Lactobacillus plantarum zjuids19, preparation and application thereof

Abstract

The application provides a lactobacillus plantarum ZJUIDS19 and preparation and application. The classification and naming of the strain is lactobacillus plantarum, and the preservation number is CGMCC NO.28018. The 16S rDNA full sequence of the lactobacillus plantarum ZJUIDS19 is shown as SEQ ID No.1. The application is based on probiotics separated from Inner Mongolia sour milk products, and the lactobacillus plantarum with antibacterial and antioxidant effects is screened out, and postbiotics is extracted, which is combined with modified lactalbumin to form a new type of hydrogel material. The material has good antibacterial property, strong antioxidant capacity, biocompatibility, and can promote wound healing as a wound dressing.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a plant lactobacillus ZJUIDS19 and its preparation and application. It is a plant lactobacillus ZJUIDS19 and its preparation of an antibacterial lactalbumin hydrogel loaded with postbiotics, and its application in biomedical materials. Background Technology

[0002] As a vital interface between the body and its surrounding environment, the skin plays a crucial role in preventing moisture loss and blocking the invasion of harmful substances and pathogenic microorganisms. Various microbial communities, including bacteria, fungi, and viruses, coexist within the skin. However, skin damage provides an opportunity for harmful bacteria to invade living tissue, leading to wound infection and even severe tissue damage. To address bacterial infections of skin wounds, novel antibiotics, antimicrobial nanoparticles, cationic polymer compounds, and antimicrobial peptides have become hot research topics in recent years.

[0003] Live bacterial therapy has garnered significant attention in recent years for treating various inflammatory and immunopathological diseases through bacterial interference and immunomodulation. Certain beneficial bacteria can create unique local microenvironments by secreting large amounts of metabolites and antimicrobial agents, which are suitable for their own survival but inhibit the growth of competing microorganisms. Therefore, beneficial bacteria that secrete bioactive substances with antibacterial, anticancer, or immunosuppressive capabilities have been widely used in diagnosis and treatment. However, the application of bacteria is largely limited due to their inherent properties, including rapid proliferation and colonization. Metabiotics, as bacterial metabolites, are safer, more controllable, and easier to store than bacteria or live bacteria; therefore, the extraction and application of metabiotics are gradually becoming a hot topic.

[0004] Hydrogels, popular biomedical polymers with 3D molecular networks, have been widely used in drug delivery, implantation, and tissue engineering. Due to their high biocompatibility and moist healing environment, hydrogels are used to promote tissue repair and regeneration. Lactalbumin (α-LA) is a small, globular protein found in the whey of all mammals. It is the second most abundant protein in bovine whey, and bovine and human lactalbumin share 74% sequence identity, similar bioactivity, and similar amino acid content. These characteristics make it a viable biomaterial, including low immunogenicity, low risk of disease transmission, and bioactivity associated with antitumor, antibacterial, antioxidant, and antihypertensive effects. However, research on the use of lactalbumin hydrogels as medical materials is currently limited. Summary of the Invention

[0005] The first objective of this invention is to provide a strain of *Lactobacillus plantarum* ZJUIDS19, which was deposited on July 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC), classified as *Lactobacillus plantarum*, with accession number CGMCC NO.28018, and located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The full 16S rDNA sequence of *Lactobacillus plantarum* ZJUIDS19 is shown in SEQ ID No. 1.

[0006] The second objective of this invention is to provide a method for preparing an antibacterial lactalbumin hydrogel loaded with post-biotic, which is achieved through the following steps:

[0007] (1) Aseptic fermentation supernatant

[0008] Lactobacillus plantarum ZJUIDS19 was inoculated into MRS liquid medium using an inoculation loop and cultured. Then, it was transferred to sterilized MRS liquid medium at an inoculation rate of 2% (v / v). After culture, the fermentation broth was obtained. The cells were collected by centrifugation. The cells were then suspended in distilled water and centrifuged to collect the supernatant. The pH was adjusted to 6.0 and filtered to obtain sterile fermentation supernatant.

[0009] The preparation of MRS liquid culture medium is as follows: Dissolve the following components in each liter of distilled water: 10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium citrate, 5 g sodium acetate, 20 g glucose, 80 ml Tween, 0.5 g magnesium sulfate, 0.25 g manganese sulfate, and 15 g agar powder.

[0010] (2) Preparation of Lactobacillus plantarum ZJUIDS19 postbiotic

[0011] The supernatant of sterile fermentation was concentrated by vacuum freezing, the supernatant was pre-frozen at ultra-low temperature, freeze-dried and vacuum-treated, and after complete freeze-drying, the metagenes were removed under reduced pressure and placed in a 4℃ refrigerator to obtain the metagenes of Lactobacillus plantarum ZJUIDS19.

[0012] (3) The extracted metagenics was added to the modified lactalbumin solution at room temperature and solidified together under ultraviolet light to form an antibacterial lactalbumin hydrogel loaded with metagenics.

[0013] The culture time in step (1) is 18 hours, the centrifugation conditions are 10000 r / min for 20 min, and the pH value is adjusted with 2M NaOH.

[0014] Step (2) The ultra-low temperature pre-freezing condition is to place it in an ultra-low temperature freezer at -80℃ for no less than 2 hours for pre-freezing.

[0015] Step (3) Ultraviolet irradiation conditions are: ultraviolet wavelength of 380-405 nm, irradiation time of 10-60 s, and ultraviolet light intensity of 30 mW / cm². 2 .

[0016] A third objective of this invention is to provide the application of the aforementioned post-biotic-loaded antibacterial milk protein hydrogel in the preparation of biomedical materials. These biomedical materials include lactic acid bacteria bacteriocins, antibacterial wound dressings, antibacterial and antioxidant live bacterial preparations, and bacterial powders with antibacterial and antioxidant functions.

[0017] The lactic acid bacteria bacteriocins can be used as preservatives in meat products, dairy products, and alcoholic beverages, or combined with other preservative technologies as a fence technology in food processing and storage.

[0018] The culture medium of *Lactobacillus plantarum* ZJUIDS19 provided by this invention shows no antibiotic resistance, indicating that the strain does not carry drug resistance genes. This invention also provides a hydrogel loaded with biogenic antimicrobial proteins; this strain exhibits antibacterial properties, anti-inflammatory and antioxidant effects, shows no antibiotic resistance in the culture medium, and promotes skin wound healing. Therefore, *Lactobacillus plantarum* ZJUIDS19 of this invention can be widely used in biomedical and other fields.

[0019] This invention is based on probiotics isolated from yogurt products in Inner Mongolia. *Lactobacillus plantarum* with antibacterial and antioxidant effects was screened from these probiotics, and its post-biotic components were extracted. These components were then combined with modified lactic acid to form a novel hydrogel material. This material exhibits good antibacterial properties, strong antioxidant capacity, and biocompatibility, and can promote wound healing when used as a wound dressing. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of *Lactobacillus plantarum* ZJUIDS19, which is the present invention.

[0021] Figure 2 This is a Gram-stained image of the cell morphology of *Lactobacillus plantarum* ZJUIDS19, as described in this invention.

[0022] Figure 3 This is an electrophoretic identification diagram of the 16S rDNA of *Lactobacillus plantarum* ZJUIDS19, as described in this invention.

[0023] Figure 4 This is a hydrogel diagram of the lactalbumin loaded with postbiotics according to the present invention.

[0024] Figure 5 The rheological properties of the prepared postbiotic-loaded lactalbumin hydrogel are shown.

[0025] Figure 6To investigate the microstructure of the lactalbumin hydrogel loaded with post-biotic.

[0026] Figure 7 This is a diagram showing the in vitro antibacterial results of lactalbumin hydrogel loaded with biotics.

[0027] Figure 8 This is a diagram showing the in vivo antibacterial results of lactalbumin hydrogel loaded with postbiotics.

[0028] Figure 9 To test the wound healing morphology of mice in each group.

[0029] Figure 10 HE staining images of mouse wounds in each group of this invention.

[0030] Figure 11 The images show Masson staining of mouse wounds in each group of mice according to this invention. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0032] Example 1: Screening, identification, and preparation of metabiotics for *Lactobacillus plantarum* ZJUIDS19

[0033] 1. Screening of Lactobacillus plantarum ZJUIDS19

[0034] 1.1 Sample Source

[0035] The bacterial strain used in this invention was derived from samples of fermented mare's milk from Inner Mongolia. A total of 20 samples were collected.

[0036] 1.2 Isolation and purification of strains

[0037] Approximately 5g of sample was collected in a sterile tube and immediately sent to the laboratory for bacterial isolation and purification. For isolation, 1g of sample was placed in 9mL of MRS liquid medium, vortexed, and incubated at 37℃ for 48h. Then, 1mL of the culture medium was taken in a laminar flow hood and serially diluted tenfold with sterile physiological saline. 10... -5 10 -6 10 -7 Three dilution gradients were used, with 100 μL of bacterial suspension from each gradient plated onto MRS solid medium and incubated at 37°C for 48 h. After incubation, plates with 30–300 single colonies were selected from the MRS solid medium, and typical colonies were picked and streaked multiple times on MRS agar plates until the colonies on the entire plate had a uniform morphology. Single colonies were then transferred to MRS liquid medium for further culture. The resulting strains were then permanently frozen at -80°C in MRS liquid medium containing 40% (w / v) glycerol.

[0038] Preparation of liquid MRS medium: Dissolve the following components in each liter of distilled water: 10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium citrate, 5 g sodium acetate, 20 g glucose, 80 ml Tween, 0.5 g magnesium sulfate, and 0.25 g manganese sulfate.

[0039] Preparation of solid MRS medium: Dissolve the following components in each liter of distilled water: 10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium citrate, 5 g sodium acetate, 20 g glucose, 80 ml Tween, 0.5 g magnesium sulfate, 0.25 g manganese sulfate, and 15 g agar powder.

[0040] 2. Identification of Lactobacillus plantarum ZJUIDS19

[0041] 2.1 Colony characteristics

[0042] After culturing Lactobacillus plantarum ZJUIDS19 on MRS solid medium for 48 hours, the surface edges were smooth, the shape was regular and round, and the color was milky white. Figure 1 .

[0043] 2.2 Microscopic morphology

[0044] Lactobacillus plantarum ZJUIDS19 colony smear: Gram-positive, non-spore-forming, rod-shaped, see Figure 2 .

[0045] 2.3 16S rDNA Identification

[0046] Genomic DNA was extracted from the target strain using the Ezup column-based bacterial genomic DNA extraction kit. The extracted lactic acid bacteria genomic DNA was used as a template for PCR amplification. PCR experiments were performed using universal bacterial primers 27F and 1492R for 16S rDNA. After the PCR reaction, the PCR product was examined and photographed on an agarose gel. The amplified fragment length was approximately 1500 bp. (See attached image.) Figure 3 The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The results are shown in SEQ ID NO.1. BLAST sequence alignment was performed on the NCBI website, and the results showed that the sequence had more than 99% homology with the 16S rDNA sequence identified by Lactobacillus plantarum.

[0047] The *Lactobacillus plantarum* ZJUIDS19 was deposited on July 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC), classified as *Lactobacillus plantarum*, with accession number CGMCCNO.28018. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The full 16S rDNA sequence of *Lactobacillus plantarum* ZJUIDS19 is shown in SEQ ID No. 1.

[0048] 3. Preparation of postbiotic from *Lactobacillus plantarum* ZJUIDS19

[0049] 3.1 Preparation of fermentation supernatant of Lactobacillus plantarum ZJUIDS19

[0050] Inoculate *Lactobacillus plantarum* ZJUIDS19 into 100 mL of MRS liquid medium using an inoculation loop and incubate at 37°C for 18 h. Then, transfer 2% (v / v) of the inoculum to 250 mL of sterilized MRS liquid medium and incubate at 37°C for 18 h. Centrifuge the resulting fermentation broth at 10000 rpm for 20 min and collect the bacterial cells. Resuspend the bacterial cells in distilled water and centrifuge at 10000 rpm for 20 min to collect the supernatant. Adjust the pH of the sample to 6.0 with 2M NaOH, then filter through a 0.45 μm filter to obtain a sterile fermentation supernatant, which is stored at 4°C for later use.

[0051] 3.2 Freezing Concentration

[0052] The aseptic fermentation supernatant was concentrated using vacuum freeze-drying. After pre-freezing the aseptic fermentation supernatant at -80°C for at least 2 hours, it was quickly removed and placed in a vacuum freeze dryer for vacuum treatment, maintaining a vacuum level of approximately 0.1P. After complete freeze-drying, the metagene was removed under reduced pressure and placed in a 4°C freezer to obtain the metagene of *Lactobacillus plantarum* ZJUIDS19.

[0053] Example 3: Validation of the antioxidant capacity of Lactobacillus plantarum ZJUIDS19

[0054] 1. Sample preparation

[0055] The strains preserved in glycerol tubes were streaked onto MRS solid medium for isolation and incubated upside down at 37°C for 48 hours. Single colonies were picked using an inoculation loop and inoculated into sterile MRS liquid medium, then incubated statically at 37°C for 18-24 hours to obtain the culture medium. The culture medium was adjusted to a lactic acid bacteria cell concentration of 10⁻⁶ cells / mL with distilled water. 10Centrifuge at 4℃, 8000×g for 20 min, and collect the supernatant as the fermentation supernatant. Resuspend the centrifuged bacterial pellet in 0.02M PBS buffer (pH=7.4), wash, and centrifuge at 4℃, 8000×g for 20 min, repeating 3 times. Resuspend the washed bacterial cells in PBS buffer and adjust the bacterial concentration to 10. 10 CFU / mL yields the bacterial suspension.

[0056] 2. Determination of total antioxidant capacity

[0057] Total antioxidant capacity (FRAP method) requires adding 150 μL of TPTZ working solution (0.3 M acetate-sodium acetate buffer, 20 mM ferric chloride solution, and 10 mM TPTZ buffer, mixed in a V:V:V = 10:1:1, freshly prepared) and 20 μL of sample to an ELISA plate, vortexing to mix thoroughly, and reacting at 37°C for 10 min. The absorbance of the solution at 593 nm is then measured. The absorbance of the sample is substituted into the ferrous sulfate standard curve. The antioxidant capacity of the sample is expressed as ferrous sulfate equivalent (μmol FeSO4 / mL sample). Each sample is performed in triplicate, and the average value is calculated.

[0058] Ferrous sulfate standard curve: Ferrous sulfate solutions of different concentrations (0 μM, 50 μM, 100 μM, 200 μM, 400 μM, 600 μM, 800 μM) were prepared. Ferrous sulfate solutions of different molar concentrations, 10 mM TPTZ buffer, and 0.3 M acetate buffer were mixed at a ratio of V:V:V = 1:1:10. 170 μL of the mixture was added to an ELISA plate, and the plate was incubated at 37°C for 10 min. The absorbance of the solution at 593 nm was measured. A standard curve was plotted with ferrous sulfate concentration on the x-axis and absorbance on the y-axis.

[0059] 3. Reducing power determination

[0060] Take 1 mL of sample into a centrifuge tube, add 1 mL each of 0.2 M, pH 6.6 PBS solution and 1% (w / v) potassium ferricyanide solution, and mix well. Incubate at 50 °C for 20 min, then cool in an ice bath. Add 1 mL of 10% (w / v) trichloroacetic acid, centrifuge at 6000 × g for 5 min, take 1 mL of the supernatant, add 1 mL of 0.1% (w / v) ferric chloride and 1 mL of distilled water, mix thoroughly, and allow to react for 10 min. Measure the absorbance of the sample at 700 nm. Use PBS buffer or MRS liquid medium as a blank control. Perform three replicates for each sample and calculate the average value.

[0061] Reducing power (%) = [(A s -A b ) / A b In the formula ]*100%: As - Sample group absorbance; A b -Absorbance of the blank group.

[0062] 4. Determination of DPPH free radical scavenging ability

[0063] Preparing V at different concentration gradients C Solution (0-30 μg / ml). Add 100 μL of the sample to be tested (or V) to the microplate. C The standard solution and 100 μL of 0.2 mM DPPH ethanol solution (prepared with anhydrous ethanol, stored at 4℃ protected from light, freshly prepared and used immediately) were mixed and incubated at room temperature in the dark for 30 min. The absorbance of the solution at 517 nm was measured. A blank group was prepared by replacing 100 μL of DPPH ethanol solution with 100 μL of anhydrous ethanol. A control group was prepared by replacing 100 μL of the test sample with 100 μL of PBS buffer (or MRS liquid medium). The blank was zeroed using a mixture of 100 μL of PBS buffer (or MRS liquid medium) and anhydrous ethanol. Each sample was repeated in triplicate, and the average value was calculated. DPPH free radical scavenging capacity (%) = [1 - (A s -A b ) / A c In the formula ]*100%: A s - Sample group absorbance; A b -Absorbance of blank group; A c -Absorbance of the control group.

[0064] Table 1. In vitro antioxidant activity of Lactobacillus plantarum ZJUIDS19

[0065] index bacterial suspension Fermentation supernatant <![CDATA[Total antioxidant capacity / Fe2SO4 equivalent μmol·mL -1 > 0.27±0.21 0.93±0.09 Reduction ability / % 12.02±1.52 53.05±3.05 DPPH free radical scavenging rate / % 42.72±3.86 85.29±1.41

[0066] Example 5: Confirmation of antibiotic susceptibility of Lactobacillus plantarum ZJUIDS19

[0067] The antibiotic susceptibility of lactic acid bacteria strains was measured using the disk diffusion method, following the Clinical and Laboratory Standards Institute (CLSI) guidelines. MRS solid culture medium was prepared in wide-mouth conical flasks, sterilized, and then incubated in a 55°C water bath. After cooling, the medium was placed in a sterilized laminar flow hood, and the lactic acid bacteria suspension (10... 8 Add 1% (CFU / mL) of antibiotic to an Erlenmeyer flask, shake until well mixed, and then mix the lactic acid bacteria suspension with MRS solid medium. Pour the mixture into sterile Petri dishes to prepare 15 mL / plate LB agar plates. After the MRS plates solidify, gently attach two antibiotic paper discs evenly to each plate using tweezers. Incubate the plates with the antibiotic paper discs at 37°C for 24 hours. After incubation, measure and record the diameter of the inhibition zone using calipers.

[0068] The diameters of the inhibition zones of *Lactobacillus plantarum* ZJUIDS19 to antibiotic susceptibility are shown in Table 5. Referring to the CLSI (2017) antibiotic susceptibility testing standards, *Lactobacillus plantarum* ZJUIDS19 showed extremely high susceptibility to ampicillin, cefazolin, ciprofloxacin, erythromycin, and chloramphenicol, and moderate susceptibility to trimethoprim-sulfamethoxazole. The experimental results indicate that *Lactobacillus plantarum* ZJUIDS19 is sensitive to common antibiotics.

[0069] Table 2. Antibiotic susceptibility results of Lactobacillus plantarum ZJUIDS19

[0070]

[0071]

[0072] Note: S, sensitive; I, intermediate; R, drug resistant.

[0073] With the widespread use of antibiotics in clinical treatment, the drug resistance of lactic acid bacteria is becoming increasingly serious. Long-term use of drug-resistant lactic acid bacteria will bring great difficulties to clinical treatment. The *Lactobacillus plantarum* ZJUIDS19 provided by this invention is sensitive to common antibiotics and will not harm human health.

[0074] Example 6: Confirmation of the pathogen-inhibiting ability of *Lactobacillus plantarum* ZJUIDS19

[0075] The antibacterial activity of lactic acid bacteria was determined using the internationally accepted agar diffusion method. Four frozen indicator strains (Escherichia coli, Salmonella, Staphylococcus aureus, and Listeria monocytogenes) were activated 2–3 times on LB agar. Single colonies of each activated strain were picked and incubated in LB agar at 37°C for 18 h. Bacterial cells were collected by centrifugation and resuspended in saline to achieve a concentration of 10⁻⁶. 8 CFU / mL. The indicator bacterial suspension was added at 1% (v / v) to sterilized LB solid medium cooled to approximately 55°C. After thorough mixing, the mixture was poured into petri dishes (15 mL / dish), and the sterile Oxford cups were removed after cooling. The activated *Lactobacillus plantarum* ZJUIDS19 was cultured in MRS medium for 18 h, then centrifuged (8000 rpm, 5 min, 4°C) and the supernatant was collected, discarding the bacterial precipitate. The metabolic supernatant of *Lactobacillus plantarum* ZJUIDS19 was adjusted to pH 6.2 with 5M NaOH solution. The pH-adjusted supernatant was then added to the wells (200 μL / well), with uninoculated MRS medium (pH 6.2) used as a blank control. The culture was incubated at 37°C for 24 h, and the diameter of the inhibition zone was measured. Strains with a clear inhibition zone around the well were selected, and the diameter of the inhibition zone was measured, with each measurement repeated three times.

[0076] As shown in Table 6, the metabolites of *Lactobacillus plantarum* ZJUIDS19 exhibited good inhibitory effects against *Staphylococcus aureus*, *Escherichia coli*, *Salmonella typhimurium*, and *Listeria monocytogenes*. This demonstrates that the metabolites of this bacterium possess antibacterial properties.

[0077] Table 3 Results of the inhibitory effect of *Lactobacillus plantarum* ZJUIDS19 on pathogens.

[0078]

[0079]

[0080] Staphylococcus aureus is the most common pathogen causing purulent infections in humans. Some Escherichia coli strains can cause severe diarrhea and sepsis, and some Salmonella strains can cause food poisoning in humans. Bacteriocins, organic acids, hydrogen peroxide, and other antibacterial products produced by lactic acid bacteria metabolism can inhibit the growth of these pathogens, either individually or in combination. The metabolites of *Lactobacillus plantarum* ZJUIDS19 provided in this invention have a certain antagonistic effect against these four pathogens, which plays an important role in combating bacterial wound infections.

[0081] Example 7: Preparation method of post-biotic-loaded lactalbumin hydrogel

[0082] Dissolve the lactic acid protein powder thoroughly in PBS buffer (pH 7-8, i.e., 1×PBS buffer) at room temperature to prepare a lactic acid protein stock solution with a concentration of 1-10 g / 100 ml. First, add methacrylic anhydride to the lactic acid protein stock solution, wherein the ratio (v / w) of the methacrylic anhydride to the lactic acid protein powder from step 1) is 1 ml / 20 g to 2 ml / 1 g; then adjust the pH to 6.0-10.0 and stir continuously at 10-25°C for 3-24 h.

[0083] The reaction solution obtained in step 2) was diluted with deionized water and dialyzed for 48–72 h. The retentate after dialyzing was freeze-dried to obtain a lyophilized powder (methacrylamide whey protein powder). The lyophilized powder was added to deionized water and stirred until dissolved to obtain a lyophilized powder solution with a concentration of 10–20 g / 100 ml. 0.1–0.4 g / ml of the post-biotic obtained in Experimental Example 1 was dissolved in the lyophilized powder solution obtained in step 4. Then, under light-protected conditions, a photoinitiator was added until the final concentration of the photoinitiator was 0.05–0.5 g / 100 ml (i.e., 0.05%–0.5%, w / v), and mixed thoroughly. The mixture obtained in step 5) was irradiated with ultraviolet light (380–405 nm) for 10–60 s to form a hydrogel with an ultraviolet light intensity of 30 mW / cm². 2 The morphology of the resulting methacrylamide lactalbumin (α-LAMA-P) hydrogel loaded with postbiotics is as follows: Figure 4 .

[0084] Example 8: Rheological properties of post-biotic-loaded lactalbumin hydrogel

[0085] The ultraviolet light intensity obtained in Example 7 was reduced from 30 mW / cm 2 Change to 12mW / cm 2 The irradiation time ranged from 0 s to 200 s; the rest was the same as in Example 7; thereby verifying the rheological properties of the methacrylamide-coated lactalbumin hydrogel loaded with post-biotic.

[0086] The rheological properties of the methacrylamide-modified lactalbumin hydrogel loaded with the biogenerator were determined using a conventional rheometer (MCR302) with a stress of 1% and a frequency of 50 Hz. The storage modulus (G') and loss modulus (G") of the methacrylamide-modified lactalbumin hydrogel loaded with the biogenerator over time are shown in [reference needed]. Figure 5 .

[0087] according to Figure 5 It can be seen that the gel time of the methacrylamide lactalbumin aqueous solution loaded with post-generic in this example is only about 42s (the critical point time from solution to gel), and its gelation speed is relatively fast compared with other lactalbumin aqueous solutions; moreover, the hydrogel properties in this example are excellent and have good application prospects.

[0088] Example 9: Microstructure of post-biotic-loaded lactalbumin hydrogel

[0089] The post-biotic lactalbumin working solution obtained in Example 7 was photocured (UV light irradiation at 30 mW / cm² for 30 s) to prepare a hydrogel with a diameter of 8 mm and a thickness of 6 mm. The prepared post-biotic lactalbumin-loaded hydrogel was then pre-frozen at -80°C for 6 h, followed by freeze-drying at -80°C for 48 h. The freeze-dried post-biotic lactalbumin-loaded hydrogel was sectioned, and its microstructure was observed using scanning electron microscopy. Figure 6 .

[0090] The cross-sectional morphology and microstructure of the hydrogel confirmed that the loaded biogenic lactalbumin hydrogel has a dense and uniform porous structure, indicating that the loaded biogenic lactalbumin hydrogel has more cross-linking sites and higher mechanical strength.

[0091] Example 10: Confirmation of the antibacterial properties of lactalbumin hydrogel loaded with postbiotics

[0092] 1. In vitro antibacterial test of lactalbumin hydrogel loaded with biotics

[0093] Staphylococcus aureus (CMCC 26003) and Escherichia coli (ATCC 25922) were pre-amplified overnight in LB liquid medium (37°C, shaker, 180 rpm). The ZJUIDS19 postbiotic prepared in Example 1 was thoroughly dissolved in deionized water to form polymer solutions with concentrations of 0.20, 0.30, and 0.40 g / mL, diluted sequentially. The bacterial solutions were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the solutions were diluted with PBS to a final concentration of 10. 6 CFU / mL.

[0094] The loaded pamoate solution prepared in Example 7 was uniformly poured into 48-well plates, 400 μL per well. The plates were then illuminated with ultraviolet light (intensity 30 mW / cm²). 2 Irradiation for 60 seconds formed a hydrogel. Then, 10 μL of diluted bacterial solution was added to the surface of the hydrogel, and the mixture was incubated at 37°C for 6 hours. The control group contained no hydrogel, but only 10 μL of diluted bacterial solution was added. After incubation, 1 mL of PBS was added to each well, and after thorough rinsing, 100 μL of the solution was spread onto LB agar plates and incubated at 37°C for 24 hours. Finally, bacterial growth was observed and counted. The antibacterial rate was calculated and is shown in Table 4.

[0095]

[0096] Table 4. Antibacterial rate of lactalbumin hydrogel loaded with biotics

[0097]

[0098] As the concentration of metagenerium increases, the antibacterial properties of the hydrogel gradually improve. At metagenerium concentrations of 0.28 and 0.4 g / mL, the antibacterial rates against Staphylococcus aureus and Escherichia coli both reach over 90%, demonstrating its good antibacterial properties.

[0099] 2. In vivo antibacterial test of lactalbumin hydrogel loaded with biotics

[0100] Under routine isoflurane and oxygen anesthesia, the backs of mice were prepared and disinfected. The skin on the backs of the mice was gently lifted to avoid forceful traction and deformation, creating a circular skin defect with a diameter of 6 mm on the backs of the mice, extending to the full dermis. The size of the wound was confirmed to be uniform. 20 μL of 10 8 CFU Staphylococcus aureus was applied evenly to the wound.

[0101] After covering the wound with different test materials, a Tegaderm film was applied.

[0102] The post-natal lactalbumin working solution and lactalbumin solution obtained in Example 7 were used as the above test materials, thereby forming a post-natal lactalbumin loaded group (α-LAMA-P) and a lactalbumin group (α-LAMA), respectively; no treatment was used as a blank control.

[0103] Two days after infection of mouse wounds, 10 μL of exudate was collected from the wound and diluted 100-fold with sterile PBS. 100 μL of the diluted solution was then spread onto LB agar plates. The plates were incubated at 37°C for 24 hours. Finally, bacterial growth was observed and counted. The number of bacteria in the wound infection is shown in Table 5.

[0104] Table 5. Number of bacteria in infected wounds of mice

[0105] control group α-LAMA group α-LAMA-P group <![CDATA[Bacterial count (×10 5 CFU)]]> <![CDATA[8.76 a ]]> <![CDATA[8.21 a ]]> <![CDATA[1.48 b ]]>

[0106] The lactalbumin hydrogel loaded with postbiotics, when used as a wound dressing, can effectively reduce bacterial infection at the wound site in mice and has a good antibacterial effect.

[0107] Example 11: Confirmation of the skin repair performance of postbiotic-loaded lactalbumin hydrogel

[0108] Under routine isoflurane and oxygen anesthesia, the backs of mice were prepared and disinfected. The skin on the backs of the mice was gently lifted to avoid forceful traction and deformation, creating a circular skin defect with a diameter of 6 mm on the backs of the mice, extending to the full dermis. The size of the wound was confirmed to be uniform. 20 μL of 10 8 CFU Staphylococcus aureus was applied evenly to the wound.

[0109] After covering the wound with different test materials, a Tegaderm film was applied. The gross healing morphology was recorded using stereoscopic imaging at 0, 2, 4, 7, and 10 days. Figure 9 (See Table 6), and on day 10, a portion of healthy skin tissue from the base of the wound and surrounding area was also excised from the wound margin. The specimens were fixed in formalin.

[0110] The post-natal lactalbumin working solution and lactalbumin solution obtained in Example 7 were used as the above test materials, thereby forming a post-natal lactalbumin-loaded group (α-LAMA-P) and a lactalbumin group (α-LAMA), respectively; no treatment was used as a blank control (Blanck). The specific procedures for paraffin section HE staining and Masson staining photography are as follows:

[0111] (1) Tissue dehydration: The specimen tissue was dehydrated sequentially by passing 75% alcohol (4h) - 85% alcohol (2h) - 90% alcohol (1.5h) - 95% alcohol (1h) - anhydrous ethanol I (0.5h) - anhydrous ethanol II (0.5h).

[0112] (2) Tissue transparency: The dehydrated tissue was subjected to anhydrous ethanol: xylene (1:1) (10 min) - xylene I (10 min) - xylene II (7 min) to complete the tissue transparency process.

[0113] (3) Paraffin impregnation: The transparent tissue blocks were sequentially impregnated with paraffin (60℃) in three separate tanks. Paraffin I (60℃) (1h) - Paraffin II (60℃) (1h) - Paraffin III (60℃) (1h);

[0114] All of the above steps are completed inside a biological tissue dehydrator.

[0115] (4) Embedding: The temperature of the wax used for embedding should be slightly higher than the impregnation temperature to ensure that the tissue block and the embedded paraffin are completely integrated, so that the tissue block impregnated with wax is wrapped in the paraffin block.

[0116] (5) Sectioning and Preparing Slides: Before slicing, freeze the cut surface of the paraffin block on the freezing stage for several minutes. Fix the target embedded block in the specimen clips, ensuring the outer cut surface of the embedded block is parallel to the cross-section of the specimen clips and that the embedded block protrudes slightly. Push the blade stage to the outer edge, loosen the screw of the blade clip, and attach the blade, making the blade plane form an angle of approximately 15° with the tissue cut surface, with the top and bottom edges of the embedded block parallel to the blade edge. Adjust the required section thickness (4μm) on the micro-motion device, move the blade stage close to the specimen stage, and let the blade edge slightly contact the tissue cut surface to begin slicing. Use your right hand to rotate the rotary wheel evenly and at a constant speed, while your left hand holds a brush to catch the sliced ​​section slightly below the blade edge and supports the cut paraffin ribbon. Once the paraffin ribbon has formed a certain length, stop rotating with your right hand, and use another brush to gently lift the paraffin ribbon and place it flat in a water bath at approximately 42°C. The sliced ​​sections need to be flattened and attached to a glass slide. The specific procedure for the slide retrieval method is as follows: First, place the series of slides into a warm water bath at about 42°C. Once the slides float on the surface and naturally flatten due to surface tension, separate the slides with tweezers. Then, use an APES or poly-L-lysine-treated slide to tilt and insert it into the water to retrieve the slides, ensuring they adhere to the appropriate position on the slide. After the slides are properly attached, bake them in a 60°C oven for 3 hours.

[0117] (6) Dewaxing the sections: Place the paraffin sections in xylene I (10 min) - xylene II (10 min) - anhydrous ethanol I (5 min) - anhydrous ethanol II (5 min) - 95% ethanol (3 min) - 90% ethanol (3 min) - 80% ethanol (2 min) - 70% ethanol (2 min) in sequence, and then rinse with distilled water for 2 min.

[0118] (7) Staining:

[0119] HE staining: Stain with Harris hematoxylin solution for 5-7 min, then rinse with tap water to regain blue color. Differentiate sections in 1% hydrochloric acid-ethanol solution for 2-5 s, then rinse with tap water to regain blue color. Stain sections with 1% water-soluble eosin solution for 2 min, then rinse with tap water for 30 s. Dehydrate sections in anhydrous ethanol, clear with xylene, air dry, and mount with neutral resin.

[0120] Masson staining: Stain with prepared Weigert iron-hematoxylin staining solution for 8 minutes. Differentiate with acidic ethanol differentiation solution for 15 seconds, then rinse with water. Re-blue with Masson blue solution for 5 minutes, then rinse with water. Rinse with distilled water for 1 minute. Stain with Ponceau S and fuchsin for 5 minutes. Rinse with weak acid working solution for 1 minute. Rinse with phosphomolybdic acid solution for 1 minute, then with weak acid working solution for 1 minute. Stain with aniline blue solution for 2 minutes, then rinse with weak acid for 1 minute. Rapidly dehydrate with 95% ethanol for 2-3 seconds, then dehydrate three times with anhydrous ethanol for 5-10 seconds each time. Clear with xylene three times for 1-2 minutes each time, then mount with neutral resin.

[0121] (8) Microscopic photography: After staining, observe under an optical microscope as follows: Figure 10 and Figure 11 .

[0122] The lactalbumin hydrogel loaded with post-biotics has good antibacterial effect as a wound dressing, which helps wound healing. Its healing speed is higher than that of lactalbumin hydrogel and blank control, and it has no toxic side effects on wound tissue, resulting in good healing status.

[0123] Table 6. Changes in skin wound condition after treatment with different gel materials

[0124]

[0125] According to the results in Table 6, the lactalbumin hydrogel loaded with post-biotic showed a significantly higher rate of wound healing than the control group and the lactalbumin hydrogel group, with a healing rate of 97.75% after 10 days, demonstrating excellent healing effect.

Claims

1. A method for preparing an antibacterial lactalbumin hydrogel loaded with postbiotics, comprising the following steps: (1) Aseptic fermentation supernatant *Lactobacillus plantarum* ZJUIDS19 was inoculated into MRS liquid medium using an inoculation loop and cultured. Then, it was transferred to MRS liquid medium at a volume ratio of 2% for further culture. After culturing, the fermentation broth was obtained. The bacterial cells were collected by centrifugation, and the supernatant was collected by centrifugation after suspending the cells in distilled water. The pH was adjusted to 6.0, and the mixture was filtered to obtain the sterile fermentation supernatant. The taxonomic name of *Lactobacillus plantarum* ZJUIDS19 is *Lactobacillus plantarum*. (Lactobacillus plantarum The accession number is CGMCCNO.28018, and the accession address is: No.3, No.1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The full 16S rDNA sequence of the *Lactobacillus plantarum* ZJUIDS19 is shown in SEQ ID No.

1. (2) Preparation of postbiotics from Lactobacillus plantarum ZJUIDS19 The aseptic fermentation supernatant was concentrated by vacuum freezing, the supernatant was pre-frozen at ultra-low temperature, freeze-dried under vacuum, and after complete freeze-drying, the metagene was removed under reduced pressure and placed in a 4°C refrigerator to obtain the metagene of *Lactobacillus plantarum* ZJUIDS19; the ultra-low temperature pre-freezing condition was to place it in an ultra-low temperature refrigerator at -80°C for no less than 2 hours. (3) The extracted metagenics was added to the modified lactalbumin solution at room temperature and co-cured into a gel under ultraviolet light irradiation to form an antibacterial lactalbumin hydrogel loaded with metagenics. The ultraviolet light irradiation conditions were: ultraviolet wavelength of 380~405nm, irradiation time of 10~60s, and ultraviolet light intensity of 30 mW / cm². 2 .

2. The preparation method according to claim 1, characterized in that, The culture time in step (1) is 18 hours, the centrifugation conditions are 10000 r / min for 20 min, and the pH value is adjusted with 2M NaOH.

3. The preparation method according to claim 1, characterized in that, Preparation of MRS liquid culture medium in step (1): Dissolve the following components in each liter of distilled water: 10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium citrate, 5 g sodium acetate, 20 g glucose, 80 ml Tween, 0.5 g magnesium sulfate, and 0.25 g manganese sulfate.

4. The application of the antibacterial milk protein hydrogel loaded with post-biotics obtained by the preparation method of claim 1 in the preparation of biomedical materials, characterized in that, The biomedical material is an antibacterial wound dressing.

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