Probiotic mucoprotein loaded oral microsphere system as well as preparation method and application thereof

Abstract

The invention discloses a probiotic / mucoprotein loaded oral microsphere system as well as a preparation method and application thereof, and belongs to the field of compositions. The preparation method of the system comprises the following steps: dissolving probiotics, Mucin and HAMA in double distilled water containing phenyl lithium phosphate LAP and calcium chloride to prepare a gel matrix solution; adding the dissolved gel matrix liquid into an oil phase solvent, and stirring at a high speed in a container; after uniformly stirring, carrying out an ultraviolet light crosslinking reaction, and after the reaction is finished, fully cleaning the obtained microspheres to remove the redundant oil phase solution, so as to obtain microspheres; and dispersing the obtained microspheres in a sodium alginate solution under an acidic condition, uniformly mixing, carrying out a cross-linking reaction, and filtering and cleaning after the reaction is finished, so as to obtain the probiotic / mucoprotein loaded oral microspheres. According to the invention, HAMA and SA are utilized to prepare an oral microsphere system resistant to gastric acid to co-deliver Mucin and EcN, so as to realize continuous and effective regulation and control of unbalanced intestinal flora by probiotics.

Description

Technical Field

[0001] The invention relates to the field of compositions, and in particular to an oral microsphere system loaded with probiotics / mucin. Background Art

[0002] Enteric sepsis is a serious infectious disease with intestinal dysfunction as an important cause (Cell Reports Medicine, 4; 100979, 2023). The most significant pathological changes in enteric sepsis include damaged mucus layer, increased bacterial translocation, excessive inflammation level and accompanied by dysbiosis of intestinal flora. As an important regulator of the intestine, probiotics have multiple mechanisms to regulate the physiological functions of the intestine, including regulating host mucosal and systemic immune function, maintaining the balance of intestinal flora, promoting nutrient absorption, and reducing inflammation levels to maintain intestinal health and prevent further development of enteric sepsis (Burns & Trauma, 9: tkab036, 2021). In experimental models of sepsis, Lactobacillus rhamnosus GG treatment can improve intestinal permeability and regulate malnutrition of microbiota (International Journal of Molecular Medicine, 43: 1107-3756, 2019). A study showed that taking the probiotic VSL#3 could increase the survival rate of rats with sepsis and improve antioxidant and anti-inflammatory conditions (Arabian Journal of Chemistry, 17: 105590, 2024).

[0003] In order to ensure that probiotics play an effective role in the intestine, the current application difficulty that must be solved is the problem of activity loss in the process of crossing the gastric juice barrier. Gastric acid is the main factor that causes probiotics to inactivate. Therefore, how to overcome the killing effect of the gastric acid barrier on probiotics and ensure that probiotics can smoothly reach the intestine and maintain sufficient activity is one of the key challenges in its clinical application. In addition, during the onset of sepsis, the high inflammation and high oxidative stress environment in the intestine can significantly weaken the function of probiotics. This harsh intestinal environment not only prolongs the time for probiotics to exert their regulatory effects, but also may reduce their overall efficacy. Therefore, how to take measures to effectively reduce the level of inflammation and oxidative stress in the intestine while delivering probiotics to the intestine to assist probiotics in playing a normal role in the intestine is the application of probiotic therapy. Summary of the invention

[0004] The object of the present invention is to provide a probiotic / mucin-loaded oral microsphere system; another object of the present invention is to provide a method for preparing the probiotic / mucin-loaded oral microsphere system; a further object of the present invention is to provide an application of the above-mentioned probiotic / mucin-loaded oral microsphere system in biomedicine,

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] A method for preparing a probiotic / mucin-loaded oral microsphere system, the method comprising the following steps:

[0007] Step (1): dissolving probiotics, mucin and methacryloyl hyaluronic acid HAMA in double distilled water containing phenyl (2,4,6-trimethylbenzoyl) lithium phosphate LAP and calcium chloride to prepare a gel matrix solution;

[0008] Step (2): adding the gel matrix liquid into the oil phase solvent and stirring at high speed in a container;

[0009] Step (3): after stirring evenly, performing a UV light cross-linking reaction, and after the reaction is completed, fully washing the obtained microspheres to obtain microspheres;

[0010] Step (4): under acidic conditions, the obtained microspheres are dispersed in a sodium alginate solution, mixed and cross-linked, and filtered and washed after the reaction to obtain probiotic / mucin-loaded oral microspheres.

[0011] In the above preparation method, the probiotics in step (1) are obtained by transforming the pET28a plasmid into E. coli Nissle 1917 to obtain E. coli Nissle 1917 with kanamycin resistance;

[0012] Or the probiotic bacteria is obtained by transforming the pTD103luxI_sfGFP plasmid into E. coli Nissle 1917 to obtain E. coli Nissle 1917 containing kanamycin resistance and GFP (green fluorescent protein).

[0013] In the above preparation method, the molecular weight of HAMA in step (1) is 100-200 kDa, and the concentrations of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt and calcium chloride in double distilled water are 2-3 mg / mL and 0.05-0.2 mol / L, respectively.

[0014] In the above preparation method, the number of probiotics in the gel matrix liquid in step (1) is ≥1*10 9 CFU / mL, the concentration of mucin was 1-15 mg / mL, and the concentration of methacryloylated hyaluronic acid was 20-30 mg / mL;

[0015] Preferably, the concentration of mucin is 8-12 mg / mL, and the concentration of methacryloyl hyaluronic acid is 23-28 mg / mL.

[0016] In the above preparation method, the oil phase solvent in step (2) is Span 80 and liquid paraffin in a volume ratio of 0.1-1:5-15.

[0017] In the above preparation method, the amount of gel matrix liquid added in step (2) is 0.05 to 1.5 times the volume of the oil phase solvent.

[0018] In the above preparation method, the light source for the photo-crosslinking reaction in step (3) has an ultraviolet wavelength of 360-405 nm, and the crosslinking time is 30-60 seconds.

[0019] In the above preparation method, the acidic condition in step (4) is a pH value of 5.0-6.0, the concentration of the sodium alginate solution is 0.5-3 g / mL, the mass volume ratio of the microspheres and the sodium alginate solution is 5-15 mg: 80-120 mL; and the cross-linking reaction time is 0.5-1 h.

[0020] A probiotic / mucin-loaded oral microsphere system is prepared by the above method.

[0021] In the technical solution of the present invention, the probiotic / mucin-loaded oral microsphere system prepared by the method is used in regulating unbalanced intestinal flora, preferably in regulating enterogenic sepsis caused by intestinal dysfunction.

[0022] The bacteria is E. coli Nissle 1917, purchased from Changsha Abiwei Biotechnology Co., Ltd.

[0023] Beneficial effects of the present invention:

[0024] The present invention forms probiotic / mucin-loaded hyaluronic acid oral microspheres containing SA shells by means of emulsification and ion crosslinking. HAMA and mucin can effectively alleviate the inflammation and oxidative stress environment produced by immune cells due to their natural physiological functions, improve the working environment of probiotics and give full play to the ability to regulate intestinal flora. The unsaturated carbon-carbon double bonds in HAMA can form a gel network structure through free radical crosslinking. SA and Ca 2+The cross-linked structure formed by chelation does not crack in the stomach, and forms calcium hydroxide cracking in the alkaline environment of the intestine, promoting the intestinal release of mucin and probiotics in the internal HAMA microspheres. In addition, the gel shell core structure of HAMA and SA can effectively improve the more pores and poor mechanical properties of single SA microspheres, which are not conducive to the delivery of probiotics. The selected HAMA can be degraded by hyaluronidase secreted by microorganisms and tissues in the intestinal environment, so as to gradually release the internal EcN and Mucin in the intestine and play a role. Mucin, as a type of mucus protein extracted from the gastrointestinal tract, can assist the damaged intestinal mucus barrier to resist the invasion of pathogens and regulate immune activation, while the thickened mucus layer can have a stronger retention effect on the microspheres. Therefore, the present invention uses HAMA and SA to make an oral microsphere system resistant to gastric acid to co-deliver Mucin and EcN, so that probiotics can continuously and effectively regulate the imbalanced intestinal flora.

[0025] In summary, the microsphere system for treating enterogenic sepsis of the present invention has the following advantages:

[0026] 1. Co-delivery of probiotics and biomacromolecule drugs: The hydrophilic polymer gel skeleton can evenly distribute micron-sized probiotics and mucin inside the microspheres through intermolecular forces, simplifying the production process and improving drug encapsulation efficiency; 2. Enhance gastric acid barrier tolerance: By adding a coating on the surface of the microspheres, the internal bioactive substances are protected from erosion by gastric juice, ensuring their precise release in the pathological environment of the intestine;

[0027] 3. Synergistic regulation of intestinal inflammatory damage: After being released, probiotics can transmit signals with other intestinal microorganisms to regulate the species composition and function of the intestinal flora. Mucin repairs the damaged intestinal mucus barrier and reduces the invasion of bacteria and pathogens on intestinal cells. The gel material can also synergistically exert anti-inflammatory effects and effectively relieve intestinal inflammatory damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The formulation screening process of the gel matrix liquid; (AC) rheology, freeze-thaw protection and release experiments investigate the gel properties formed by methacryloyl hyaluronic acid (HAMA), mucin and EcN under different concentration conditions; (D) the proliferation of EcN under different Mucin concentrations; (E) the swelling curves of HAMA gel under different formulation compositions.

[0029] Figure 2 The protective effect of methacryloyl hyaluronic acid (HAMA) and mucin on the tight junction protein ZO-1 between Caco-2 cells. The injury treatment condition was lipopolysaccharide (LPS). The staining of ZO-1 was photographed using a laser confocal microscope (CLSM). The scale bar is 50μm. Compared with the LPS group,* P<0.05.

[0030] Figure 3 The in vitro antioxidant effect of methacryloyl hyaluronic acid (HAMA) and mucin; the reactive oxygen species produced by macrophages after hydrogen peroxide treatment were photographed using CLSM, the scale bar is 100μm. Compared with the H2O2 group, **** P<0.0001.

[0031] Figure 4 The in vitro anti-inflammatory effects of methacryloylated hyaluronic acid (HAMA) and mucin were investigated; the expression levels of inflammation-related genes in macrophages after LPS treatment were verified by qPCR. **** P<0.0001. Figure 5 The morphological characteristics of the microspheres under an optical microscope; the green fluorescent part is EcN-GFP.

[0032] Figure 6 The distribution of each component in the microspheres was investigated; (A) is the maximum cross-section of the microspheres photographed by CLSM, with a scale of 200 μm; (B) is the distribution of characteristic elements in the microspheres and the microscopic morphological characteristics of the microspheres photographed by elemental energy spectrum scanning electron microscopy (EDS-SEM), with a scale of 200 μm.

[0033] Figure 7 Encapsulation and release of mucin in microspheres; (A) is a comparison of the encapsulation efficiency between block gel and microspheres; (B) is the time-release curve of mucin under different microspheres and enzyme treatment conditions.

[0034] Figure 8 The activity of probiotics inside different microspheres was investigated after they were subjected to in vitro simulated gastric acid and in vitro simulated intestinal fluid conditions.

[0035] Fig. 9 The morphological characteristics of the microspheres under different simulated pH conditions were observed by SEM; the picture on the right is an enlarged view of the picture on the left.

[0036] Fig.10 The number of probiotics in mouse feces after oral administration of microspheres; (A) is the EcN-GFP fluorescence image in mouse feces taken by the IVIS system at different time points in different oral drug groups; (B) is the fluorescence quantification image of different groups at 24h in (A).

[0037] Fig.11The mucus layer added by the microspheres hinders the penetration of bacteria-related simulated proteins; (A) is the penetration rate of bacteria-related simulated proteins after different microspheres are mixed into the simulated mucus layer in vitro; (B) is the quantitative graph of proteins that penetrated in different groups at the 24h time point, ns means no significant difference.

[0038] Fig.12 qPCR was used to investigate the protective effect of microspheres on goblet cell mucus secretion and anti-inflammatory effects in vitro; (A) is the expression of goblet cell E12 related genes; (B) is the expression of macrophage related genes. Compared with the LPS group, **P<0.01, ***P<0.001, ****P<0.0001.

[0039] Fig.13 Body weight changes of septic mice after oral administration of microspheres, the observation period was 7 days.

[0040] Fig.14 Colon tissue sections of mice after different microsphere treatments, including hematoxylin & eosin staining (H&E) and Alcian blue-periodic acid Schiff staining (AB-PAS).

[0041] Fig.15 H&E sections of the heart, liver, spleen, lung, and kidney of mice after treatment with different microspheres; the lower right corner of each image is the global view of the image.

[0042] Fig.16 Plate spreading results of liver and kidney tissue homogenates of mice after treatment with different microspheres.

[0043] Fig.17 After treatment with different microspheres, the expression of related inflammatory genes in the small intestine and colon of mice was detected by qPCR, and the content of inflammatory cytokines in serum was detected by ELISA. Compared with the CLP group, ****P<0.0001.

[0044] Fig.18 After EcN+Muc@SHM microspheres treatment, the intestinal microbial composition of mice was compared with that of the sham group (Sham) and the surgical group (CLP); (A) is the α-diversity statistical result; (B) is the difference in microbial composition of phylum; (C) is the functional unit PCoA analysis. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto:

[0046] In this invention scheme, the bacterium is E. coli Nissle 1917, purchased from Changsha Abiwei Biotechnology Co., Ltd. Subsequently, pET28a and pTD103luxI_sfGFP plasmids were transformed into E. coli Nissle 1917, respectively, to obtain E. coli Nissle 1917 with kanamycin resistance and E. coli Nissle 1917 containing kanamycin resistance and GFP (green fluorescent protein).

[0047] Plasmid transformation was performed using the heat shock method. The specific steps are as follows:

[0048] The transformation methods of pET28a and pTD103luxI_sfGFP plasmids are the same, and the following steps are all referred to as plasmids.

[0049] (1) Place 100 μL of competent E. coli Nissle 1917 in an ice bath and thaw for 2 min.

[0050] (2) Add 100 ng of plasmid to competent E. coli Nissle 1917 and keep it on ice.

[0051] (3) The mixed solution of plasmid and E. coli Nissle 1917 was kept in an ice bath in the dark for 30 min.

[0052] (4) Place the mixture in a 42°C water bath for 90 seconds, then immediately place it in an ice water bath for 3 minutes.

[0053] (5) Add 700 μL of resistance-free LB medium and culture in a constant temperature air bath shaker at 37°C for 1 h.

[0054] (6) Centrifuge at 12000 rpm for 1 min, discard the supernatant to a total volume of 100 μL, and evenly spread on a solid plate containing kanamycin resistance, and culture in a constant temperature incubator at 37°C until obvious single colonies appear.

[0055] (7) The pET28a transformed strain was verified by colony PCR technology. The pTD103luxI_sfGFP transformed strain itself emits green fluorescence, which can be directly observed and verified. Finally, E. coli Nissle 1917 with kanamycin resistance and E. coli Nissle 1917 containing kanamycin resistance and GFP (green fluorescent protein) were obtained.

[0056] Example 1

[0057] Table 1 Main symbol table

[0058]

[0059] Table 2 Main materials and reagents

[0060]

[0061]

[0062] Cells: THP-1 mononuclear cells, Caco-2 intestinal epithelial cells and E12 intestinal goblet cells were cultured in our laboratory.

[0063] 1) Effects of different HAMA (methacryloyl hyaluronic acid), Mucin (mucin) concentrations and the presence or absence of EcN on the mechanical properties of the gel (the strain used was E. coli Nissle 1917 with kanamycin resistance):

[0064] Probiotics, mucin and methacryloyl hyaluronic acid HAMA were dissolved in double distilled water containing phenyl (2,4,6-trimethylbenzoyl) lithium phosphate LAP and calcium chloride to prepare a gel matrix solution, in which the concentration range of HAMA (150kDa) was 5-40mg / mL, the concentration range of Mucin was 0.25mg / mL-10mg / mL, and the concentration of EcN was guaranteed to be 1*10 9 CFU / mL, the solvent of the system is double distilled water containing 2.5 mg / mL LAP and 0.1 mol / L CaCl2 (hereinafter referred to as double distilled water system). After determining the linear viscoelastic region using a rotational rheometer, the stress was set to 1% and the frequency sweep range was 0.1-100 rad / s to test the storage modulus and loss modulus of different combination gels, such as Figure 1 As shown in AC, under the premise that the gel is not easy to break, the HAMA concentration of EcN and Mucin is determined to be 20-30 mg / mL. The gel formulation with a Mucin concentration of 0.5 mg / mL is excluded, and the remaining concentrations need to be further screened.

[0065] 2) Effects of different concentrations of mucin on EcN activity (the strain used was E. coli Nissle 1917 with kanamycin resistance)

[0066] A double distilled water system containing EcN was divided into 7 equal parts, 6 of which were mixed with Mucin to form Mucin-EcN mixed solutions with concentrations of 0.25, 0.5, 1, 2, 5, and 10 mg / mL, and the double distilled water system was used as the control group. After incubation at 37°C on a shaker for 12 h, the mixture was diluted with sterile water for 10 min. 5 100 μL of each group was plated on a solid plate and cultured for 12 h. The results were as follows: Figure 1 As shown in D, EcN showed a Mucin concentration-dependent increase in proliferation.

[0067] 3) Cryoprotective effect of different HAMA and Mucin concentrations on EcN (the strain used was E. coli Nissle 1917 with kanamycin resistance)

[0068] Probiotics, mucin and methacryloyl hyaluronic acid HAMA were dissolved in double distilled water containing phenyl (2,4,6-trimethylbenzoyl) lithium phosphate LAP and calcium chloride to prepare a gel matrix solution. The concentrations of HAMA were set to 20 mg / mL, 25 mg / mL, and 30 mg / mL, and the concentrations of Mucin were set to 5 mg / mL, 10 mg / mL, and 20 mg / mL, and they were mixed with an equal amount of EcN to prepare a gel system. The final concentration of EcN was 1*10 9 CFU / mL, the solvent system was double distilled water system (2.5 mg / mL LAP and 0.1 mol / L CaCl2). After three cycles of cooling and heating at 37°C and -80°C, the gel was fully crushed with double distilled water system, centrifuged at 2000 rpm for 1 min, and diluted with sterile water for 10 5 After 100 μL of the supernatant was taken for solid plate coating and cultured for 12 h. Figure 1 When the gel ratio shown in E is HAMA25 mg / mL-Mucin10 mg / mL and HAMA25 mg / mL-Mucin 10 mg / mL, the cryoprotection ability for EcN is the best.

[0069] 4) EcN release in gel (the strain used was E. coli Nissle 1917 with kanamycin resistance)

[0070] Probiotics, mucin and methacryloyl hyaluronic acid HAMA were dissolved in double distilled water containing phenyl (2,4,6-trimethylbenzoyl) lithium phosphate LAP and calcium chloride to prepare a gel matrix solution. The gel system with HAMA concentration of 20mg / mL, 25mg / mL, and Mucin concentration of 10mg / mL was investigated. Each system contained EcN with OD600=0.45. Each gel was divided into two groups with or without HAase. The solvent was a double distilled water system (2.5mg / mL LAP and 0.1mol / LCaCl2). Each group of gels was prepared with 200μL of mother liquor and soaked in 2mL of double distilled water system solution with or without HAase. The OD600 of the supernatant was measured and plotted at 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h and 24h after placement. The results are shown in the figure. Figure 1 As shown in F, the gel system of 25 mg / mL HAMA + 10 mg / mL Mucin can better release EcN and show a certain ability to promote proliferation.

[0071] 5) Gel swelling (the strain used was E. coli Nissle 1917 with kanamycin resistance)

[0072] Probiotics, mucin and methacryloyl hyaluronic acid HAMA were dissolved in double distilled water containing phenyl (2,4,6-trimethylbenzoyl) lithium phosphate LAP and calcium chloride to prepare a gel matrix solution. The gel system with HAMA concentration of 20mg / mL, 25mg / mL, and 30mg / mL and Mucin concentration of 10mg / mL was investigated. Each system contained 1*10 9 CFU / mL EcN, the solvent is double distilled water system. Weigh the prepared gel system, locate the initial weight, and then measure the weight of the gel system when immersed in an excess solution environment at each time point, and calculate the swelling ratio according to the formula: W Time point Refers to the weight of the gel at each time point, W Initial Refers to the initial weight of the gel. Figure 1 As shown in G, the swelling ratios of 20 mg / mL HAMA + 10 mg / mL Mucin and 30 mg / mL HAMA + 10 mg / mL Mucin are similar and large, indicating that the gel system prepared according to this ratio can release the internal substances faster through swelling and has the potential to be absorbed and fused with intestinal mucus and other tissues, thereby increasing its colonization ability in special parts.

[0073] Combined with the above results, the final composition of the prescription was determined to be 25mg / mL HAMA + 10mg / mL Mucin and >1*10 9 CFU / mL of EcN.

[0074] 6) Verification of the intestinal barrier effects of HAMA and Mucin in vitro

[0075] In the cell experiment, HAMA and Mucin were kept in solution and directly prepared and swollen in sterile complete culture medium. The final concentrations of HAMA and Mucin were kept at 25 mg / mL and 10 mg / mL.

[0076] Effects of HAMA and Mucin on intestinal endothelial cells

[0077] 2×10 5After Caco-2 cells were plated in a confocal dish and cultured for 24 hours, the original solution was replaced with a culture medium containing HAMA, Mucin or a mixture of the two, and LPS was added to continue induction for 12 hours. The final concentration of LPS was 100 ng / mL. The solution in the confocal dish was discarded, washed three times with PBS and fixed with 4% paraformaldehyde. ZO-1 antibody and the corresponding AF488 secondary antibody and DAPI were used for staining, and finally CLSM was used for imaging. The results are shown in Figure 2 As shown, HAMA and Mucin can effectively alleviate the effect of LPS on tight junction proteins and protect the intestinal barrier.

[0078] Antioxidant Effects of HAMA and Mucin

[0079] 3×10 5 After THP-1 cells were plated in a confocal dish and cultured for 48 hours, PMA was added and THP-1 was continuously induced to differentiate into macrophages within 48 hours. The amount of PMA used was 100 ng / mL. When THP-1 was fully attached to the wall, the original solution was replaced with a culture medium containing HAMA, Mucin or a mixture of the two, and H2O2 was added for induction for 4 hours. The final concentration of H2O2 was 400 μM. After washing with PBS three times, 10 μM DCFH-DA solution was added and incubated at 37°C for 1 hour. After washing again with PBS, the cells were fixed with 4% paraformaldehyde and the cell nuclei were stained with DAPI, and finally imaged with CLSM. The results are shown in Figure 3 As shown in Figure 2, HAMA and Mucin can effectively alleviate the oxidative stress of macrophages and play an antioxidant role.

[0080] 3×10 5 After THP-1 cells were plated in a 6-well plate and cultured for 48 hours, PMA was added and THP-1 was continuously induced to differentiate into macrophages within 48 hours. The amount of PMA used was 100 ng / mL. When THP-1 was fully attached to the wall, the original solution was replaced with a culture medium containing HAMA, Mucin or a mixture of the two, and LPS was added to continue induction for 12 hours. The final concentration of LPS was 100 ng / mL. After washing three times with enzyme-free PBS, the RNA in the cells was extracted using a total RNA extraction kit, and the corresponding cDNA was obtained using a reverse transcription kit. qPCR experiments were performed using the designed primers, and the expression of genes such as TNF-α, IFN-γ, NF-κB, IL-1β, and IL-6 in THP-1 was investigated using SYBR green dye and a fluorescent quantitative PCR instrument. The results are shown in the figure. Figure 4 As shown, HAMA and Mucin can effectively alleviate the inflammatory activation state of macrophages caused by LPS and play an anti-inflammatory role.

[0081] Preparation of probiotic / mucin loaded oral microspheres:

[0082] Gel matrix solution configuration:

[0083] (1) Phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt LAP and anhydrous calcium chloride are added to 100 mL of deionized water and heated and stirred until fully dissolved, wherein the concentrations of LAP and calcium chloride in double distilled water are 2.5 mg / mL and 0.1 mol / L, respectively.

[0084] (2) Weigh 2.5 g of methacryloyl hyaluronic acid HAMA, 1 g of mucin and E. coli Nissle 1917 containing kanamycin resistance, and dissolve them in the mixed solution of LAP and calcium chloride in step (1) to make the solution clear and transparent, wherein the final concentration of probiotics is 1*10 9 CFU / mL, and the gelling liquid was obtained.

[0085] Example 1

[0086] Cultivation and acquisition of probiotics:

[0087] (1) The strains used in the examples are E. coli Nissle 1917 with kanamycin resistance and E. coli Nissle 1917 containing kanamycin resistance and GFP (green fluorescent protein), which are hereinafter referred to as EcN.

[0088] (2) Weigh 10 g of sodium chloride, 10 g of tryptone, and 5 g of yeast powder in 1000 mL of deionized water. Weigh 1 g of sodium chloride, 1 g of tryptone, 0.5 g of yeast powder, and 1.8 g of agar powder in 100 mL of deionized water. Sterilize the above prepared solutions in an autoclave at 121°C for 20 min to obtain sterile LB liquid medium and solid medium.

[0089] (3) Use a sterile inoculation loop to pick up a small amount of EcN cryopreserved solution, transfer it to solid culture medium by plate streaking method, and culture it at 37°C for 12 hours for recovery. Pick up a single colony after recovery and expand it in liquid culture medium. When the OD600 is about 1.5, EcN with good activity can be obtained.

[0090] (4) Take 1 mL of EcN medium and dilute it by 10 under sterile conditions. 6 Times, take 100 μL of it and evenly spread it on the surface of the solid plate, and count the CFU after 24 hours to determine the number of EcN required for the subsequent preparation.

[0091] Take an appropriate amount of liquid paraffin and add Span 80 to configure the oil phase. The volume ratio of Span 80 to liquid paraffin is 0.5:10. Add the gel matrix solution to the rapidly stirred oil phase according to 0.1 times the volume of the oil phase, and continue to stir in the dark for 30 minutes. After the mixed system is stationary, quickly crosslink it with a 405nm ultraviolet light source for 30 seconds to obtain a solidified gel microsphere core. The obtained gel microsphere core is placed on a 30-mesh and 80-mesh sieve for filtration to remove larger and smaller particles, and the microsphere core is quickly and fully washed with anhydrous ethanol to remove excess oil phase to obtain gel microspheres (EcN+Muc@HM microsphere system without SA shell). Weigh 1g of sodium alginate and dissolve it in 100mL of an aqueous solution with a pH of 5.5. After it is fully swollen to colorless and transparent, mix the above 10mg of the gel microsphere core into 100mL of sodium alginate solution and mix thoroughly. After cross-linking for 1 h, the mixed system was placed on an 80-mesh sieve, and then the excess sodium alginate solution was rinsed with purified water to obtain a sodium alginate-encapsulated probiotic / mucin oral microsphere system with a core-shell structure, which was called EcN+Muc@SHM.

[0092] Characterization of EcN+Muc@SHM

[0093] Microsphere morphology

[0094] The EcN+Muc@HM microsphere system without SA shell (same as EcN+Muc@SHM microsphere system except no cross-linking with sodium alginate) and the EcN+Muc@SHM microsphere system with SA shell were prepared, dispersed in pure water and observed with an inverted fluorescence microscope for bright field and green fluorescence, where EcN was EcN-GFP with kanamycin resistance and GFP. The results are shown in Figure 5 As shown, the outside of the microspheres with SA shells clearly has a double-layer structure, and EcN can be fully encapsulated inside the microspheres without being exposed.

[0095] Distribution of components in microspheres

[0096] According to the method of claim 1, the EcN+Muc@SHM microsphere system with SA shell was prepared. Before preparation, WGA-Rho was added to mark mucin, and finally SA was marked with mitochondrial deep red fluorescent probe. Finally, the distribution of each component in the microsphere was observed by CLSM, wherein EcN was EcN-GFP with kanamycin resistance and GFP. The results are as follows Figure 6 As shown in A, SA is distributed outside the microspheres, and EcN and mucin are also mainly distributed in the core of the microspheres. Since the SA detection probe has a strong positive charge, it has a strong penetration effect on the negatively charged microspheres, so the core also produces strong fluorescence.

[0097] In order to more accurately verify the distribution of each component in the microsphere, SEM with EDS module was used to more intuitively analyze the EcN+Muc@SHM microsphere system, where Ca indicates the location of SA, N indicates the location of Mucin, and P indicates the location of EcN. The results are shown in Figure 2. Figure 6 As shown in B, SA is distributed on the outside of the microspheres, and Mucin and EcN are distributed on the inside of the microspheres.

[0098] Encapsulation and release of mucin in microspheres

[0099] Muc@SHM microsphere system with SA shell, and also used the same ratio of HAMA and Mucin direct mixed gel system and free Mucin as control. After the bulk gel (Bulk formation) and microsphere system (Spheroid formation) were fully crushed, 1000U / mL HAase was added and treated for 24h. Free Mucin was also treated in the same way. The sample was centrifuged at 12000rpm for 3min and the supernatant was obtained. Finally, BCA was used to measure the Mucin content in the supernatant of the bulk and microsphere system. The results are shown in Figure 7 As shown in A, both the block (84.34%) and spherical (73.69%) forms have a high encapsulation efficiency for Mucin.

[0100] Simulated gastric acid conditions (SGF) were set up: deionized water was adjusted to pH 1.5 with 4 M hydrochloric acid at 37 °C.

[0101] Simulated intestinal fluid conditions (SIF): deionized water at 37°C, adjusted to pH 7.4 with sodium hydroxide and HAase added to a concentration of 300 U / mL. Note: Phosphate is not used here to prevent phosphate from ionizing the Ca in the formulation. 2+ Displaced, rather than OH in alkaline environment - , affecting the observation of release results.

[0102] The Muc@HM microsphere system without SA shell and the Muc@SHM microsphere system with SA shell were first placed in SGF for 2 hours and then placed in SIF for 48 hours. The control group without HAase was also compared. The microspheres treated with 1000U / mL HAase after being fully crushed were used as the complete release group (i.e., 100% content), and the release results were measured using BCA. The results are shown in Figure 7 As shown in Figure B, in SGF, the release rate of Mucin in Muc@SHM is relatively slow. When placed in SIF environment, the release of Muc@HM and Muc@SHM is similar and can reach about 80% within 48 hours. When there is no HAase, the release rate of both is significantly reduced.

[0103] Microspheres protect EcN from gastric acid

[0104] The EcN+Muc@HM microsphere system without SA shell and the EcN+Muc@SHM microsphere system with SA shell, where EcN is EcN-GFP with kanamycin resistance and GFP. The microspheres were placed in SGF for 2 h, and then the two types of microsphere systems were fully crushed, centrifuged at 2000 rpm for 1 min, and diluted with sterile water for 10 6 After 100 μL of the supernatant was taken for solid plate coating and cultured for 12 h. An equal amount of free EcN was used as a control and subjected to the same treatment process. Figure 8 As shown, more EcNs retained better activity in the EcN+Muc@SHM microsphere system with SA shell.

[0105] Morphological changes of microspheres under gastric acid

[0106] EcN+Muc@SHM microsphere system with SA shell, the same batch of microspheres were placed in pH 1.5 and pH 7.0 solution environment for more than 2h, and SEM photography was performed, among which EcN was EcN with only kanamycin resistance. The results are shown in Fig. 9 As shown in the figure, under low pH conditions, the surface of the microspheres is compact, while under neutral pH conditions, the outside of the microspheres presents a loose structure, indicating that the SA shell can protect the internal core by structural compaction under low pH conditions.

[0107] Protective effect of microspheres on EcN in vivo

[0108] EcN@H microsphere system without SA shell and Mucin, EcN+Muc@HM microsphere system without SA shell, EcN+Muc@SHM microsphere system with SA shell, and mixed non-crosslinked solution of several materials, among which EcN is EcN-GFP with kanamycin resistance and GFP. . With equal amount of free EcN as control group, healthy C57BL / 6 male mice were gavaged, and then the mouse feces were collected at 2h, 4h, 8h, and 24h, and the EcN-GFP signal was collected by IVIS system and recorded and analyzed. The results are shown in Fig.10 As shown, the EcN+Muc@SHM microsphere system administration group showed higher EcN signals at each time point. This result indirectly proves that the constructed shell core microsphere system can effectively help EcN pass through the gastric acid barrier, colonize in the intestine and maintain a high proliferation activity.

[0109] Permeation inhibition of microspheres on bacteria-associated proteins

[0110] The EcN+Muc@HM microsphere system without SA shell and the EcN+Muc@SHM microsphere system with SA shell were mixed into the simulated mucus layer (80 mg / mL mucin solution), and GFP protein was used as the bacteria-related simulated protein, where EcN was EcN with only kanamycin resistance. The mixed mucus was added to a Transwell with a pore size of 0.4 μm, and then the GFP protein solution was added, and 1 mL of deionized water was added to the lower chamber. At regular intervals, 100 μL of the solution in the lower chamber was aspirated and an equal volume of deionized water was added in time, and the results were measured using a fluorescent microplate reader. The control solution replaced the mucus layer with deionized water, and the rest of the operations were the same. The results are shown in Fig.11 As shown, each type of microspheres can play a role in permeation inhibition of bacteria-related mimic proteins, but there is no significant difference between them.

[0111] Protective and anti-inflammatory effects of microspheres on mucus cells in vitro

[0112] An in vitro cellular 3D intestinal model was constructed. E12 cells that secrete mucin were laid on the upper layer of the Transwell, and immune cells THP-1 were laid on the lower layer and induced into macrophages with PMA.

[0113] According to the method of claim 1, H microspheres without Mucin and SA shells (EcN and Mucin are not added in step 1, and step 4 is not performed, and the rest remains unchanged), Muc@HM microsphere system without SA shell (EcN is not added in step 1, and step 4 is not performed, and the rest remains unchanged) and Muc@SHM microsphere system with SA shell (EcN is not added in step 1, and the rest remains unchanged) are prepared, and the microsphere mother liquor is filtered through a 0.22μm water system membrane twice. After the prepared microspheres are transferred to a sterile centrifuge tube, they are fully dried and sterilized by ultraviolet for 1h. The microspheres are swollen with DMEM culture medium for 4h before administration, and then administered to the upper layer E12. Finally, LPS is added to the upper layer of Transwell at a concentration of 100ng / mL, and 300U / mL of HAase is treated for 24h. RNA was extracted and the expression of genes such as MUC2, MUC5AC in E12 and IL-10, IL-1β, IL-6 in THP-1 was verified by qPCR. The results are as follows Fig.12 As shown in the figure, the Muc@SHM microsphere system with SA shell can effectively alleviate the mucus secretion damage and high inflammation caused by LPS.

[0114] Application Example 1

[0115] Example 1 Treatment of CLP-induced enterogenic sepsis in mice with probiotic / mucin-loaded oral microsphere system

[0116] 1 Experimental animals

[0117] SPF-grade C57BL / 6J male mice aged about 6-8 weeks were purchased from Shanghan Bikai Keyi Biotechnology Co., Ltd.

[0118] 2 Experimental methods

[0119] 2.1 Construction of a mouse model of enterogenic sepsis

[0120] At least 14 days after the mice arrived at the breeding environment, all mice were given analgesics 5 mg / kg, meloxicam injection, subcutaneous injection, starting 30 minutes before CLP and then every 12 hours until the third day. The mice were anesthetized by inhalation of isoflurane (1% to 3%), and a 1 to 2 cm incision was made in the midline of the anterior abdomen to expose the cecum. The distal half of the cecum was ligated (moderate CLP), and a 22G needle was used to puncture the ligated segment once, and a small drop of cecal contents was squeezed out (the sham group did not perform the ligation and puncture step). The cecum was returned to the abdomen, and 1 ml of sterile saline (pyrogen-free 0.9% NaCl) was injected into the abdominal cavity (inner layer of the abdominal cavity) (adjusted according to actual conditions, about 1 to 2 drops), and the internal peritoneum was ligated with sterile sutures. Finally, the incision was closed with a 9 mm steel wound clip and antibacterial ointment was applied.

[0121] 2.2 Dosage regimen

[0122] 7 days before CLP modeling, mice in each group were given oral administration at around 5-6 pm for prevention. From the day of modeling, drugs were given every two days, at 5-6 pm, and the microspheres administered were microspheres made from 200 μL of gel mother solution. The sham group and the CLP modeling group were given an equal volume of normal saline, in which EcN was EcN with only kanamycin resistance.

[0123] Grouping: 28 mice were randomly divided into 7 groups, each with 4 mice: sham group, CLP group, free EcN, EcN@H microsphere group, EcN+Mucin@HM microsphere group, EcN+Mucin@SHM microsphere group and mixed material group Mix. The concentration of EcN was kept at 1*10 9 -5*10 9 CFU / mL.

[0124] 2.3 Recording of mouse weight changes and feces collection

[0125] The weight changes were recorded from the first modeling, and the records were kept every day for seven consecutive days. In order to make the results more intuitive, the data used the initial weight of each mouse as the benchmark, and the daily weight change rate compared to the initial weight was recorded.

[0126] 2.4 Collection of mouse intestinal tissue, major organs and serum

[0127] 7 days after modeling (i.e., 8th day), blood was collected after intragastric administration and euthanasia was performed to collect serum, all intestinal segments from the stomach to the anus, and heart, liver, spleen, lung, kidney and other tissues. A 3 cm segment of jejunum and a 1 cm segment of colon were fixed with 4% paraformaldehyde. Half of the heart and spleen were fixed and half were frozen by longitudinal section. One lobe of the liver and lung was fixed and the rest was frozen. One lobe of each kidney was fixed and the other was frozen.

[0128] 2.5 Biochemical index detection

[0129] 2.5.1 Tissue sections

[0130] The jejunum and colon were stained with H&E and AB-PAS, and the tissue was stained with H&E.

[0131] 2.5.2 Bacterial counts in liver and kidneys

[0132] The liver and kidney of each group of mice were soaked in 75% ethanol solution for 5 minutes to remove bacteria attached to the surface. Under a sterile environment, 50 mg of tissue was placed in a sterile tube and 1 mL of deionized water was added to fully grind. After the contents were naturally settled, the supernatant was diluted 10 times, and 100 μL was used for plate coating and cultured for 12 hours. The number of bacteria in the liver and kidney of each group was observed. 2.5.3 Levels of inflammatory factors in serum, small intestine and colon

[0133] A small section of jejunum and colon was taken, the intestinal tissue was cut longitudinally and the intestinal contents were slowly rinsed with cold PBS, and the intestinal mucus layer was gently scraped and collected. RNA in the tissue was extracted using Trizol solution in an enzyme-free environment, and the expression of IL-1β and IL-6 genes was verified by qPCR. ELISA kits were used to detect the levels of IL-1β and IL-6 in the serum of each group.

[0134] 2.5.4 Intestinal flora detection

[0135] In order to detect the changes of EcN in EcN+Muc@SHM on the microbial composition of the intestinal environment under disease conditions, 15 mice were divided into 3 groups, with n=5 in each group. The groups were divided into Sham group (NC), CLP group (C) and EcN+Muc@SHM group (MS). The drugs were administered according to 2.2. When the modeling mice were in a depressed state, fresh feces were collected as soon as possible, 5 pellets per mouse, of which EcN was EcN with kanamycin resistance only. Fresh feces were collected from mice in good condition 7 days after modeling. 16S microbiome was tested and analyzed by Shanghai Hongxu Company.

[0136] 3. Results and Discussion

[0137] 3.1 Changes in mouse body weight

[0138] exist Fig.13The weight of the Sham group changed steadily within seven days; the weight of the CLP group dropped significantly on the second day after modeling, and died on the second and third days, and finally all died on the sixth day; the weight of the free EcN group rose and fell, and died on the 3rd and 6th days, and two reached the end point; the mice in the EcN@H, EcN+Muc@HM, and EcN+Muc@SHM groups all reached the end point of the study, among which the weight of the mice in the EcN+Muc@SHM group basically showed an upward trend in the later period; the weight of the Mix group all dropped, and one died on the second day, and the rest reached the end point of the study. In general, the EcN microsphere preparation groups can effectively alleviate the survival of mice with enterogenic sepsis.

[0139] 3.2 Small Intestine Sectioning

[0140] exist Fig.14 In the H&E results of the small intestine sections, it can be clearly seen that the addition of EcN+Muc@HM and EcN+Muc@SHM groups can effectively alleviate the damage to the small intestinal villi and maintain the integrity of the tissue. AB-PAS staining mainly stains neutral and acidic mucins. From the section results, it can be seen that the EcN+Muc@SHM group can effectively restore the mucus secretion on the small intestinal villi, indicating that the intestinal mucus barrier has obvious signs of recovery.

[0141] 3.3 Sections of major organs

[0142] exist Fig.15 There was no significant difference between the heart and spleen. The CLP in the liver had dark purple staining suspected of necrosis. The free EcN group showed obvious liver damage, the EcN@H group showed more inflammatory cell infiltration, and the Mix group also had more inflammatory cell infiltration. The other groups including EcN+Muc@SHM were similar to the Sham group and did not show obvious similar phenomena. The CLP group showed obvious consolidation of the lungs, which was alleviated after the microsphere system treatment. The changes in the renal tubules were mainly observed in the kidneys, and it was found that the renal tubules in the CLP group showed severe edema, resulting in the almost disappearance of the internal cavity and the shedding of renal tubular endothelial cells, which improved after microsphere treatment. This shows that the EcN+Muc@SHM microsphere treatment system can effectively alleviate multi-organ lesions caused by enterogenic sepsis.

[0143] 3.4 Bacterial counts in liver and kidneys

[0144] exist Fig.16In the results, it can be observed that there are different degrees of bacteria in the liver and kidney of each group, among which the CLP group has the largest number of bacteria and the highest density, and the EcN@H and Mix groups also have high density of bacteria. Compared with other groups, the number of bacteria in the EcN+Muc@SHM group is close to that in the Sham group, indicating that EcN+Muc@SHM can alleviate the intestinal bacterial translocation during enterogenic sepsis.

[0145] 3.5 Levels of inflammatory factors in serum, small intestine, and colon

[0146] exist Fig.17 In the experiment, the expression levels of IL-1β and IL-6, the hallmark inflammatory factors during sepsis, were expressed at high levels in the small intestine and colon tissues, and after treatment, they were downregulated to varying degrees, with EcN+Muc@SHM downregulating the most significantly. This result corresponds to the levels of IL-1β and IL-6 in serum, indicating that the level of inflammation in the body has been alleviated to a certain extent, indicating that EcN+Muc@SHM can effectively alleviate the high inflammatory activation in enterogenic sepsis.

[0147] 3.6 Regulation of intestinal flora

[0148] exist Fig.18 In the experiment, the addition of EcN+Muc@SHM reduced the α-diversity of intestinal flora to a certain extent, which may be related to the use of a single probiotic. However, after the administration of EcN+Muc@SHM, the phylum of microorganisms in the intestinal tract of mice basically returned to a level similar to that of the Sham group, and there was a significant difference in the composition of the CLP group. In addition, in the functional classification of microorganisms, the intestinal flora function of the EcN+Muc@SHM group was closest to that of the Sham group, indicating that EcN+Muc@SHM can effectively regulate the imbalanced intestinal flora to a normal state.

[0149] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a probiotic / mucin-loaded oral microsphere system, characterized in that: The method comprises the following steps: Step (1): dissolving probiotics, mucin and methacryloyl hyaluronic acid HAMA in double distilled water containing phenyl (2,4,6-trimethylbenzoyl) lithium phosphate LAP and calcium chloride to prepare a gel matrix solution; Step (2): adding the gel matrix liquid into the oil phase solvent and stirring at high speed in a container; Step (3): after stirring evenly, performing a UV light cross-linking reaction, and after the reaction is completed, fully washing the obtained microspheres to obtain microspheres; Step (4): under acidic conditions, the obtained microspheres are dispersed in a sodium alginate solution, mixed and cross-linked, and filtered and washed after the reaction to obtain probiotic / mucin-loaded oral microspheres.

2. The method for preparing the probiotic / mucin-loaded oral microsphere system according to claim 1, characterized in that: The probiotics described in step (1) are obtained by transforming the pET28a plasmid into E. coli Nissle 1917 to obtain E. coli Nissle 1917 with kanamycin resistance; Or the probiotic is obtained by transforming the pTD103luxI_sfGFP plasmid into E. coli Nissle 1917 to obtain E. coli Nissle 1917 containing kanamycin resistance and GFP (green fluorescent protein).

3. The method for preparing the probiotic / mucin-loaded oral microsphere system according to claim 1, characterized in that: The molecular weight of HAMA in step (1) is 100-200 kDa, and the concentrations of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt and calcium chloride in double distilled water are 2-3 mg / mL and 0.05-0.2 mol / L, respectively.

4. The method for preparing the probiotic / mucin-loaded oral microsphere system according to claim 1, characterized in that: The number of probiotics in the gel matrix solution in step (1) is ≥1*10 9 CFU / mL, the concentration of mucin was 1-15 mg / mL, and the concentration of methacryloylated hyaluronic acid was 20-30 mg / mL; Preferably, the concentration of mucin is 8-12 mg / mL, and the concentration of methacryloyl hyaluronic acid is 23-28 mg / mL.

5. The method for preparing the probiotic / mucin-loaded oral microsphere system according to claim 1, characterized in that: In step (2), the oil phase solvent is Span 80 and liquid paraffin in a volume ratio of 0.1-1:5-15.

6. The method for preparing the probiotic / mucin-loaded oral microsphere system according to claim 1, characterized in that: In step (2), the amount of gel matrix liquid added is 0.05 to 1.5 times the volume of the oil phase solvent.

7. The method for preparing the probiotic / mucin-loaded oral microsphere system according to claim 1, characterized in that: The light source for the photo-crosslinking reaction in step (3) has an ultraviolet wavelength of 360-405 nm and a crosslinking time of 30-60 s.

8. The method for preparing the probiotic / mucin-loaded oral microsphere system according to claim 1, characterized in that: In step (4), the acidic condition is a pH value of 5.0-6.0, the concentration of the sodium alginate solution is 0.5-3 g / mL, the mass volume ratio of the microspheres to the sodium alginate solution is 5-15 mg: 80-120 mL; and the cross-linking reaction time is 0.5-1 h.

9. A probiotic / mucin-loaded oral microsphere system, characterized in that: The system is prepared by the method described in any one of claims 1 to 8.

10. Use of the probiotic / mucin-loaded oral microsphere system prepared by the method of claim 1 in treating or regulating unbalanced intestinal flora; preferably: use of the probiotic / mucin-loaded oral microsphere system prepared by the method in treating or regulating enterogenic sepsis with intestinal dysfunction.

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