Preparation method and application of hydrogel encapsulating functionalized probiotics

Abstract

The invention discloses a preparation method and application of hydrogel encapsulating functionalized probiotics, live lactobacillus rhamnosus is wrapped by a lipid membrane containing ceramide and then encapsulated in PMBV / PVA hydrogel containing sodium selenite, and the hydrogel system can release selenium nanoparticles, antibacterial substances secreted by bacteria and ceramide; according to the invention, a hydrogel system is constructed, probiotics in the hydrogel system can be protected from being influenced by complex external conditions, the activity of the probiotics can be maintained, and various active substances released by the hydrogel can synergistically treat infectious wounds of skin, promote wound healing and protect skin micro-ecology; the hydrogel has potential application prospects in the fields of antibacterial wound dressing, infectious wound treatment and viable bacteria therapy.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of a hydrogel for encapsulating functionalized probiotics. Background Art

[0002] When the skin is damaged, it cannot prevent harmful bacteria from invading the tissue, causing wound infection, or even severe tissue damage and even life-threatening. Open wounds can be contaminated or colonized by bacteria, which can cause pain, erythema, fever, swelling, purulent secretions, and delayed wound healing longer than expected. Therefore, open wounds need to be covered with medical dressings to achieve the effects of sterilization and promoting wound healing. At present, there are many wound dressings used to promote wound healing, such as hydrogels, hydrocolloids, foams, film dressings, etc. The ideal wound dressing needs to meet the following properties: good biocompatibility; good moisture retention; good antibacterial properties; sufficient mechanical strength; appropriate surface microstructure and biochemical properties to promote cell adhesion, proliferation and differentiation, so as to achieve the effect of promoting wound healing.

[0003] Lactobacillus rhamnosus is a probiotic that is safe for the human body. It has beneficial effects on digestive system diseases, cancer, immunity, urinary tract infections and the reconstruction of healthy intestinal and vaginal microbiota. It can secrete antibacterial substances such as organic acids and antimicrobial peptides to inhibit pathogenic bacteria including drug-resistant bacteria. In addition, Lactobacillus rhamnosus has been shown to be able to convert high-valent selenium elements into zero-valent selenium nanoparticles on the cell surface and inside. Selenium nanoparticles have good bioavailability and safety. They have antioxidant, anticancer, anti-inflammatory, antibacterial and wound healing activities.

[0004] Ceramide is a lipid naturally present in the skin, accounting for about half of the total lipid content of human skin, and plays an important role in repairing the skin barrier and promoting wound healing. Ceramide, phospholipids and cholesterol are prepared into a lipid membrane form and wrapped on Lactobacillus rhamnosus, which can play a similar role as ceramide liposomes and increase the skin utilization rate of ceramide. Not only that, the active substances of ceramide and Lactobacillus rhamnosus can act synergistically on human skin.

[0005] Traditional antibacterial hydrogel dressings usually use chemical drugs to inhibit bacteria, which may not be able to eliminate drug-resistant pathogens and will eliminate harmless probiotics, destroy skin flora, and have poor effects in promoting wound healing. Summary of the invention

[0006] The present invention provides a preparation method and application of a hydrogel encapsulating functionalized probiotics. Live Lactobacillus rhamnosus is wrapped in a lipid membrane containing ceramide, and then encapsulated in a PMBV / PVA hydrogel containing sodium selenite. Lactobacillus rhamnosus produces selenium nanoparticles and secretes antibacterial substances in a sodium selenite environment, which diffuse to the surface of infected wounds through the hydrogel, cooperate with ceramide to treat wound infection, promote wound healing, and protect skin microecology.

[0007] The specific technical solutions of the present invention are as follows:

[0008] A method for preparing a hydrogel encapsulating functionalized probiotics, the specific steps are as follows:

[0009] (1) 880-900 mg of 2-methacryloyloxyethyl phosphorylcholine (MPC), 140-150 mg of n-butyl methacrylate (BMA), 140-150 mg of p-vinylphenylboronic acid (VPBA) and 40-50 mg of azobisisobutyronitrile (AIBN) were dissolved in 6-10 mL of anhydrous ethanol in a round-bottom flask, nitrogen was bubbled into the solution for 5-10 minutes to eliminate oxygen, and then the round-bottom flask was sealed and stirred at 50-70° C. for 1-2 hours. After the contents were cooled to room temperature, the contents were poured into a 15-fold volume of a mixed solvent of ether and chloroform, the precipitate was collected by filtration and vacuum dried overnight, and then the dried product was dissolved in distilled water, dialyzed in pure water for one week using a dialysis bag, and finally the distilled water was removed by freeze drying to obtain a white polymer powder, namely PMBV;

[0010] (2) washing 2-3 mL of Lactobacillus rhamnosus bacterial solution by centrifugation with PBS buffer, and resuspending the solution in 2-3 mL of ice-cold PBS containing 12-13 mM CaCl2 to obtain a PBS bacterial solution, dissolving 0.02-0.03 mol of dioleoylphosphatidic acid monosodium salt (DOPA), 0.005-0.006 mol of cholesterol, and 0.005-0.006 mol of ceramide in 2-3 mL of chloroform, and drying the resulting solution at room temperature using a rotary evaporator to obtain a ceramide lipid film, hydrating the obtained ceramide lipid film in the above 2-3 mL of PBS bacterial solution, vortexing for 15-30 minutes, and then centrifuging, and resuspending the precipitate in 2-3 mL of MRS culture medium to obtain a probiotic bacterial solution wrapped with a ceramide lipid film;

[0011] (3) Sodium selenite and polyvinyl alcohol are added to MRS medium to obtain solution A, and sodium selenite, PMBV, and a probiotic solution wrapped in a ceramide lipid membrane are added to MRS medium to obtain solution B. After equal volumes of solution A and solution B are mixed, a hydrogel encapsulating functionalized probiotics is obtained.

[0012] In step (1), the volume ratio of ether to chloroform in the mixed solvent of ether and chloroform is 8:2.

[0013] In step (1), the molecular cutoff of the dialysis bag is 3500.

[0014] In step (2), Lactobacillus rhamnosus can also be replaced by other functional beneficial bacteria.

[0015] In step (2), the concentration of Lactobacillus rhamnosus in the PBS bacterial solution is 10 9 CFU / mL.

[0016] In step (3), the degree of polymerization of polyvinyl alcohol in the solution A is 1000-1700, the mass concentration of polyvinyl alcohol is 2.5%-10%, and the concentration of sodium selenite is 100-300 mg / L.

[0017] In step (3), in the solution B, the mass concentration of PMBV is 2.5%-10%, and the concentration of Lactobacillus rhamnosus is 10 8 CFU / mL, sodium selenite concentration is 100-300mg / L.

[0018] The present invention also provides application of the hydrogel prepared by the preparation method in preparing medical dressings for antibacterial and wound healing promotion.

[0019] The invention loads beneficial bacteria Lactobacillus rhamnosus in PMBV / PVA hydrogel to prepare bacteria-loaded hydrogel. The hydrogel has high biocompatibility and no toxic side effects. In the sodium selenite environment of the hydrogel, the live Lactobacillus rhamnosus therein converts the sodium selenite into selenium nanoparticles. The selenium nanoparticles have anti-inflammatory, antioxidant and antibacterial effects, and the antibacterial effects are also effective against drug-resistant bacteria. Meanwhile, the Lactobacillus rhamnosus grows and reproduces in the hydrogel, and produces bacteriocins, lactic acid and other antibacterial active substances. These antibacterial active substances can selectively eliminate pathogenic bacteria without eliminating probiotics. The lipids coated on the surface of the Lactobacillus rhamnosus contain ceramides, which are also released as the bacteria grow and reproduce, and can repair the skin barrier and promote wound healing. These active substances are released to the wound surface through the hydrogel, and synergistically treat wound infections caused by drug-resistant bacteria and promote wound healing.

[0020] The hydrogel encapsulating functional live bacteria prepared by the present invention utilizes live bacteria therapy, encapsulates live probiotics encapsulating ceramide in the PMBV / PVA hydrogel, and causes them to produce selenium nanoparticles and secrete antibacterial substances. These substances and ceramide are controllably released into infected wounds through the hydrogel, inhibiting the growth of pathogenic bacteria and promoting wound healing. Meanwhile, beneficial bacteria are not inhibited, the skin surface flora can be adjusted, and the probiotics are effective against drug-resistant bacteria.

[0021] Beneficial effects of the present invention:

[0022] (1) The present invention constructs a novel bacteria-loaded hydrogel, the loaded probiotics have no toxic side effects and have the function of selectively eliminating drug-resistant pathogenic bacteria, and the culture scheme is mature and can be sustainably produced and utilized.

[0023] (2) The present invention is different from many existing technologies for synthesizing selenium nanoparticles by chemical methods. The selenium nanoparticles in the hydrogel are produced by synthesizing selenium nanoparticles through probiotics. The method is simpler, greener and more environmentally friendly. The obtained selenium nanoparticles have anti-inflammatory, antioxidant and highly effective antibacterial effects.

[0024] (3) The lipids coated on the surface of the probiotics of the present invention contain ceramide, which is also released as the beneficial bacteria grow and multiply, and can repair the skin barrier and promote wound healing.

[0025] (4) During the treatment process, the active substances in the hydrogel of the present invention are released to the wound surface through the hydrogel, synergistically treating wound infections caused by drug-resistant bacteria and promoting wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 For PMBV 1 H-NMR spectrum;

[0027] Figure 2 is the FTIR spectrum of PMBV;

[0028] Figure 3 is the GPC spectrum of PMBV;

[0029] Figure 4 Electron microscopic images of probiotics wrapped and not wrapped with ceramide lipid membrane;

[0030] Figure 5 SEM images and elemental analysis of selenium nanoparticles;

[0031] Figure 6 The scanning electron microscope image and elemental analysis of selenium-enriched probiotics;

[0032] Figure 7 is the visible-UV spectrum of extracellular Se nanoparticles;

[0033] Figure 8 The changes of bacterial activity in the hydrogel;

[0034] Fig. 9 To evaluate the antioxidant capacity of selenium nanoparticles;

[0035] Fig.10 To evaluate the antibacterial ability of bacteria-loaded hydrogels in vitro;

[0036] Fig.11 Photo of bacteria-loaded hydrogel promoting wound healing. DETAILED DESCRIPTION

[0037] The technical scheme of the present invention is described in detail below in conjunction with specific embodiments. The experimental conditions and operations not mentioned in the embodiments of the present invention are carried out according to the conventional methods in the art or the conditions recommended by the manufacturer, and the protection scope of the present invention is by no means limited to the embodiments. The probiotics selected in the embodiments of the present invention are Lactobacillus rhamnosus, and Lactobacillus rhamnosus can also be replaced by other functionalized beneficial bacteria.

[0038] The phosphate buffer PBS buffer and MRS culture medium used in the present invention are conventional commercial products.

[0039] Example 1

[0040] The preparation method of PMBV comprises the following specific steps:

[0041] 886 mg of 2-methacryloyloxyethyl phosphorylcholine (MPC), 142 mg of n-butyl methacrylate (BMA), 148 mg of p-vinylphenylboronic acid (VPBA) and 40 mg of azobisisobutyronitrile (AIBN) were dissolved in a round-bottom flask with 6 mL of anhydrous ethanol, nitrogen was bubbled into the solution for 5 minutes to eliminate oxygen, and then the round-bottom flask was sealed and stirred at 60°C for 1.5 hours. After the contents were cooled to room temperature, the contents were poured into a mixture of 15 times the volume of ether and chloroform (the volume ratio of ether to chloroform was 8:2), the precipitate was collected by filtration and vacuum dried overnight, and then the polymer was dissolved in distilled water and dialyzed in pure water for one week using a dialysis bag (molecular cutoff 3500). Finally, the distilled water was removed by freeze drying to obtain a white polymer powder, namely PMBV, whose reaction equation is shown below:

[0042]

[0043] Figure 1 is the one-dimensional H NMR spectrum of PMBV, according to Figure 1 As a result, the MPC unit integral value is 3.19ppm(-N + (CH3)3, 9H), the integral value of BMA unit is 1.34-1.54ppm (-CH2-, 4H), and the integral value of VPBA unit is 6.93-7.64ppm (-C6H4-, 4H). The composition of each monomer is calculated based on the integral values ​​of the above peaks, and the molar ratio of each unit is obtained: MPC:BMA:VPBA=55:17:28.

[0044] Figure 2 The Fourier infrared spectrum of PMBV shows that -1 、1720cm -1 、1385cm -1 and 965cm-1 -CH2-, C=O, BO and -N + The infrared absorption of (CH3)3 further proves that PMBV has been successfully synthesized.

[0045] Figure 3 This is the gel permeation chromatogram of PMBV. It can be seen from the figure that the number average molecular weight of PMBV is 93813, the weight average molecular weight is 95751, the molecular weight distribution index (PDI) is 1.02, which is less than 1.1. The molecular weight distribution is narrow and the molecular weight reaches the designed target value.

[0046] Depend on Figure 1-3 It can be seen that PMBV was successfully synthesized.

[0047] Example 2

[0048] The preparation method of the probiotic liquid wrapped with ceramide lipid membrane comprises the following specific steps:

[0049] Wash with 2 mL of 10% phosphate buffered saline (PBS) 9 CFU / mL of Lactobacillus rhamnosus bacterial liquid is prepared and resuspended in 2 mL of ice-cold PBS (about 4°C) containing 12.5 mM CaCl2 to obtain a PBS bacterial liquid, 0.02 mol of dioleoylphosphatidic acid monosodium salt (DOPA), 0.005 mol of cholesterol and 0.005 mol of ceramide are dissolved in 2 mL of chloroform, and the resulting solution is dried at room temperature using a rotary evaporator to obtain a ceramide lipid film, the obtained film is hydrated in 2 mL of the above PBS bacterial liquid, vortexed for 15 minutes, and then centrifuged, and the bacterial precipitate is resuspended in 2 mL of MRS culture medium to obtain a probiotic bacterial liquid wrapped in a ceramide lipid film.

[0050] The probiotics without ceramide lipid membrane and the probiotics with ceramide lipid membrane were fixed, stained, and dried. The samples were observed on a copper grid using a transmission electron microscope. The results are as follows: Figure 4 As shown, the edge shadow of the probiotics not wrapped in the ceramide lipid membrane is thinner, and the junction is clear and sharp, while the edge of the probiotics wrapped in the ceramide lipid membrane is thicker, the junction is rougher, and there are obvious lipids, indicating that the ceramide lipid membrane is successfully wrapped on the surface of the probiotics.

[0051] Example 3

[0052] The preparation method of bacteria-loaded hydrogel comprises the following specific steps:

[0053] Sodium selenite and polyvinyl alcohol (PVA) were added to MRS medium to obtain solution A, in which the degree of polymerization of polyvinyl alcohol in solution A was 1000-1700, the mass concentration of polyvinyl alcohol was 5%, and the concentration of sodium selenite was 200 mg / L; sodium selenite, PMBV and probiotic bacterial solution wrapped with ceramide lipid membrane were added to MRS medium to obtain solution B, in which the mass concentration of PMBV in solution B was 2.5%, the concentration of Lactobacillus rhamnosus was 10 8 CFU / mL, the concentration of sodium selenite was 200 mg / L, and solution A and solution B were stirred and mixed in equal volumes to obtain the bacteria-loaded hydrogel.

[0054] The degree of polymerization of polyvinyl alcohol in solution A is 1000-1700, the mass concentration of polyvinyl alcohol is between 2.5% and 10%, and the concentration of sodium selenite is between 100 and 300 mg / L; in solution B, the mass concentration of PMBV is between 2.5% and 10%, and the concentration of Lactobacillus rhamnosus is 10 8 CFU / mL, and the sodium selenite concentration is between 100-300 mg / L, and bacteria-loaded hydrogels can be prepared.

[0055] Example 4

[0056] The preparation method of the bacteria-free hydrogel comprises the following specific steps:

[0057] Sodium selenite and polyvinyl alcohol are added to MRS culture medium to obtain solution A, in which the degree of polymerization of polyvinyl alcohol in solution A is 1000-1700, the mass concentration of polyvinyl alcohol is 5%, and the concentration of sodium selenite is 200 mg / L; sodium selenite and PMBV are added to MRS culture medium to obtain solution B, in which the mass concentration of PMBV in solution B is 2.5%, and the concentration of sodium selenite is 200 mg / L. After equal volumes of solution A and solution B are mixed, a bacteria-free hydrogel is obtained.

[0058] Example 5

[0059] The ability of Lactobacillus rhamnosus to produce nanoparticles was characterized as follows:

[0060] (1) Add Lactobacillus rhamnosus to a 200 mg / L sodium selenite MRS medium at a concentration of 10 8CFU / L, after culturing at 37°C for 24 hours, centrifuged at 12000 rpm for 5 minutes, washed the precipitate twice with 10 mM Tris-HCl (pH 7.4), and resuspended in a lysis buffer containing 2% (w / v) sodium dodecyl sulfate (SDS) and 0.2 M NaOH, then, in an ice bath, further treated the cells with an ultrasonic cell disruptor at a power output of 120 w for 10 minutes, and finally the homogenate was centrifuged at 12000 rpm for 5 minutes to collect the precipitate, which contained nano-selenium and was washed three times with deionized water, and freeze-dried using a freeze dryer to obtain selenium nanoparticles;

[0061] The selenium nanoparticles obtained in step (1) were sprayed with gold and observed using a scanning electron microscope. The elemental composition was analyzed by energy spectrum scanning using the scanning electron microscope. The results are as follows: Figure 5 As shown, SEM and EDX images show that a large number of particles composed of carbon (C), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P) and zero-valent selenium are encapsulated into spheres, indicating that Lactobacillus rhamnosus can produce selenium nanoparticles in a sodium selenite environment.

[0062] The selenium nanoparticles obtained in step (1) were sprayed with gold and then observed using a scanning electron microscope. The scanning electron microscope was used to perform an energy spectrum scan to analyze whether the selenium nanoparticles were present on the outer surface of the bacteria. The results were as follows: Figure 6 As shown, from the electron microscope image, it can be clearly seen that some spheres exist on the outer surface of the bacteria, and the results of the energy spectrum scan show that these spheres are enriched with selenium, so selenium nanoparticles exist on the outer surface of the bacteria, indicating that selenium nanoparticles are released through the hydrogel.

[0063] In the sodium selenite MRS medium with a concentration of 200 mg / L, Lactobacillus rhamnosus was added to a concentration of 10 8 CFU / L, cultured at 37°C for 0, 8, 16, 24, and 48 hours, took the bacterial solution at different time points, filtered it with a 0.22μm bacterial filter, and recorded its external-visible spectrum using a visible spectrophotometer. The results are shown in Figure 7 As shown, the highest absorption peak is at 306 nm, which is a typical plasma resonance band of selenium nanoparticles, proving the existence of selenium nanoparticles outside the bacteria and the content of selenium nanoparticles outside the bacteria increases with time.

[0064] Example 6

[0065] The hydrogel properties were characterized as follows:

[0066] 1. After 0, 12, and 24 hours of preparation of the bacteria-loaded hydrogel, the bacteria-loaded hydrogel was incubated in a PBS buffer containing a live / dead bacterial staining reagent (SYTO@9 / PI) at 37°C for 20 minutes, then washed in a PBS buffer to remove excess staining reagent, and then observed using a laser confocal microscope. The green fluorescence area and the red fluorescence area were quantitatively analyzed to calculate the bacterial activity. The results are shown in Figure 8 As shown, the bacterial activity at 0 hour was higher than 90%. Although it decreased to a certain extent after 24 hours, the bacterial activity was still higher than 80%. The hydrogel has high biocompatibility and can allow probiotics to survive in it.

[0067] 2. Antioxidant capacity of hydrogel

[0068] Macrophages were plated on 24-well plates for 10 5 The number of cells per well was calculated. After 24 h of culture, selenium nanoparticles were added to the experimental group for 1 h of pretreatment, and then hydrogen peroxide with a final concentration of 2.0 mmol / L was added. The negative control group was only added with hydrogen peroxide with a final concentration of 2.0 mmol / L. The blank control group was added with MRS culture medium, followed by the addition of fluorescent red dye, and continued to incubate for 30 min. The cells were washed 3 times with PBS, fixed, and stained with DAPI for 10 min. Then, a laser confocal microscope was used to observe the red fluorescence intensity under different treatment conditions at an excitation wavelength of 570 nm. The results are shown in Fig. 9 As shown, there was little difference in red fluorescence intensity between the negative control group and the blank control group, while the red fluorescence intensity of the experimental group was the smallest, proving that selenium nanoparticles play a protective role in hydrogen peroxide-induced cell damage.

[0069] 3. In vitro antibacterial effect

[0070] The bacteria-loaded hydrogel and the bacteria-free hydrogel were placed in a 48-well plate at a volume of 280 μL per well to form a hydrogel coating, and 300 μL of sterile water was added to each well. 300 μL of PBS buffer, 300 μL of probiotic supernatant and 300 μL of oxacillin sodium solution were also placed in the 48-well plate. The drug-resistant Staphylococcus aureus (MRSA) was activated overnight for 12 h to obtain 2*10 9 CFU / mL, diluted 100 times to 2*10 7CFU / mL of bacterial suspension, and then 100 μL of the bacterial suspension was added to the above 48-well plate: A) 300 μL PBS buffer; B) 300 μL bacterial supernatant; C) 300 μL sterile water and bacteria-loaded hydrogel coating; D) 300 μL sterile water and bacteria-free hydrogel coating (280 μL); E) 300 μL oxacillin sodium solution (5 μg / mL). After incubation at 37°C for 5 hours and 24 hours, the incubated liquid was diluted 1000 times with PBS buffer, and then 50 μL of the bacterial solution was spread on a Luria Bertani (LB) agar plate and maintained at 37°C for 24 hours. The colonies were observed and counted. The results are shown in Fig.10 As shown, compared with the control group (A), the antibiotic group (E) and the blank hydrogel group (D) had no antibacterial effect within 24 hours, which also shows that the resistant Staphylococcus aureus showed resistance to 4μg / mL of benzylpenicillin sodium. However, the bacteria-loaded hydrogel (C) significantly inhibited the proliferation of bacteria within 24 hours. After incubation with the bacteria-loaded hydrogel, the number of MRSA colonies decreased rapidly, and a 71% inhibition rate was obtained within 5 hours, and a 100% inhibition rate was obtained within 24 hours. The bacterial solution group (B) had a more efficient antibacterial effect, and the inhibition rate reached 100% within 5 hours. This can indicate that the bacterial solution contains antibacterial substances secreted by Lactobacillus rhamnosus, which can effectively inhibit resistant Staphylococcus aureus, and these substances can achieve the antibacterial effect through hydrogel sustained release.

[0071] 4. Healing-promoting effect in C57BL mice

[0072] Eight-week-old male C57BL mice were randomly divided into a control group, a blank hydrogel (i.e., non-bacteria-loaded hydrogel), and a bacteria-loaded hydrogel group, with 8 mice in each group. After one week of adaptive feeding, the mice were anesthetized with isoflurane, and a circular hole was punched on the back of the mice with a hole puncher. The full-thickness skin with a diameter of 8.0 mm was cut, and 50 μL of resistant Staphylococcus aureus liquid was injected into the wound. Subsequently, the bacteria-loaded hydrogel was applied to the wound. The control group was applied with PBS buffer, and the blank hydrogel group was applied with non-bacteria-loaded hydrogel. Each group was given the drug once every other day, and the wounds were photographed at preset time intervals. The results are shown in Figure 2. Fig.11 As shown, the photos showed that the bacteria-loaded hydrogel group significantly promoted the wound healing of mice compared with the control group. On the 7th day, the wound healing rate of the bacteria-loaded hydrogel group was 38%, while the wound healing rates of the control group and the blank hydrogel group were 11% and 17%; on the 14th day, the wound healing rates of the bacteria-loaded hydrogel group and the bacterial liquid group were 92%, while the wound healing rates of the control group and the blank hydrogel group were 67% and 71%, respectively. The results were statistically significant, indicating that the bacteria-loaded hydrogel can accelerate wound healing, while the blank hydrogel is to provide a suitable growth and reproduction environment for probiotics, as well as to serve as a drug delivery platform, but it itself has no antibacterial effect.

[0073] The bacteria-free hydrogel prepared by the present invention can be used as a drug carrier to load selenium nanoparticles, ceramide, various functional probiotics (including but not limited to the beneficial bacteria Lactobacillus rhamnosus) or their bacteriocins or other antibacterial active substances produced by probiotics, etc., and other functional hydrogels can also be obtained. The present invention provides a new application idea for hydrogels.

[0074] The specific implementation modes of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above implementation modes, and various changes can be made within the knowledge scope of ordinary technicians in this field without departing from the purpose of the present invention.

Claims

1. A method for preparing a hydrogel encapsulating functionalized probiotics, characterized in that: The specific steps are as follows: (1) 880-900 mg of 2-methacryloyloxyethyl phosphorylcholine, 140-150 mg of n-butyl methacrylate, 140-150 mg of p-vinylphenylboronic acid and 40-50 mg of azobisisobutyronitrile were dissolved in 6-10 mL of anhydrous ethanol, nitrogen was bubbled into the solution for 5-10 minutes and then sealed, stirred at 50-70° C. for 1-2 hours, and after the contents were cooled to room temperature, poured into a 15-fold volume of a mixed solvent of ether and chloroform, the precipitate was collected by filtration and vacuum dried overnight, the dried product was dissolved in distilled water, dialyzed in pure water for one week using a dialysis bag, and freeze-dried to obtain a white powder, namely PMBV; (2) washing 2-3 mL of Lactobacillus rhamnosus bacterial solution by centrifugation with PBS, and then resuspending it in 2-3 mL of ice-cold PBS with a concentration of 12-13 mmol / LCaCl2 to obtain a PBS bacterial solution, dissolving 0.02-0.03 mol of dioleoylphosphatidic acid monosodium salt, 0.005-0.006 mol of cholesterol and 0.005-0.006 mol of ceramide in 2-3 mL of chloroform, and drying the solution at room temperature to obtain a ceramide lipid film, hydrating the ceramide lipid film in the PBS bacterial solution, vortexing for 15-30 minutes, and then centrifuging, and resuspending the precipitate with 2-3 mL of MRS culture medium to obtain a probiotic bacterial solution wrapped with a ceramide lipid film; (3) Sodium selenite and polyvinyl alcohol are added to MRS medium to obtain solution A, sodium selenite, PMBV and probiotic bacterial liquid wrapped with ceramide lipid membrane are added to MRS medium to obtain solution B, and solution A and solution B are mixed in equal volumes to obtain a hydrogel encapsulating functionalized probiotics.

2. The method for preparing the hydrogel encapsulating functionalized probiotics according to claim 1, characterized in that: In step (1), the volume ratio of ether to chloroform in the mixed solvent of ether and chloroform is 8:

2.

3. The method for preparing the hydrogel encapsulating functionalized probiotics according to claim 1, characterized in that: In step (1), the molecular cutoff of the dialysis bag is 3500.

4. The method for preparing the hydrogel encapsulating functionalized probiotics according to claim 1, characterized in that: In step (2), Lactobacillus rhamnosus is replaced by other functionalized beneficial bacteria.

5. The method for preparing the hydrogel encapsulating functionalized probiotics according to claim 1, characterized in that: In step (2), the concentration of Lactobacillus rhamnosus in the PBS bacterial solution is 10 9 CFU / mL.

6. The method for preparing the hydrogel encapsulating functionalized probiotics according to claim 1, characterized in that: In step (3), the degree of polymerization of polyvinyl alcohol in the solution A is 1000-1700, the mass concentration of polyvinyl alcohol is 2.5%-10%, and the concentration of sodium selenite is 100-300 mg / L.

7. The method for preparing the hydrogel encapsulating functionalized probiotics according to claim 1, characterized in that: In step (3), in the solution B, the mass concentration of PMBV is 2.5%-10%, the concentration of Lactobacillus rhamnosus is 108 CFU / mL, and the concentration of sodium selenite is 100-300 mg / L.

8. Use of the hydrogel encapsulating functionalized probiotics prepared by the preparation method of claim 1 in preparing medical dressings for antibacterial and wound healing promotion.

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