Probiotic delivery system based on heterologous expression of S-layer protein as well as preparation method and application of probiotic delivery system

Abstract

The invention provides an S-layer protein heterologous expression-based probiotic delivery system as well as a preparation method and application thereof, and relates to the technical field of probiotic delivery. The probiotic delivery system provided by the invention comprises probiotics, wherein the probiotics carry S-layer protein displayed on the surface. The probiotic delivery system is subjected to genetic modification, so that the probiotics can stably express S-layer protein, the S-layer protein is secreted out of cells, the activity of the S-layer protein is stably displayed on the surfaces of probiotic thalli, and a probiotic expression system is formed and has the functions of enhancing tolerance, adhesion and long-term colonization; the industrial bottleneck that the naturally extracted SLP is low in yield, high in cost and tedious in process is solved, the defects that the SLP embedded on the surface of the probiotics cannot be continuously generated and cannot continuously play a role after being digested are overcome, and a low-cost and high-yield SLP recombinant expression platform is established.

Description

Technical Field

[0001] This invention belongs to the field of probiotic delivery technology, specifically relating to a probiotic delivery system based on heterologous expression of S-layer proteins, its preparation method, and its application. Background Technology

[0002] Probiotics are a class of live microorganisms that provide health benefits to the host. Their efficacy depends on a sufficient number of live bacteria successfully reaching the intestines and colonizing effectively. However, current probiotic preparations face severe challenges in practical applications, significantly limiting their sustained probiotic effects. This stems primarily from two obstacles: First, probiotics encounter extreme environments such as stomach acid and bile salts during their journey through the upper digestive tract, leading to a sharp decline in survival rates. Second, even if some bacteria survive to reach the colon, their colonization time within the intestines is extremely short (usually only a few hours to a few days), making it difficult to sustain their effects in regulating gut microbiota balance, enhancing barrier function, and modulating the immune system.

[0003] To address these issues, various probiotic delivery strategies have been proposed in recent years. For example, patent CN120617198A uses thiohyaluronic acid hydrogel as a carrier for the encapsulation and delivery of probiotics, thereby improving the survival rate of probiotics in the gastrointestinal environment. Patent CN115381101A presents a scheme for preparing probiotic microcapsules based on a composite aggregation method of modified soybean protein and polysaccharides. This method produces probiotic microcapsules with advantages such as minimal loss of probiotic activity, long duration of activity maintenance, and effective delivery of probiotics to the intestines to exert their beneficial effects. However, the above microcapsule technologies generally suffer from insufficient precision in controlling intestinal targeted release and low colonization efficiency, which restricts the full realization of their beneficial effects. Patent CN113230280B describes the preparation of multilayer encapsulated microcapsules for probiotics using electrostatic spraying of sodium alginate, pectin, and soy protein isolate. However, multilayer encapsulation technology generally suffers from problems such as low biocompatibility of encapsulation materials, loose encapsulation layer structure, and easy dissociation. Furthermore, multilayer encapsulation wall materials may prevent bacterial release and inhibit bacterial bioactivity. Therefore, there is an urgent need to develop novel wall materials with multiple functions, including enhancing gastrointestinal tolerance, intestinal adhesion, and intestinal colonization ability, to construct probiotic delivery systems. Summary of the Invention

[0004] To address the aforementioned technical problems, the primary objective of this invention is to provide a probiotic delivery system based on heterologous expression of S-layer proteins. This probiotic delivery system enhances gastrointestinal tolerance, intestinal adhesion, and intestinal colonization capabilities, thus possessing enhanced tolerance, adhesion, and long-term colonization functions.

[0005] The second objective of this invention is to provide a method for preparing a probiotic delivery system based on heterologous expression of S-layer proteins.

[0006] A third objective of this invention is to provide the application of the above-described probiotic delivery system or preparation method in the preparation of products that improve the gastrointestinal tolerance of probiotics.

[0007] A fourth objective of this invention is to provide the application of the above-described probiotic delivery system or preparation method in the preparation of products that enhance the intestinal adhesion and colonization capabilities of probiotics.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a probiotic delivery system based on heterologous expression of S-layer protein, comprising probiotics carrying S-layer protein displayed on their surface.

[0009] In one embodiment, the probiotics include any species of the genus Lactococcus.

[0010] The present invention also provides a method for preparing the above-mentioned probiotic delivery system based on S-layer protein recombinant plasmid, comprising the following steps: introducing the S-layer protein recombinant plasmid into probiotics, and then inducing the expression of S-layer protein in an induction medium to obtain a probiotic delivery system loaded with S-layer protein.

[0011] As one embodiment, the nucleotide sequence of the S-layer protein recombinant plasmid is shown in SEQ ID No. 1.

[0012] In one implementation, the induction temperature is 28~35℃ and the duration is 22~26 h.

[0013] In one embodiment, the induction culture medium includes an inducer, which is nisin, and the concentration of nisin is 80~100 ng / mL.

[0014] In one embodiment, the expression vector of the S-layer protein recombinant plasmid includes pNZ8148-PASPA.

[0015] As one implementation method, the importation method includes electroconversion.

[0016] The present invention also provides the application of the above-described probiotic delivery system or the above-described preparation method in the preparation of products that improve the gastrointestinal tolerance of probiotics.

[0017] The present invention also provides the application of the above-described probiotic delivery system or the above-described preparation method in the preparation of products that enhance the intestinal adhesion and colonization ability of probiotics.

[0018] The advantages of this invention compared to existing technologies are as follows: This invention provides a probiotic delivery system based on heterologous expression of S-layer protein. By secreting SLP protein extracellularly and expressing S-layer protein on the surface of probiotics, a probiotic expression system is formed. This probiotic delivery system improves the structural stability of probiotics in the complex gastrointestinal fluid environment, enhances their tolerance to gastrointestinal digestion, strengthens their colonization performance in the intestine, and prolongs their colonization time in vivo, exceeding 16 days. It also exhibits enhanced tolerance, adhesion, and long-term colonization functions. Furthermore, this probiotic delivery system demonstrates biocompatibility in the intestine. This invention overcomes the industry bottlenecks of low yield, high cost, and cumbersome processes associated with naturally extracted SLP, and overcomes the drawbacks of encapsulating SLP on the surface of probiotics, which results in continuous production after digestion and thus lack of sustained efficacy. It establishes a low-cost, high-yield SLP recombinant expression platform. Attached Figure Description

[0019] Figure 1 Transmission electron microscopy characterization of a probiotic delivery system for heterologous SLP expression was performed, with NZ9000 / E as comparative example 1 and NZ9000 / SLP as example 1. Figure 2 The results of the single-factor experiments for induced expression are shown in Figure 1. A represents the SDS-PAGE spectrum, A1 represents the optimization of induction temperature, A2 represents the optimization of bacterial concentration before induction, A3 represents the optimization of nisin induction concentration, A4 represents the optimization of induction time, and B represents the Imagej quantitative analysis of the SLP target protein band. Figure 3 To compare the viable bacterial counts in simulated dynamic gastrointestinal digestion using a probiotic delivery system that constructs a heterologous SLP expression model. Figure 4 To improve the adhesion rate of intestinal epithelial cells to a probiotic delivery system that expresses SLP heterologously; Figure 5 In vitro fluorescence imaging of a probiotic delivery system for heterologous SLP expression in the mouse digestive tract. Detailed Implementation

[0020] This invention provides a probiotic delivery system based on an S-layer protein recombinant plasmid, comprising probiotics carrying an S-layer protein displayed on their surface. As an optional embodiment, the probiotics include any species of the genus *Lactococcus*, preferably *Lactococcus lactis* NZ9000.

[0021] The present invention also provides a method for preparing the above-mentioned probiotic delivery system based on S-layer protein recombinant plasmid, comprising the following steps: introducing the S-layer protein recombinant plasmid into probiotics, and then inducing the expression of S-layer protein in an induction medium to obtain a probiotic delivery system loaded with S-layer protein; the nucleotide sequence of the S-layer protein recombinant plasmid is shown in SEQ ID No. 1.

[0022]

[0023] This invention uses the S-layer protein (SLP) gene (SEQ ID No. 2) as a template, and performs PCR amplification using forward primer 8PSLPF (SEQ ID No. 3) and reverse primer 8PSLPR (SEQ ID No. 4) as specific primers. The SLP gene is then recovered and purified. The product obtained by enzyme digestion of the SLP gene with an expression vector, preferably pNZ8148-PASPA, is then transformed into competent cells, and the recombinant plasmid of the S-layer protein is obtained after screening and extraction.

[0024]

[0025] SEQ ID No. 3: CGCGGATCCAAATCATATGCCAAA.

[0026] SEQ ID No. 4:TTTGTAGAACGATAAGGTACCTAT.

[0027] Then, this invention constructs probiotic competent cells. The preparation method of the probiotic competent cells includes transferring the probiotics to a glycine medium and culturing for 5-7 hours. The glycine medium contains 1-4% glycine, preferably at a concentration of 1%, 2%, 3%, or 4%. This invention weakens the bacterial cell wall by adding glycine to the medium, making the cell wall more porous and easier to absorb exogenous plasmids. The cells are then obtained by centrifugation and resuspended in electroporation washing buffer. This invention pre-treats the cells with electroporation washing buffer, reducing ionic strength, maintaining osmotic pressure and low temperature, preserving cell viability, increasing cell density, increasing contact opportunities with recombinant plasmids, and improving the conversion efficiency of electroporation. In this invention, the probiotics include *Lactococcus*, *Lactobacillus*, *Streptococcus*, and *Leuconostoc*. This invention uses *Lactococcus lactis* NZ9000 as the probiotic; *Lactococcus lactis* is a mature model bacterium.

[0028] This invention constructs an S-layer protein recombinant plasmid and probiotic competent cells, and then introduces the S-layer protein recombinant plasmid into the probiotic competent cells. The introduction method includes electroporation. The voltage of the electroporation method is 1000~1500 V, the capacitance is 20~30 µF, and the resistance is 150~250 Ω. This invention introduces exogenous S-layer protein recombinant plasmids into probiotics via electroporation to obtain recombinant expression strains.

[0029] This invention induces the expression of S-layer protein by culturing recombinant expression strains. In this invention, the recombinant expression strain is inoculated into an induction medium, wherein the bacterial concentration at inoculation is OD0.05. 600 The OD is 0.4~0.8. 600The preferred concentrations are 0.5, 0.6, or 0.7. The induction medium includes an inducer, which is nisin, at a concentration of 60-100 ng / mL, preferably 70 ng / mL, 80 ng / mL, or 90 ng / mL. The induction temperature in this invention is 28-35°C, preferably 30°C, 32°C, or 34°C, and the induction time is 22-26 h, preferably 22 h, 24 h, or 25 h. The pNZ8148-PASPA vector of this invention contains a USP45 signal peptide, which can secrete SLP protein extracellularly, expressing S-layer protein on the surface of probiotics to form a probiotic expression system. Under the induction conditions of this invention, the SLP expression level can be maximized, thereby improving the tolerance of the prepared probiotic delivery system to gastrointestinal digestion, as well as its adhesion ability and colonization time in the intestine.

[0030] Based on the fact that the probiotic delivery system of this invention can improve the tolerance of probiotics to gastrointestinal digestion, this invention also provides the application of the above-mentioned probiotic delivery system or the above-mentioned preparation method in the preparation of products with improved probiotic gastrointestinal tolerance. Through simulated gastrointestinal digestion experiments, this invention demonstrates that the probiotic delivery system of this invention improves the high structural stability of probiotics in the complex gastrointestinal fluid environment and enhances the tolerance of probiotics to gastrointestinal digestion.

[0031] Based on the fact that the probiotic delivery system of this invention can enhance the intestinal adhesion and colonization ability of probiotics, this invention also provides the application of the above-mentioned probiotic delivery system or the above-mentioned preparation method in the preparation of products that enhance the intestinal adhesion and colonization ability of probiotics. Through in vivo experiments on mice, this invention verifies that the probiotic delivery system of this invention improves the colonization performance of probiotics in the intestine, prolongs the colonization time of probiotics in vivo (exceeding 16 days), and is harmless to mice, demonstrating biosafety.

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of this invention.

[0033] Unless otherwise specified, the materials, reagents, etc. used in the following examples are commercially available. Unless otherwise specified, they are generally used under conventional conditions or under conditions recommended by the company.

[0034] M17 liquid culture medium (lactic acid bacteria electroconversion wash): contains 42.3 g / L M17 broth, 0.5 mol / L sucrose, 0.5% glucose, 2 mmol / L CaCl2 solution and 20 mmol / L MgCl2 solution.

[0035] GM17 liquid medium (Lactococcus lactis electroconversion resuscitation solution): 42.3 g / L M17 liquid medium and 0.5% glucose.

[0036] Example 1: Preparation of a probiotic delivery system 1. Constructing S-layer protein recombinant plasmids Amplification of the full-length S-layer protein (SLP) gene: Using the SLP gene (SEQ ID No. 2) as a template, and with forward primer 8PSLPF (SEQ ID No. 3) and reverse primer 8PSLPR (SEQ ID No. 4) as specific primers, PCR amplification was performed according to the PCR amplification system (see Table 1) and amplification program (see Table 2). The PCR amplification products were recovered using the TIANGEN DNA gel extraction kit and purified using the TIANGEN DNA product purification kit to obtain the SLP gene.

[0037] Table 1. PCR amplification system of the target gene

[0038] Table 2 PCR amplification procedure for the target gene

[0039] Enzyme digestion and ligation: *E. coli* containing the pNZ8148-PASPA plasmid (containing the USP45 secretion signal peptide) were cultured. Plasmids were extracted using a TIANGEN plasmid miniprep kit. The extracted pNZ8148-PASPA plasmid and the purified SLP gene were subjected to a double enzyme digestion reaction at 37°C for 2 h. The digestion sites were BamHI and KpnI. The digestion system is shown in Tables 3 and 4. The digested products were purified by agarose gel electrophoresis. The purified products were ligated at 25°C for at least 1 h. The ligation system is shown in Table 5. 10 μL of the ligation product was added to... E. coli In MC1061 competent cells, the cells were incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, and then incubated on ice for 3 min. 1 mL of LB liquid medium was added, and the cells were incubated at 37℃ with shaking at 200 r / min for 1 h. 100 μL of the transformed bacterial culture was evenly spread onto a plate containing chloramphenicol (Cm) and incubated at 30℃ for 12 h. Single-clone verification and sequence comparison were performed. Single colonies were picked for colony PCR verification, and agarose gel electrophoresis was used to verify the transformation results. Successfully ligated positive single-clone colonies were cultured, and the S-layer protein recombinant plasmid pNZ8148-PASPA-SLP (SEQ ID No. 1) was extracted and finally sent to Genewiz for sequencing.

[0040] Table 3. Enzyme digestion system for plasmid double digestion

[0041] Table 4. Enzyme digestion system for SLP double digestion

[0042] Table 5 Connection System

[0043] 2. Preparation of probiotic competent cells Lactococcus lactis NZ9000 ( L.lactis NZ9000 was streaked onto GM17 plates and incubated at 30°C for 24 h. A single colony was picked and placed in 5 mL of GM17 liquid medium, then incubated overnight at 30°C for activation. The activated strain was then transferred at a 1% inoculation ratio to 100 mL of M17 liquid medium containing 10% glycine (M17 liquid medium with 10% glycine added), and incubated at 30°C for 5–7 h (OD measured every h). 600 (Value). When OD 600 When the concentration reaches 0.5-0.6, add ampicillin (Amp) to a final concentration of 20 µg / mL and incubate at 30°C for 1 h. Aliquot the bacterial culture into ice-bathed centrifuge tubes and centrifuge at 5000 rpm for 10 min at 4°C. Remove the supernatant and add an equal volume of lactic acid bacteria electroporation washing buffer (after ice bath treatment). Resuspend the bacteria by shaking and centrifuge at 6000 rpm for 10 min at 4°C, then remove the supernatant. Add 1 / 2 volume of lactic acid bacteria electroporation washing buffer (after ice bath treatment), resuspend the bacteria by shaking and centrifuge at 6000 rpm for 10 min at 4°C, then remove the supernatant. Add 1 / 4 volume of lactic acid bacteria electroporation washing buffer (after ice bath treatment), resuspend the bacteria by shaking and centrifuge at 7000 rpm for 10 min at 4°C, then remove the supernatant. Finally, add 1% of the lactic acid bacteria electroconversion washing buffer by volume, mix the bacterial cells thoroughly, and dispense 50 µL / tube into 1.5 mL EP tubes to obtain competent cells, which are then stored at -80℃.

[0044] 3. Electroconversion The successfully constructed S-layer protein recombinant plasmid was transferred into [the target protein] using electroporation. L.lactis NZ9000 competent cells. The specific steps are: […]. L.lactisNZ9000 competent cells and pNZ8148-PASPA-SLP plasmid were placed in an electroporation cuvette and electroporated using an electroporator at 1200 V, 25 µF, and 200 Ω. The cells were then resuspended in 1 mL of Lactococcus lactis electroporation resuscitation medium (on ice). The resuscitation solution was transferred to a 1.5 mL centrifuge tube and incubated at 30°C with shaking for 2 h. The cells were collected by centrifugation at 8000 r / min for 1 min, and approximately 100 µL of supernatant was used to resuspend the cells. The bacterial suspension was then plated onto GM17 agar plates containing 10 µg / mL chloramphenicol and incubated at 30°C until single colonies appeared, approximately 12–24 h. Positive clones were selected for colony PCR identification. Correct fragment size detection by agarose gel electrophoresis indicated the successful acquisition of the pNZ8148-PASPA-SLP strain (NZ9000 / SLP) recombinantly expressed in lactic acid bacteria. Its transmission electron microscopy characterization is as follows: Figure 1 As shown.

[0045] 4. Induced expression The strains that have completed electroporation were transferred to induction medium (GM17 liquid medium with added nisin) to induce SLP expression. The induction conditions were: culture temperature of 30℃ and pre-induction bacterial concentration of OD0.05. 600 The concentration of nisin was 0.6, the nisin inducer concentration was 80 ng / mL, and the induction culture time was 24 hours.

[0046] Example 2 The difference from Example 1 is that the SLP-induced expression conditions are a culture temperature of 30°C and a pre-induction bacterial concentration of OD0.05. 600 The concentration of nisin was 0.5, the induction concentration was 80 ng / mL, the induction culture time was 20 hours, and other steps were the same as in Example 1.

[0047] Example 3 The difference from Example 1 is that the SLP-induced expression conditions are a culture temperature of 30°C and a pre-induction bacterial concentration of OD0.05. 600 The concentration of nisin was 0.7, the induction concentration was 100 ng / mL, the induction culture time was 24 hours, and other steps were the same as in Example 1.

[0048] Comparative Example 1 The difference from Example 1 is that the pNZ8148-PASPA plasmid (empty vector) was electrospun to... L.lactis In NZ9000 competent cells, the other steps were the same as in Example 1 to obtain the pNZ8148-PASPA strain (NZ9000 / E) expressed in lactic acid bacteria. Its transmission electron microscopy characterization is as follows: Figure 1 As shown.

[0049] Example 1: Single-factor experiment on induced expression Comparison of different induction temperatures (16, 25, 30, 37℃) and pre-induction bacterial concentration (OD). 600 Under various combinations of nisin inducer concentrations (0.4, 0.6, 0.8 ng / mL) and induction times (16, 20, 24, 28 h), Imagej quantitative analysis was performed on the SDS-PAGE images of the SLP target protein bands. The optimal conditions for SLP expression in the strain were determined based on the relative intensities. The experimental results are shown in Tables 6-9 below. Figure 2 .

[0050] Table 6. Gray values ​​of SLP protein at different temperatures

[0051] Table 7. Gray values ​​of SLP protein at different bacterial concentrations

[0052] Table 8. Gray values ​​of SLP protein at different inducer concentrations

[0053] Table 9. Gray values ​​of SLP protein at different inducer concentrations

[0054] As can be seen from the experimental results in Tables 6-9 above, induction temperature, pre-induction bacterial concentration, nisin inducer concentration, and induction time all affect the expression of the SLP target protein. When the culture temperature is 30℃ and the pre-induction bacterial concentration is OD... 600 The optimal relative strength of SLP expression was achieved with a concentration of 0.6, a nisin inducer concentration of 80 ng / mL, and an induction culture time of 24 hours.

[0055] Experiment Example 2: Gastrointestinal Dynamic Digestion Experiment Simulated gastric fluid preparation (300 mL): 0.6 g pepsin (15000 U) was dissolved in 0.9% NaCl solution, pH was adjusted to 2.0 (±0.02) using HCl, sterilized by 0.22 µm filtration, and preheated at 37℃ for 1 h.

[0056] Simulated intestinal fluid preparation (400 mL): 0.8 g trypsin, 1.4 g ox bile salt, and 2 g KH2PO4 were dissolved in 0.9% sodium chloride solution, and the pH was adjusted to 7.4 (±0.02) using NaOH. The solution was sterilized by 0.22 µm filtration and preheated at 37℃ for 1 h.

[0057] The survival rate of Comparative Example 1 and Example 1 within 2 hours under gastric emptying was determined using an in vitro dynamic human stomach-intestinal digestion system (Dynamic Human Stomach-Intestine IV+, DHSI-IV+). Before the experiment, the heating switch was turned on to preheat the equipment and simulated digestion solution to 37°C. Two different bacterial suspensions of 130 mL each were prepared, and the OD600 was adjusted to 0.6–0.8. These suspensions were added to the inlet, and the equipment was immediately turned on to simulate digestion. 10 mL of digested samples were collected from the stomach and small intestine sampling ports at 1 hour and 2 hours of digestion, respectively. After vortexing the continuously digested stomach and small intestine samples, 100 µL of the sample solution was added to 900 µL of sterile physiological saline for serial dilution. Plate counts were performed on the digested samples at 0, 1, and 2 hours of digestion (e.g., ...). Figure 3 As shown in the figure, after 2 hours of gastric digestion, the viable bacterial concentration of the NZ9000 / SLP strain reached 7.91 log CFU / mL, which is 32.36 times that of the NZ9000 / E strain; after 2 hours of small intestinal digestion, the viable bacterial concentration of the NZ9000 / SLP strain was 6.5 log CFU / mL, which is 30.20 times that of the NZ9000 / E strain.

[0058] The results showed that in the simulated gastrointestinal digestion test, the gastrointestinal tolerance of the probiotics in Example 1 of the present invention was significantly enhanced, with the number of live bacteria increasing by approximately 30.20 times compared to Comparative Example 1, and the survival rate reaching 78%. This indicates that the probiotic delivery system of the present invention improves the high structural stability of probiotics in the complex gastrointestinal fluid environment and enhances the tolerance of probiotics to gastrointestinal digestion.

[0059] Experiment Example 3: Cell Adhesion Experiment Caco-2 cells in good condition (2.5 × 10⁻⁶) 5 (Number of cells / well) were inoculated into 6-well plates and incubated at 37°C and 5% CO2 for 24 hours to form a cell monolayer. After removing the culture medium, 1 mL of the probiotic delivery system from Example 1 and Comparative Example 1 was added to each 6-well plate. L.lactis NZ9000 competent cells were used as a blank control group (NZ9000), and then 2 mL of DMEM medium was added for co-incubation for 2 hours. After incubation, the cells were gently washed three times with PBS buffer to remove any non-adherent bacteria. Subsequently, Caco-2 cells and adherent probiotics were digested with trypsin (Seven Biotechnology Co., Ltd., Beijing, China). Cells were collected, resuspended in physiological saline, and then inoculated onto GM 17 agar medium to calculate the adhesion rate of probiotics.

[0060] Results of in vitro bacterial adhesion experiments are as follows Figure 4As shown, compared to Comparative Example 1, Example 1 showed an approximately 4-fold increase in adhesion to Caco-2 cells, reaching 1.5%. This indicates that the probiotic delivery system of the present invention improves the adhesion of probiotics in the gut.

[0061] Experiment Example 4: Intestinal Colonization Experiment The probiotic delivery systems of Example 1 and Comparative Example 1 were used in in vivo intestinal colonization experiments in mice, respectively, at a concentration of 1×10⁻⁶. 9 Mice in each group were administered 200 μL of CFU / 200 μL via gavage once. In vitro intestinal fluorescence imaging and fecal bacterial count analysis were performed on days 1, 2, 4, 8, and 16. The experimental results are as follows: Figure 5 As shown, NZ9000 / E without S-layer protein coating was almost undetectable in feces on day 16, while the engineered bacteria NZ9000 / SLP containing S-layer protein had a content of 5.27 log CFU / g in feces, confirming that the engineered probiotic colonized in the intestine for more than 16 days. The results indicate that the probiotic delivery system of this invention improves the colonization performance of probiotics in the intestine, prolongs the colonization time of probiotics in vivo (exceeding 16 days), and the obtained probiotic delivery system is harmless in mice, demonstrating its biosafety.

[0062] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A probiotic delivery system based on heterologous expression of S-layer proteins, characterized in that, This includes probiotics carrying S-layer proteins displayed on their surface.

2. The probiotic delivery system based on heterologous expression of S-layer proteins according to claim 1, characterized in that, The probiotics include any species of the genus Lactococcus.

3. A method for preparing a probiotic delivery system based on heterologous expression of S-layer protein as described in claim 1 or 2, characterized in that, Includes the following steps: The S-layer protein recombinant plasmid was introduced into probiotics, and then the S-layer protein was induced to express in an induction medium to obtain a probiotic delivery system with S-layer protein attached.

4. The preparation method according to claim 3, characterized in that, The nucleotide sequence of the S-layer protein recombinant plasmid is shown in SEQ ID No.

1.

5. The preparation method according to claim 3, characterized in that, The induction temperature was 28~35℃, and the duration was 22~26 h.

6. The preparation method according to claim 3, characterized in that, The induction culture medium includes an inducer, which is nisin, and the concentration of nisin is 80~100 ng / mL.

7. The preparation method according to claim 4, characterized in that, The expression vector for the S-layer protein recombinant plasmid includes pNZ8148-PASPA.

8. The preparation method according to claim 4, characterized in that, The importation method includes electroconversion.

9. The use of the probiotic delivery system according to any one of claims 1 to 3 or the preparation method according to any one of claims 4 to 8 in the preparation of products that improve the gastrointestinal tolerance of probiotics.

10. The use of the probiotic delivery system according to any one of claims 1 to 3 or the preparation method according to any one of claims 4 to 8 in the preparation of products that enhance the intestinal adhesion and colonization ability of probiotics.

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