colon tissue; Figure 3 shows the quantitative analysis of tight junction proteins and mucin expression in colon tissue; Figure 4 shows the detection of mRNA expression levels of intestinal barrier-related genes; Figure 5 shows the detection of mRNA expression levels of related pathway genes.

[0041] Figure 20 The graphs shown are: A) the abundance of gut microbiota in Example 2; B) the Chao1 detection graph; C) the Simpson detection graph; D) the Shannon detection graph; E) the principal coordinate analysis graph; F) the non-metric multidimensional scaling analysis graph; and G) the genus-level analysis graph of gut microbiota. Detailed Implementation

[0042] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0044] The mice used in the following examples were male C57BL / 6 mice aged 6-8 weeks, purchased and housed in a specific pathogen-free (SPF) barrier environment (room temperature: 22℃-25℃, relative humidity: 40%-60%, lighting: 12 h / 12 ​​h circulating lighting) at Harbin CAS Biotechnology Co., Ltd. All animal experiments were approved by the Animal Ethics Committee of Northeast Agricultural University (ethics number: NEAUEC 20240435), and all personnel involved in the experiments were trained and strictly adhered to animal experiment ethics principles.

[0045] Example 1: Preparation of recombinant Lactococcus lactis

[0046] 1. Preparation of pNZ8149-CATSOD recombinant plasmid

[0047] By integrating the fusion gene encoding catalase (CAT) and superoxide dismutase (SOD) (nucleotide sequence shown in SEQ ID NO.27), optimizing the gene sequence according to the codon preference of Lactococcus lactis, adding a 6×His tag to the C-terminus of the tandem sequence, and sending it to Hangzhou Baosai Biotechnology Co., Ltd. for full sequence synthesis, the CATSOD-pET28a gene synthesis plasmid was obtained.

[0048] The fusion gene encoding catalase and superoxide dismutase (CATSOD gene) and the gene sequence of pNZ8149 were synthesized by Hangzhou Baosai Biotechnology Co., Ltd. Primers were designed using Primer 5.0, as shown in Table 1. All primers were synthesized by Shanghai Jierui Biotechnology Co., Ltd.

[0049] Table 1

[0050]

[0051] Using the CATSOD-pET28a gene-synthesized plasmid as a genomic template, PCR amplification was performed using the primers described in Table 1. The PCR amplification system is shown in Table 2. The amplification program was as follows: 94℃ pre-deformation for 5 min, 94℃ deformation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, 30 cycles, and a final extension at 72℃ for 10 min. The PCR products were identified by 1% agarose gel electrophoresis and then purified using a purification kit for later use.

[0052] like Figure 1 As shown, an approximately 2000 bp target fragment was detected by electrophoresis, indicating that the amplified CATSOD gene was correct.

[0053] Table 2

[0054]

[0055] Using the pNZ8149 vector as a template, the pNZ8149 vector was amplified in reverse by polymerase chain reaction. The polymerase chain reaction system is shown in Table 3. The reaction conditions were: 94℃ pre-deformation for 5 min, 94℃ deformation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, 32 cycles, and 72℃ final extension for 10 min.

[0056] like Figure 2As shown, the size of the polymerase chain reaction reverse amplification product was 2550 bp as detected by electrophoresis, indicating that the linearized vector obtained by polymerase chain reaction reverse amplification was correct.

[0057] Table 3

[0058]

[0059] After the polymerase chain reaction amplified fragment and the pNZ8149 vector were recovered from the solution, they were seamlessly cloned and ligated according to the following system to obtain the pNZ8149-CATSOD recombinant plasmid. The ligation system is shown in Table 4.

[0060] Table 4

[0061]

[0062] 2. Preparation of recombinant Lactococcus lactis

[0063] Lactococcus lactis NZ3900 competent cells (purchased from Hangzhou Baosai Biotechnology Co., Ltd.) were activated and cultured at 30℃. Single colonies were picked and inoculated into 5 mL of GM17 medium (liquid: M7 broth was autoclaved at 121℃ for 15 min, sterilized, cooled, and then 0.5% glucose was added; solid: M7 agar was autoclaved at 121℃ for 15 min, sterilized, cooled to 55℃, and then 0.5% glucose was added). The culture was statically incubated overnight. The overnight culture was further expanded to OD. 600 The bacterial cells were washed three times with pre-cooled washing buffer I (400 mL 0.5 M sucrose, 10% glycerol) and stored at 4°C. The bacterial cells were then resuspended in 4 mL washing buffer II (200 mL 0.5 M sucrose, 10% glycerol, 50 mM EDTA) and stored at 4°C. The resuspended cells were then aliquoted into pre-cooled 0.5 mL EP tubes (40 µL per tube) and stored at -80°C.

[0064] Pre-cool the electroporation cuvette and resuscitation medium on ice for 15 min. Remove the activated Lactococcus lactis NZ3900 competent cells and thaw them on ice. After thawing, add the pNZ8149-CATSOD recombinant plasmid and mix well. Incubate on ice for 5 min to obtain a mixture. Transfer the mixture to an electroporation cuvette and perform electroporation (the electroporator settings are: voltage 2000 V, resistance 200 Ω, pulse 25 μF, electroporation time 4.5-5 s). After treatment, transfer the mixture from the electroporation cuvette to a resuscitation medium containing GM17 broth with 20 mmol·L⁻¹. -1 MgCl2 and 2 mmol·L -1The culture was placed in EP tubes containing CaCl2 (autoclaved at 121℃ for 30 min) and incubated statically at 30℃ for 3 h. After centrifugation, the culture was spread onto selective medium (composition: 20 g casein peptone, 5 g yeast extract, 2.5 g gelatin, 5 g D-glucose, 5 g lactose, 5 g sucrose, 4 g sodium chloride, 1.5 g sodium acetate, and 0.5 g ascorbic acid vitamin C, diluted to 1000 mL with distilled water, adjusted to pH 6.8, autoclaved at 121℃ for 15 min; after sterilization and cooling, 0.5% lactose and 0.004% bromocresol purple were added) and incubated statically at 30℃.

[0065] Single colonies were picked from the overnight culture plates and identified by colony PCR. Each single colony was mixed in 10 μL of sterile water, and 0.5 μL was used as a template. The specific PCR validation system is shown in Table 5. The reaction conditions were: 94℃ pre-deformation for 5 min, 94℃ deformation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles, and a final extension at 72℃ for 10 min. The PCR products were validated using 1.0% agarose gel. For positive results, the PCR product (2359 bp) was sequenced for identification.

[0066] like Figure 3 As shown, a specific band of 2359 bp was detected by electrophoresis, indicating that the electroconversion was successful and Lactococcus lactis containing recombinant plasmid was obtained, abbreviated as: LL-SC.

[0067] Table 5

[0068]

[0069] (1) Drawing the growth curve:

[0070] To investigate the effect of antioxidant enzyme expression on the growth process of *Lactococcus lactis*, LL (*Lactococcus lactis*), LL-pNZ8149 (*Lactococcus lactis* containing the empty vector pNZ8149), and LL-SC were inoculated into fresh M17 liquid medium at a 2% inoculum. The effects of LL-pNZ8149 and LL-SC expression on the growth process were investigated at OD... 600 When the OD value reaches 0.3-0.4, the inducer Nisin is introduced. The change in OD value over a day is measured using a growth curve analyzer, with measurements taken every hour, and then the growth curve of the strain is plotted.

[0071] like Figure 4 As shown, LL grows the fastest, which may be because plasmid replication and expression require energy, while LL's energy is only used for growth; however, the growth trends of the three are roughly the same, indicating that the synthesis of antioxidant enzymes does not affect the normal growth of the strain, that is, protein expression has no effect on the growth of the strain.

[0072] (2) Expression and identification of recombinant proteins

[0073] The successfully identified recombinant lactococcus LL-SC was inoculated into GM17 liquid medium (M17 broth with 0.5 mol·L⁻¹ sucrose, 0.5% glucose and 2.5% glycine added, autoclaved at 115℃ for 15 min), and pNZ8149 empty vector was inoculated into 5 mL of MRS medium and incubated overnight at 30℃ until OD₀. 600 =0.3-0.4; Nisin was added to the culture medium at concentrations of 0 ng / mL, 1 ng / mL and 5 ng / mL respectively for induction, resulting in pNZ8149 empty vector group, pNZ8149-1 empty vector group and pNZ8149-5 empty vector group respectively.

[0074] The successfully identified recombinant lactococcus LL-SC was inoculated into GM17 liquid medium (M17 broth with 0.5 mol·L⁻¹ sucrose, 0.5% glucose, and 2.5% glycine, autoclaved at 115℃ for 15 min), and SODCAT recombinant bacteria were inoculated into 5 mL of MRS medium and incubated overnight at 30℃ until OD₂O₃. 600 =0.3-0.4; Nisin was added to the culture medium at concentrations of 0 ng / mL, 1 ng / mL and 5 ng / mL respectively for induction to obtain SODCAT recombinant bacteria, SODCAT-1 recombinant bacteria and SODCAT-5 recombinant bacteria respectively.

[0075] When the OD reached 0.8, the cultures were stored separately. 1.5 mL of each of the pNZ8149 empty vector group, pNZ8149-1 empty vector group, pNZ8149-5 empty vector group, SODCAT recombinant bacteria, SODCAT-1 recombinant bacteria, and SODCAT-5 recombinant bacteria were centrifuged (12000 r / min, 3 min), and then 400 μL of 10 mM pH 7.0 Tris-HCl buffer was added for sonication (400 W, 9 s for lysis, 15 s for rest, 3 min). 40 μL of the supernatant from the centrifuged culture (12000 r / min, 10 min) was added, and 10 μL of 5×SDS-loading buffer was added. The mixture was boiled for 5 min, and Western blotting was performed using a His-tagged mouse monoclonal antibody for identification.

[0076] like Figure 5As shown, the supernatant of the bacterial culture induced by Nisin showed a specific band at approximately 84 kDa, which was consistent with the expected size. However, the supernatant of the pNZ8149 empty vector and the uninduced LL-SC bacterial culture showed no band. This indicates that recombinant lactococcus can express the target protein in NZ3900 cells after Nisin induction, and it is expressed in the form of a soluble protein.

[0077] 3. Preparation of recombinant Lactococcus lactis microcapsules

[0078] Sodium alginate (2 mg / mL) and ethylene glycol chitosan (2 mg / mL) were dissolved in 0.5 M sodium chloride solution. The pH of the solution was then adjusted to 6.0 using NaOH or HCl. The obtained LL-SC was first washed with NaCl (0.5 M) solution and resuspended in chitosan solution for 30 min to obtain mixture 1, i.e., recombinant Lactococcus lactis microcapsules, abbreviated as LL-SC-C1. Mixture 1 was then washed a second time with NaCl (0.5 M) solution and resuspended in sodium alginate solution for 30 min to obtain mixture 2, i.e., recombinant Lactococcus lactis microcapsules, abbreviated as LL-SC-C1A1. Mixture 2 was then washed a third time with NaCl (0.5 M) solution and resuspended in chitosan solution for 30 min to obtain mixture 3, i.e., recombinant Lactococcus lactis microcapsules, abbreviated as LL-SC-C2A1. Mixture 3 was washed a fourth time with NaCl (0.5 M) solution and resuspended in sodium alginate solution for 30 min to obtain mixture 4, which is the recombinant Lactococcus lactis microcapsule, abbreviated as LL-SC-C2A2. Mixture 4 was washed a fifth time with NaCl (0.5 M) solution and resuspended in chitosan solution for 30 min to obtain mixture 5, abbreviated as LL-SC-C3A2; mixture 5 was washed a sixth time with NaCl (0.5 M) solution and resuspended in sodium alginate solution for 30 min to obtain mixture 6, which is the recombinant Lactococcus lactis microcapsule, abbreviated as LL-SC-C3A3.

[0079] (1) Potential analysis

[0080] Take 1 mL of fresh LL-SC, LL-SC-C1, LL-SC-C1A1, LL-SC-C2A1, and LL-SC-C2A2 prepared above and dilute them in 9 mL of deionized water. Gently vortex to disperse them evenly. Use a Malvern laser particle size analyzer to measure the potential of LL-SC, LL-SC-C1, LL-SC-C1A1, LL-SC-C2A1, and LL-SC-C2A2 respectively.

[0081] like Figure 6As shown, the voltage of LL-SC is negative (-23.14 ± 0.40 mV), which may be attributed to the presence of anionic groups on the bacterial surface. The surface potential of LL-SC-C1 becomes slightly positive (+6.37 ± 0.29 mV), indicating that this cationic polymer forms a coating around them. The surface potential of LL-SC-C1A1 becomes negative (-20.73 ± 0.83 mV), indicating that anionic polymers form an additional coating around LL-SC-C1A1.

[0082] (2) Determination of growth curve

[0083] To evaluate the effect of microcapsule coating on the activity and growth of Lactococcus lactis, the test bacteria were cultured in M17 broth at an inoculum of 2% for 16 h. The OD value changes within 0-16 h were measured using an enzyme-linked immunosorbent assay (ELISA) reader, with measurements taken every two hours, and the growth curves of the strains were plotted.

[0084] like Figure 7 As shown, LL-SC-C1A1 (double-layer coating) and LL-SC-C2A2 (quadruple-layer coating) exhibited similar growth trends to LL-SC, indicating that the double-layer and quadruple-layer coatings did not affect the growth of probiotics, while LL-SC-C3A3 prepared by the six-layer coating process showed a phenomenon of delayed probiotic growth.

[0085] Based on existing technology, the integrity, stability, density, and cross-linking sufficiency of the polyelectrolyte composite protective layer depend on two core parameters: charge matching degree (whether each coating layer can neutralize the opposite charge exposed in the previous layer) and interchain diffusion-interpenetration depth (whether adjacent polyelectrolyte chains can fully entangle at the interface, form hydrogen bonds and van der Waals contact, and form a physical-chemical "interlock"). Specifically, the double-coated LL-SC-C1A1 only completes "one charge reversal and one interpenetration," leaving unpaired charge defects within the membrane. The four-coated LL-SC-C2A2, through "two charge reversals and two interpenetrations," completely repairs residual charges, porosity, and mechanically weak areas, forming a three-dimensional interlocked polyelectrolyte composite network. Therefore, the four-coated LL-SC-C2A2 exhibits a more complete, stable, denser, and more fully cross-linked polyelectrolyte composite protective layer.

[0086] (3) Micromorphological analysis

[0087] The surface morphology of LL-SC-C2A2 was observed using TEM (transmission electron microscopy). The coated LL-SC-C2A2 samples were centrifuged (6000 r / min, 4 min), and the supernatant was removed. The precipitate was washed three times with PBS buffer and fixed overnight with 2.5% glutaraldehyde solution. The next day, the samples fixed the previous night were rinsed with 0.1 M phosphate buffer (pH 7.2), then fixed with 1% osmium tetroxide fixative, and rinsed again. The samples were dehydrated sequentially at 4°C using 50%, 70%, and 90% ethanol solutions, each for 8-10 min. Dehydration was then performed twice at 4°C using 100% ethanol (10 min each time). Dehydration was then performed at 4°C using a 1:1 solution of 100% ethanol and 100% acetone for 10 min. Finally, the samples were incubated with 100% acetone at room temperature for 5 minutes. The final solution was then treated with a 1:1 solution of pure acetone and Eponine. The 812 embedding solution was soaked overnight, and the cells were embedded the following morning. Polymerization was carried out for 3-9 days, followed by trimming, sectioning, and staining. Finally, the cells were observed.

[0088] like Figure 8 As shown, under a transmission electron microscope, LL-SC-C2A2 is covered by a smooth and transparent shell, and the surface of Lactococcus lactis is not visible.

[0089] (4) Evaluation of tolerance to simulated digestion

[0090] Gastric juice tolerance was evaluated according to T / CNHFA435-2024 published by the China Nutrition and Health Food Association. Take 8.0 mL of electrolyte solution A (composition: weigh 0.064 g potassium chloride, 0.015 g potassium dihydrogen phosphate, 0.263 g sodium bicarbonate, 0.345 g sodium chloride, 0.003 g magnesium chloride hexahydrate, 0.006 g ammonium carbonate, 1.5 g tryptone, and 0.05 g L-cysteine ​​hydrochloride monohydrate, dissolve thoroughly in 95 mL distilled water, adjust pH to 3.0 with concentrated hydrochloric acid or sodium hydroxide solution, bring volume to 100 mL, and autoclave at 121℃ for 15 min) and 1.0 mL of electrolyte solution B (composition: weigh 0.022 g calcium chloride dihydrate, dissolve in water and bring volume to 100 mL, autoclave at 121℃ for 15 min) into a beaker, add the equivalent of 4000 U of pepsin, adjust pH to 3.0 with 1 mol / L hydrochloric acid solution or sodium hydroxide solution, bring volume to 10 mL, mix well, and then autoclave at 0.22... The simulated gastric juice (SGJ) was prepared by filtration through a μm sterile filter membrane and used immediately after preparation.

[0091] Dissolve 3.4 g potassium dihydrogen phosphate in 125 mL of distilled water, then add 95 mL of 0.1 M sodium hydroxide, and bring the volume up to 500 mL with distilled water. Adjust the pH of the mixture to 7.5 using 0.1 M sodium hydroxide solution, and sterilize before adding 3 g of bile salts to prepare simulated intestinal fluid (SIJ).

[0092] Recombinant Lactococcus lactis cultured to the third generation (LL-SC) and recombinant Lactococcus lactis microcapsules (LL-SC-C2A2) were inoculated into SGJ at a ratio of 1:9 and incubated at 37°C with constant stirring (150 rpm) for 2 h. Then, they were transferred to SIJ and incubated with continuous stirring (150 rpm) for 4 h. The total viable count of the sample bacterial culture was determined at 0, 2 h, 4 h and 6 h. The total viable count was determined using the M17 agar pour plate method and expressed as log 10 CFU / g.

[0093] like Figure 9 As shown, in a simulated gastric fluid environment, the survival rate of embedded LL-SC-C2A2 was consistently higher than that of unembedded LL-SC, and the difference was significant; in a simulated intestinal fluid environment, the total number of viable bacteria in embedded LL-SC-C2A2 was consistently higher than that in unembedded LL-SC, and the difference was significant.

[0094] (5) Antioxidant capacity determination

[0095] LL-SC and LL-SC-C2A2 were inoculated into fresh M17 liquid medium at a 2% inoculum and cultured until OD500. 600 When the concentration reached 0.3-0.4, the inducer Nisin was introduced, and the mixture was washed twice with PBS buffer, sonicated, and the supernatant was collected. The content of each indicator was determined using SOD and CAT kits, respectively.

[0096] like Figure 10 As shown, there was no significant difference in SOD activity when the culture time was 6 or 10 h; however, the SOD activity was lower at 8 h than at 6 / 10 h. Since the supernatant of the culture medium dilutes the concentration of proteins secreted by the bacteria, intracellular protein content is more favorable for in vitro enzyme activity determination. Therefore, the supernatant after ultrasonic disruption was used for measurement. There were no significant differences when the ultrasonic conditions were 200 W for 10 min, 400 W for 3 min, or 5 min. Based on these results, the conditions selected for subsequent experiments in this invention were: a culture time of 6 hours, ultrasonic power of 400 W, duration of 3 min, and a 4 s working interval followed by a 9 s rest interval.

[0097] Based on the above-mentioned ultrasound conditions, the effects of inducers and coatings on antioxidant capacity were determined. Figure 11As shown, the antioxidant gene on the plasmid could only be expressed in the presence of the inducer (x-axis 1 and 2 represent LL-SC-C2A2 and LL-SC with the inducer added, respectively). Furthermore, the expression of antioxidant capacity in the empty vector (x-axis 4 represents LL-p8149 with the inducer added) was low even in the presence of the inducer (p<0.001). This result is consistent with the Western blotting results, and the presence of the coating does not affect protein expression.

[0098] (6) In vivo safety evaluation

[0099] This invention uses the NICE system for induced expression, and the *Lactococcus lactis* NZ3900 used is... lacF Defective strains, lacF The LL-SC-C2A2 gene was used as a selection marker in an in vivo study to evaluate its biocompatibility. Fourteen female C57BL / 6 mice (6-8 weeks old) were divided into two groups (n=7 per group) and orally administered either PBS or LL-SC-C2A2 for 7 consecutive days (acute toxicity test). Serum was collected for biochemical analysis. Major organs were harvested and analyzed by HE staining; staining results are presented at 100 μm. Mice were observed daily, and their coat, activity level, movement, and fecal condition were recorded and evaluated. On day 10, mice were euthanized by enucleation, and blood samples were collected for complete blood count and liver function enzyme measurements. Major organs were collected for histopathological evaluation by hematoxylin and eosin (HE) staining. Liver function enzymes included alanine aminotransferase (ALT / GPT), aspartate aminotransferase (AST / GOT), blood urea nitrogen (BUN), and albumin (ALB). The mouse condition assessment criteria are shown in Table 6.

[0100] Table 6

[0101]

[0102] like Figure 12As shown, mice given and not given LL-SC-C2A2 exhibited similar blood biochemistry, organ morphology, and living conditions, indicating that the toxicity of LL-SC-C2A2 to mice is negligible and it has high safety. No histological damage or morphological differences were observed in sections of major organs (i.e., colon, liver, kidney, and spleen); the blood cell parameters of mice, including white blood cells (WBC, a), red blood cells (RBC, b), hemoglobin (HGB, c), hematocrit (HCT, d), mean corpuscular hemoglobin (MCH, e), mean corpuscular hemoglobin concentration (MCHC, f), and platelets (PLT, g), were consistent with those of healthy mice; liver and kidney function parameters, including alanine aminotransferase (ALT, h), aspartate aminotransferase (AST, i), blood urea nitrogen (BUN, j), and albumin (ALB, k), were also within the normal range, further confirming the biocompatibility of LL-SC-C2A2. Furthermore, compared with mice not given LL-SC-C2A2, mice given LL-SC-C2A2 showed good condition in all aspects, including coat, activity level, movement status, and fecal condition, with no significant difference between the two groups (mice condition score during feeding, l). These results indicate that LL-SC-C2A2 did not induce any adverse side effects.

[0103] Example 2: Application of recombinant Lactococcus lactis microcapsules in the preparation of drugs for treating or alleviating inflammatory bowel disease

[0104] 1. In this embodiment, to reproduce the pathological characteristics of human ulcerative colitis, an experimental colitis model was established by injecting mice with 3% DSS (e.g., Figure 13 (As shown). Specifically: Forty female C57BL / 6 mice (6-8 weeks old) were divided into 5 groups of 8 mice each, and allowed to acclimatize to the environment for 1 week before further experiments. For the DSS-induced mouse IBD model, mice were fed drinking water containing 3% DSS for 5 days, then switched to regular water. Control group (NC) healthy mice received only regular water. Then, on predetermined dates, mice were gavaged with PBS, Lactococcus lactis encapsulated with empty vector (LL-p8149), recombinant Lactococcus lactis microcapsules (LL-SC-C2A2), and recombinant Lactococcus lactis (LL-SC). Nisin was added to the drinking water to induce the expression of CAT and SOD in the gastrointestinal tract. The concentration and volume of bacteria administered were 1×10⁻⁶. 9 (CFU / mL, 200 μL / mouse / day), mouse weight was recorded daily; at the end, mice were euthanized by ocular blood collection, colon tissue was quickly excised and fixed with 4% paraformaldehyde, and all dissected tissues were cryopreserved at -80°C for further analysis.

[0105] like Figure 14As shown in Part A, at the end of the modeling process, mice treated with LL-SC-C2A2, LL-SC, LL-p8149, and DSS all experienced varying degrees of weight loss; LL-SC and LL-pNZ8149 had a slight alleviating effect on DSS-induced weight loss, while LL-SC-C2A2 significantly alleviated DSS-induced weight loss. p <0.001).

[0106] (1) Disease Activity Index Score

[0107] The Disease Activity Index (DAI) scoring system serves as a comprehensive quantitative indicator for assessing the severity of IBD in models of disease. Mice induced by DSS exhibit typical characteristics of IBD, including severe weight loss, consistency in liquid feces, and significant bloody stools. In this embodiment, the weight of mice in each group was recorded daily from the start of DSS modeling (days 7-21), and the Disease Activity Index (DAI) was assessed. The DAI was evaluated based on the feces, bloody stools, and degree of weight loss, and is the average of these three factors. The DAI evaluation indicators are shown in Table 7.

[0108] like Figure 14 As shown in Part B, the DAI score of the LL-SC-C2A2 treatment group (1.61±0.33) was significantly lower than that of the DSS-induced group (2.92±0.17). p <0.001); it can be seen that the intervention in the LL-SC-C2A2 treatment group significantly reduced the severity of DSS-induced colitis.

[0109] Table 7

[0110]

[0111] (2) Measurement of colon length and intestinal permeability

[0112] Mice in each group were fasted for 6 hours and then administered FITC-Dextran (500 mg / kg) by gavage. Blood was collected 4 hours later, and serum was obtained by centrifugation in the dark. The absorbance of the serum was measured using a multi-functional microplate reader (excitation wavelength 492 nm, emission wavelength 525 nm). A standard curve of FITC-Dextran concentration-absorbance was constructed to obtain the concentration of FITC-Dextran in the serum of each group of mice. On day 21, mice in each group were sacrificed to obtain colon tissue for colon length measurement.

[0113] like Figure 15As shown in section A, the NC group mice had the longest colon length (6.90±0.30 cm). After DSS induction, the colon wall of the mice became thinner and the length significantly shortened (3.90±0.12 cm). The colon length of the LL-SC treatment group was 4.23±0.31 cm, and the colon length of the LL-pNZ8149 treatment group was 3.93±0.38 cm. It can be seen that compared with the LL-SC and LL-pNZ8149 treatment groups, the LL-SC-C2A2 treatment group significantly improved DSS-induced colonic injury (5.07±0.22 cm).

[0114] like Figure 15 As shown in Section B, the serum FITC-Dextran concentration in the DSS group was significantly higher than that in the NC group, confirming that DSS caused significant intestinal damage. The serum FITC-Dextran levels in the LL-SC and LL-SC-C2A2 treatment groups were significantly lower than those in the DSS group, and the LL-p8149 treatment group was also significantly lower than that in the DSS group. This demonstrates that the introduction of antioxidant genes plays a protective role for the intestinal mucosa. This invention protects intestinal integrity through the synergistic effect of recombinant Lactococcus lactis carrying antioxidant genes and polysaccharide encapsulation.

[0115] (3) Detection of ROS scavenging ability

[0116] To detect reactive oxygen species (ROS) production, the DCFH-DA fluorescent probe method was used, and the fluorescence intensity was positively correlated with the ROS level. Frozen sections of colon tissue from mice in each group were thawed at room temperature, dried, stained with ROS, and incubated in the dark (37°C for 30 min), then washed; the dye was then added again and incubated in the dark (room temperature for 10 min), washed with PBS, mounted, and then detected and quantified by flow cytometry.

[0117] like Figure 16 As shown, there were significant differences in colonic ROS levels among different groups. The ROS level in the NC group was 4.65% ± 0.05%, while the ROS level in the DSS-induced colitis group was significantly increased to 22.63% ± 1.24%. The ROS levels in the intervention groups were: 9.46% ± 0.46% in the LL-SC-C2A2 group, 14.11% ± 0.35% in the LL-SC group, and 16.10% ± 0.34% in the LL-pNZ8149 group. It can be seen that the LL-SC-C2A2 group showed a stronger ROS clearance capacity, with a clearance efficiency 1.5 times higher than that of the LL-SC group.

[0118] (4) Organizational morphology analysis

[0119] Colonic tissues obtained from mice sacrificed on day 21 were subjected to histopathological analysis (HE staining and AB-PAS staining) and apoptosis assessment (TUNEL staining). 2-4 mm mouse colonic tissues were fixed with 4% neutral paraformaldehyde, rinsed with running water, dehydrated with graded ethanol, cleared, paraffin-embedded, sectioned, baked, rehydrated, and stained with HE. After mounting and drying, the sections were scanned, observed, and photographed. The fixed colonic tissue sections were processed strictly according to the instructions of the AB-PAS staining kit. Goblet cells in the intestinal sections were assessed using Image-J software. Following the manufacturer's instructions, TUNEL staining was also applied using a TUNEL assay kit to evaluate apoptosis in the colonic tissues.

[0120] like Figure 17 As shown, DSS induction leads to significant pathological changes in colonic tissue, including epithelial cell necrosis and shedding, tissue structure destruction and loosening, crypt loss, submucosal edema, extensive inflammatory cell infiltration, and a severe reduction in goblet cells. The LL-SC-C2A2 group showed significantly improved colonic tissue pathology, characterized by compact epithelial structure, only sporadic inflammatory cells in the mucosal layer, intact glandular structure, and almost complete recovery of goblet cell numbers. TUNEL staining revealed only trace amounts of red fluorescence in the NC group (indicating baseline physiological apoptosis); conversely, the DSS group showed a significant increase in red fluorescence, an abnormality attributable to increased apoptosis rate due to stimulus-induced cell damage; the LL-SC-C2A2 group showed a reduced number of apoptotic cells, with apoptosis levels approaching those of the NC group. This indicates that the recombinant Lactococcus lactis microcapsules provided by this invention offer a certain degree of protection against DSS-induced cell damage.

[0121] (5) Cytokine level detection

[0122] Colon tissues were collected from mice in each group, and inflammatory cytokines were detected using an ELISA kit. All procedures were strictly performed in accordance with the manufacturer's instructions. The absorbance (450 nm) was measured using a multi-functional microplate reader, and the cytokine content was calculated based on the standard curve.

[0123] like Figure 18As shown, the levels of pro-inflammatory cytokines were significantly increased in the DSS group: IL-6 (73.84 ± 4.20 pg / mL), IL-1β (97.07 ± 5.34 pg / mL), TNF-α (684.55 ± 35.40 pg / mL), and TGF-β (258.76 ± 9.33 pg / mL). Conversely, the level of the anti-inflammatory cytokine IL-10 (44.19 ± 2.50 pg / mL) was significantly decreased. The LL-SC-C2A2 group significantly reduced the levels of all pro-inflammatory cytokines and significantly reduced MPO activity to 0.39 ± 0.05 U / mg. Therefore, LL-SC-C2A2 significantly alleviated DSS-induced colonic inflammation, as evidenced by a significant decrease in the levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, TGF-β), a significant increase in the level of the anti-inflammatory cytokine IL-10, and effective inhibition of MPO activity.

[0124] (6) Extraction of mRNA from colon tissue and RT-PCR analysis

[0125] 20-30 mg of colon tissue from each group of mice was cut into enzyme-free tubes, 1 mL of RNAiso plus was added, and the mixture was vortexed and incubated at room temperature for 5 min. The mixture was then centrifuged at 12000 rpm / min for 5 min at 4°C. The supernatant was carefully transferred to another enzyme-free tube, 200 µL of chloroform was added, the mixture was vortexed, incubated for 5 min, and then centrifuged for 15 min. The supernatant was transferred to another centrifuge tube. 1 mL of isopropanol was added, the mixture was mixed, and incubated for 10 min. After centrifugation at 4°C for 10 min, the supernatant was carefully poured off. The white precipitate at this point is RNA. The precipitate was washed with 75% ethanol, inverted, and centrifuged (4°C, 7500 rpm / min, 5 min), repeated twice. The ethanol was removed, the precipitate was retained, and the enzyme-free tube was rapidly dried under aseptic conditions until translucent. An appropriate amount of DEPC water was added to dissolve the RNA. The RNA concentration and purity were measured using a nucleic acid protein analyzer. When the OD... 260 / OD 280 When the value is in the range of 1.8-2.1, it is used for subsequent tests.

[0126] RNA was reverse transcribed into cDNA according to the PrimeScript™ RT kit instructions. The cDNA was then stored at -20°C for subsequent experiments. Finally, gene-specific primers were designed using Primer 5.0 software and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences for the target gene are shown in Table 8. Real-time RT-PCR reaction systems for cDNA were prepared according to the TB Green® Premix Ex Taq™ II kit instructions. Real-time RT-PCR reactions were performed using the QuantStudio® 3 system, with β-actin as the internal control gene. Each reaction was repeated at least three times.

[0127] Table 8

[0128]

[0129] Mucin-2 (MUC-2) is the main mucin secreted by goblet cells and constitutes the main chemical barrier of the colonic mucosa; as a complement to this protective layer, tight junction (TJ) proteins maintain the integrity of the physical barrier by regulating paracellular permeability. To assess the recovery of intestinal barrier function in DSS-induced inflammatory mice under different treatment groups, the expression of MUC-2 and TJ proteins was investigated.

[0130] like Figure 19 As shown in section A, immunofluorescence (IF) staining revealed that, compared to the NC group, DSS induction significantly disrupted the integrity of the intestinal barrier and impaired tight junctions between cells. The LL-SC-C2A2 group mice recovered tissue structure and significantly increased the expression levels of these four proteins, bringing them close to the levels in the NC group.

[0131] Quantitative analysis results as follows Figure 19 As shown in the BE study, the mean optical density (MOD) values ​​of MUC-2, Claudin-1, Occludin-1, and ZO-1 in the DSS-induced group were significantly reduced, with the LL-SC-C2A2 group exhibiting the most significant repair effect. This indicates that LL-SC-C2A2 alleviates the loss of MUC-2 and tight junction proteins in mouse colon tissue, thereby stabilizing the mucosal barrier.

[0132] like Figure 19 As shown in section FI, compared with the DSS group, the LL-SC-C2A2, LL-SC, and LL-p8149 treatment groups all showed varying degrees of upregulation of MUC-2 expression. This restoration of mucus secretion is consistent with the previous AB-PAS staining results, confirming its role in maintaining mucosal integrity and protecting goblet cell function.

[0133] In summary, improvements in MUC-2 and tight junction proteins may alleviate DSS-induced inflammatory bowel disease (IBD) inflammation in mice by enhancing the protective effects of the epithelial and mucosal barriers; the LL-SC-C2A2 treatment group showed the best effect in upregulating MUC-2 and TJ proteins.

[0134] like Figure 19 As shown in the JM section, the levels of Keap1 protein were significantly increased and the levels of Nrf2 protein were significantly decreased in mice induced by DSS, indicating that the colitis mouse model was successfully established and that DSS induction inhibited the Nrf2-Keap1 signaling pathway. In the LL-SC-C2A2 treatment group, the levels of Keap1 protein were significantly decreased and the levels of Nrf2 protein were significantly increased, indicating that LL-SC-C2A2 treatment significantly alleviated the inhibitory effect on the Nrf2-Keap1 signaling pathway.

[0135] NADPH quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1) are downstream effector molecules of the Nrf2-Keap1 pathway; after DSS induction, the expression of NQO1 and HO-1 genes in mice was significantly inhibited. Compared with the DSS treatment group, the mRNA levels of NQO1 and HO-1 were significantly increased in the LL-SC-C2A2 treatment group. Therefore, the recombinant Lactococcus lactis microcapsules (LL-SC-C2A2) provided by this invention inhibit oxidative stress by regulating the expression levels of related mRNAs.

[0136] (7) Determination of gut microbiota abundance

[0137] To investigate the ability of LL-SC-C2A2 to regulate the gut microbiota, 16S rDNA analysis was performed on the intestinal contents of different groups of mice. DNA was extracted from the intestinal contents of each group of mice using the CTAB method. PCR amplification was performed on the V3-V4 region of the 16S rDNA samples using primers shown in SEQ ID NO. 25-26. To determine whether the PCR products could be used for subsequent experiments, the products were purified by gel extraction and analyzed using an Agilent 2100 bioanalyzer and an Illumina platform. Statistical analysis was performed using GraphPad Prism 10, and data are expressed as mean ± standard deviation. One-way ANOVA was used for statistical significance analysis. * : P <0.05; ** : P <0.01; *** : P <0.001; **** : P <0.0001.

[0138] like Figure 20 As shown in section AC, there were significant differences between the DSS group and the NC group in α-diversity indices (including Chao1, Simpson, and Shannon). In the LL-SC-C2A2 treatment group, all of these indices recovered to levels comparable to those in the NC group. This demonstrates that LL-SC-C2A2 has a significant effect on improving α-diversity of the gut microbiota.

[0139] like Figure 20 As shown in Section D, the spatial distribution of the samples is visualized using principal coordinate analysis (PCoA) based on weighted UniFrac distance. The percentages on the horizontal axis (PCoA1, 55.12%) and the vertical axis (PCoA2, 25.71%) represent the explanatory power of each principal coordinate axis for sample differences. The NC group samples exhibited tight intragroup clustering, indicating a high degree of structural homogeneity; while the DSS group samples were distributed across different quadrants, showing severe dysbiosis; the LL-SC-C2A2 group restored the microbial community structure, and its composition was most similar to that of the NC group.

[0140] like Figure 20 As shown in section E, the results of nonmetric multidimensional scaling (NMDS) are consistent with those of PCoA. The DSS group samples are far from the NC group and have a certain degree of dispersion within the group, further confirming that DSS induces changes in the composition of the mouse gut microbiota. The changes in the microbiota structure of the LL-SC-C2A2 treatment group are improved, and the diversity tends to be restored.

[0141] Gate horizontal analysis results are as follows Figure 20 As shown in section F, DSS-induced treatment significantly reduced Firmicutes (Firmwallis) and Bacteroidetes The abundance of (Bacteroidetes) was significantly increased, and at the same time, the abundance of (Bacteroidetes) was significantly increased. Proteobacteria (Proteobacteria) Campylobacterota (Campylobacteria) and Actinobacteria The abundance of (Actinomycetes) was increased in the LL-SC-C2A2 treatment group. Firmicutes , Bacteroidetes and Verrucomicrobia The abundance of (verrucous microbes) was significantly reduced, while the abundance of (verrucous microbes) was also significantly reduced. Proteobacteria The abundance; and, the LL-SC-C2A2 treatment group Firmicutes and Proteobacteria The proportion is closest to that of the NC group.

[0142] The changes in the abundance of Firmicutes and Bacteroidetes reflected the DSS-induced gut microbiota dysbiosis in mice, which disrupted intestinal homeostasis and led to intestinal dysfunction. After intervention with different treatment groups, the gut microbiota of mice showed varying degrees of improvement at the phylum level, with the LL-CS-C2A2 treatment group showing the most significant effect. This may be because the components in the embedding material not only protect the bacteria but also act as prebiotics, promoting the proliferation of dominant probiotics.

[0143] Results of genus-level analysis of gut microbiota as follows Figure 20 As shown in section G, DSS induction significantly increased the abundance of pathogenic genera, while the abundance of pathogenic genera decreased significantly in the LL-SC-C2A2 group, and the abundance of beneficial genera increased. Among them, although LL-SC also showed a positive regulatory effect, its efficacy was weaker than that of LL-SC-C2A2.

[0144] As can be seen, the LL-SC-C2A2 provided by this invention, with its recombinant Lactococcus lactis carrying antioxidant genes and polysaccharide nanocoating working synergistically, provides prebiotic polysaccharides while preventing the recombinant Lactococcus lactis from releasing antioxidant enzymes before reaching the intestines. Thus, the LL-SC-C2A2 provided by this invention, with its polysaccharide nanocoating, protects the recombinant Lactococcus lactis carrying antioxidant genes through a physical barrier, enabling it to successfully reach the intestines and continuously release antioxidant enzymes; simultaneously, the coating material itself can act as a prebiotic, jointly promoting the proliferation of beneficial bacteria and restoring the homeostasis of the microbial community.

[0145] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A method for preparing recombinant lactococcus microcapsules, characterized in that, The preparation method includes the following steps: S1: By seamless cloning, the fusion gene encoding catalase and superoxide dismutase was ligated to plasmid pNZ8149 to obtain a recombinant plasmid; S2: The recombinant plasmid in S1 was introduced into the competent cells of Lactococcus lactis NZ3900 by electroporation to obtain recombinant Lactococcus lactis expressing antioxidant enzymes; S3: Resuspend the pretreated recombinant Lactococcus lactis in chitosan solution for 30 min to obtain mixture 1; resuspend the pretreated mixture 1 in sodium alginate solution for 30 min to obtain mixture 2; resuspend the pretreated mixture 2 in chitosan solution for 30 min to obtain mixture 3; resuspend the pretreated mixture 3 in sodium alginate solution for 30 min to obtain mixture 4, which is the recombinant Lactococcus lactis microcapsule; The nucleotide sequence of the fusion gene described in S1 is shown in SEQ ID NO.

27.

2. The preparation method according to claim 1, characterized in that, The electroporation steps described in S2 are as follows: Pre-cool the electroporation cuvette and recovery medium on ice for 15 min, remove Lactococcus lactis NZ3900 competent cells, thaw them on ice, add the recombinant plasmid after thawing, mix well, and incubate on ice for 5 min to obtain a mixture; transfer the mixed solution to the electroporation cuvette and place it in an electroporator for electroporation treatment; after treatment, transfer the mixed solution in the electroporation cuvette to an EP tube containing recovery medium, and incubate at 30℃ for 3 h to obtain a bacterial culture; centrifuge the bacterial culture and spread it onto a selective medium plate, and incubate at 30℃.

3. The preparation method according to claim 2, characterized in that, The electric shock device is configured with the following settings: voltage of 2000 V, resistance of 200 Ω, pulse of 25 μF, and shock duration of 4.5-5 s.

4. The preparation method according to claim 2, characterized in that, The recovery culture medium consisted of GM17 liquid culture medium with 20 mmol·L⁻¹ added. -1 MgCl2 and 2 mmol·L -1 CaCl2, 121℃, high pressure for 30 min.

5. The preparation method according to claim 4, characterized in that, The GM17 liquid culture medium is obtained by autoclaving M7 broth at 121°C for 15 minutes, sterilizing and cooling it, and then adding 0.5% glucose.

6. The preparation method according to claim 2, characterized in that, The selective culture medium consists of: 20 g casein peptone, 5 g yeast extract, 2.5 g gelatin, 5 g D-glucose, 5 g lactose, 5 g sucrose, 4 g sodium chloride, 1.5 g sodium acetate, and 0.5 g ascorbic acid vitamin C. The medium is brought to a final volume of 1000 mL with distilled water, adjusted to pH 6.8, autoclaved at 121°C for 15 min, sterilized, and cooled. Then, 0.5% lactose and 0.004% bromocresol purple are added.

7. The preparation method according to claim 1, characterized in that, The pretreatment step described in S3 is: washing 2-3 times with 0.5 M NaCl solution.

8. A recombinant lactococcus microcapsule, characterized in that, The recombinant lactococcus microcapsules are obtained by the preparation method according to any one of claims 1 to 7.

9. The application of recombinant lactococcus microcapsules, characterized in that, The application is to use the recombinant lactococcus microcapsules of claim 8 to prepare a drug for treating or alleviating inflammatory bowel disease.

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