Lactoferrin biological membrane containing milk-derived antibacterial peptide as well as preparation and application of lactoferrin biological membrane

Abstract

The invention relates to a preparation method and application of an antibacterial bone regeneration promoting biological membrane which takes lactoferrin as a base material and is grafted with milk-derived antibacterial peptide. The preparation method is characterized in that lactoferrin is taken as an initial raw material, a hydrogel biological membrane is prepared by crosslinking grafted milk-derived antibacterial peptide KHPIKHQGLP and alginic acid through an electrostatic complexing method, and the obtained composite gel membrane has good antibacterial property and biological activities of promoting osteogenic activity expression of osteogenic-like cells and the like. The method is simple in preparation process and green and environment-friendly in process; the water vapor permeability, the light permeability, the mechanical tension and the tensile tension performance are good, the biological safety is high, and the degradable property is realized.

Description

Technical Field

[0001] The present invention relates to a method for preparing a lactoferrin biofilm containing a milk-derived antimicrobial peptide and its application. The method uses lactoferrin as the starting material, and then cross-links and grafts the milk-derived antimicrobial peptide KHPIKHQGLP and alginate via electrostatic complexation to produce a hydrogel biofilm. More specifically, the method relates to the use of a biofilm containing lactoferrin and antimicrobial peptides in wound repair and targeted intervention for orthodontic relapse.

[0002] Background

[0003] Hydrogels are highly hydrated polymer materials that ensure their structural integrity through physical and chemical interactions between molecules. They can absorb wound exudate, isolate wounds, and prevent bacterial infection in the air. The antibacterial activity of simple hydrogel dressings is slightly insufficient. Therefore, the use of antibacterial active materials combined with antibiotics or antibacterial active substances to improve the antibacterial ability of dressings is a widely studied and effective method in this field. Antibiotics can kill or inhibit the growth and reproduction of certain microorganisms and are the preferred strategy for treating non-viral infections. However, antibiotics have caused many problems in clinical use, such as allergies, intestinal flora disorders, superinfections, multidrug resistance and other side effects. Therefore, the search for alternative substances to antibiotics has always been a research hotspot in this field.

[0004] Lactoferrin (LF) is one of the most important active substances in milk. It is primarily synthesized and secreted by mammalian mucosal epithelial cells. With a molecular weight of approximately 80 kDa, it possesses a potent ability to transport and absorb iron ions, earning it the reputation of "the first line of defense for health." LF's most notable characteristic is its diverse biological activities, which have garnered widespread attention in the industrial scientific community. Research has shown that LF possesses not only antibacterial, antioxidant, antiviral, and immunomodulatory activities but also plays a role in iron absorption and transport. Lactoferrin can also act as a transcription factor and possess enzyme activities such as RNase, ATPase, and amylase. Furthermore, lactoferrin can promote bone growth and wound healing. Numerous studies have demonstrated that LF can inhibit the proliferation of pathogens by absorbing iron from microorganisms or interacting with cytolytic pathogens. Furthermore, naturally occurring lactoferrin is positively charged, making it susceptible to interaction with negatively charged bacteria, affecting bacterial metabolism and thereby inhibiting their growth. Therefore, LF holds great potential for development in the biomedical field.

[0005] While lactoferrin capsules are clinically used for antiviral treatment and rehabilitation of patients undergoing chemotherapy and radiotherapy, their antibacterial and osteoblastic cell proliferation-promoting biological activities hold greater application and development value. Antimicrobial peptides, as a novel agent for treating bacterial infections, have attracted widespread attention due to their natural origin, broad-spectrum antimicrobial properties, safety, efficacy, and resistance to drug resistance, offering promising research and application prospects.

[0006] Based on this, a hydrogel biofilm was prepared using lactoferrin as the starting material, cross-linked with the lactoferrin antimicrobial peptide KHPIKHQGLP and alginate via electrostatic complexation. This composite biofilm, containing the antimicrobial peptide, exhibited excellent water vapor permeability, film-forming properties, and mechanical properties. The synergistic effect of the antimicrobial peptide and lactoferrin significantly enhanced the biofilm's antimicrobial properties. Furthermore, the biofilm exhibited bioactivity, such as promoting the expression of osteoblast-like cell osteogenic activity. The biofilm, whose raw materials are all food-derived, offers advantages such as ease of preparation, safety, non-toxicity, and controlled degradation.

[0007] A method for preparing a lactoferrin biofilm containing milk-derived antimicrobial peptides and its application, comprising the following steps in sequence:

[0008] (1) Lactoferrin and alginate powder were weighed separately and dissolved in water. After swelling, they were stirred until completely dissolved. The lactoferrin concentration was 0.5-8%, the molecular weight (Mw) was 78-80 kD, and the Fe saturation was 5%-16%. The source was natural bovine lactoferrin. The alginate concentration was 0.5-2%. Both solutions were sterile filtered through a 0.22 μm filter membrane.

[0009] (2) mixing the lactoferrin solution obtained in step (1) and the alginic acid solution in a volume ratio of 10:1 to 1:10 (v / v) and allowing to stand for degassing;

[0010] (3) adding antimicrobial peptide and plasticizer to the mixed film-forming solution prepared in step (2), and evenly coating 1 mL on a horizontal glass plate, allowing it to flow naturally until it is flat, and allowing it to stand for 5 to 20 minutes to dry;

[0011] (4) peeling off the dried gel film prepared in step (3).

[0012] The antimicrobial peptide sequence in step (3) is KHPIKHQGLP, and its amino acid sequence is SEQ ID NO: 1.

[0013] Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro. White powder, soluble in water, with strong inhibitory effects on the growth and activity of various pathogens. Concentration ranges from 0.01% to 1%.

[0014] The antimicrobial peptide fragment obtained in step (3) has an isoelectric point of 10.6, a molecular weight of 1154.36 Da, and 10 amino acids. Biological information of the amino acid sequence shown in SEQ ID NO: 1 obtained using an online tool indicates that the peptide fragment has an average hydrophobicity GRAVY of -1.3, an instability coefficient of 16.89, and an Aliphatic index of 78.

[0015] Information on SEQ ID No. 1

[0016] (a) Sequence characteristics

[0017] *Length: 10 amino acids

[0018] *Type: Amino Acid

[0019] (b) Molecule type: protein

[0020] Sequence description: KHPIKHQGLP

[0021] The plasticizer in step (3) is one of sorbitol, mannitol, glycerol, ethylene glycol, and polyethylene glycol.

[0022] The concentration of the plasticizer in step (3) is 0.05% to 1%.

[0023] The cross-linking film-forming temperature in step (4) is 20 to 37°C.

[0024] The mass ratios of the components in the cross-linked film in step (4) are as follows: lactoferrin: sodium alginate: antimicrobial peptide:

[0025] Plasticizer = 0.5-80: 0.5-20: 0.01-1: 0.05-1

[0026] This lactoferrin biofilm can be used for wound repair such as cuts, burns / scalds, diabetic foot, pressure sores, and targeted intervention of orthodontic recurrence.

[0027] The present invention has the following advantages:

[0028] 1. The electrostatic complexation reaction is used to separate the lactoferrin biofilm containing antimicrobial peptides. The preparation conditions are mild and the process is simple. Through synergistic effects, the antibacterial properties of the organisms are significantly improved.

[0029] 2. All raw materials are derived from food, and no organic solvents or cross-linking agents are introduced during the preparation process. The resulting biofilm has excellent biocompatibility and safety, and exhibits excellent water vapor permeability, film-forming properties, and mechanical properties. It also offers numerous advantages, including antibacterial properties and the ability to promote osteoblast precursor cell proliferation, and can be used for wound repair and bone formation. Furthermore, topical percutaneous application can avoid the first-pass effect in the liver and gastrointestinal digestion and enzymatic degradation of lactoferrin or antimicrobial peptides, thereby better preserving biofilm activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The antibacterial results of lactoferrin are as follows: + represents chlorhexidine, + represents chlorhexidine, - represents sterile water, 25 represents 25 mg / mL lactoferrin, and 50 represents 25 mg / mL lactoferrin. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below with reference to specific embodiments. The following examples will help those skilled in the art and researchers to further understand the present invention, but do not constitute any limitation of the present invention. Any modifications made by anyone within the scope of the claims of the present invention are still within the scope of protection of the claims of the present invention.

[0032] Example 1

[0033] Antibacterial activity of peptides

[0034] Nanjing Jiepeptide Biotechnology Co., Ltd. was commissioned to synthesize the peptide KHPIKHQGLP using the solid phase method with a purity of 96.71%.

[0035] The antimicrobial peptide has an isoelectric point of 10.6, a molecular weight of 1154.36 Da, and 10 amino acids. Biological information of the amino acid sequence represented by SEQ ID NO: 1 was obtained using online tools. The peptide had an average hydrophobicity of -1.3, an instability coefficient of 16.89, and an Aliphatic Index of 78.

[0036] Information on SEQ ID No. 1

[0037] (a) Sequence characteristics

[0038] *Length: 10 amino acids

[0039] *Type: Amino Acid

[0040] (b) Molecule type: protein

[0041] Sequence description: KHPIKHQGLP

[0042] The antibacterial activity of the peptide KHPIKHQGLP was investigated using Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Porphyromonas gingivalis and Streptococcus mutans as target bacteria.

[0043] (1) Strains and recovery

[0044] Escherichia coli (CICC10899), Staphylococcus aureus (CICC10384), Staphylococcus epidermidis (CICC10398), Porphyromonas gingivalis (ATCC33277), and Streptococcus mutans (ATCC25175) were stored at -80°C using glycerol. Before the experiment, the bacteria were inoculated into liquid culture medium (LB broth for Escherichia coli, TSB broth for Staphylococcus aureus and Staphylococcus epidermidis, and BHI broth for Porphyromonas gingivalis and Streptococcus mutans) and cultured at 37°C for 12 h to resuscitate the strains and restore their vitality, thus obtaining the restored bacterial solution.

[0045] (2) Antibacterial test

[0046] Take 1 mL of the revitalized bacterial solution and wash it twice with PBS buffer and then dilute it to 10 7 CUF / mL bacterial suspension was used to obtain bacterial suspensions of Staphylococcus aureus, Escherichia coli, Porphyromonas gingivalis, Staphylococcus epidermidis, and Streptococcus mutans. The antimicrobial peptide KHPIKHQGLP samples were dissolved in the culture media corresponding to the above bacterial cultures (LB broth for Escherichia coli, TSB broth for Staphylococcus aureus and Staphylococcus epidermidis, and BHI broth for Porphyromonas gingivalis and Streptococcus mutans) to prepare 50 mg / mL antimicrobial peptide solutions. The solutions were sterilized by passing through a 0.22 μm membrane and then used to obtain peptide solutions corresponding to different culture media. 100 μL of the above-mentioned concentration of antimicrobial peptide solution or culture medium corresponding to different bacteria was mixed with 100 μL of the above-mentioned corresponding bacterial solution (LB broth medium or the bacterial solution obtained after dissolving the sample for Escherichia coli, TSB broth medium or the bacterial solution obtained after dissolving the sample for Staphylococcus aureus and Staphylococcus epidermidis, BHI broth medium or the bacterial solution obtained after dissolving the sample for Porphyromonas gingivalis and Streptococcus mutans) and added to different wells of a 96-well cell culture plate in sequence. Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis were cultured under aerobic conditions at 37°C in an incubator, while Porphyromonas gingivalis and Streptococcus mutans were cultured under anaerobic conditions at 37°C in an anaerobic incubator. After the above bacterial solutions were cultured for 12 hours, the absorbance was measured at a wavelength of 600 nm. The antibacterial rate of the samples corresponding to different bacteria was calculated according to the following formula:

[0047] Inhibition rate % = (OD1-OD2) / OD2×100%

[0048] OD1 control group: 100 μL bacterial solution + 100 μL culture medium corresponding to bacterial culture

[0049] OD2 experimental group: 100 μL bacterial solution + 100 μL antimicrobial peptide solution

[0050] The absorbance OD value of the blank culture medium (the culture medium corresponding to the bacterial culture) at a wavelength of 600nm is the background value

[0051] (3) Experimental results

[0052]

[0053] Example 2

[0054] Antimicrobial activity of lactoferrin

[0055] Lactoferrin was commercially available bovine milk lactoferrin with a purity of >95%, a molecular weight (Mw) of 78-80 kD, and an Fe saturation of 5%-16%. It was derived from natural bovine lactoferrin. The antibacterial activity of bovine lactoferrin was evaluated against Gram-negative bacteria (Escherichia coli and Porphyromonas gingivalis) and Gram-positive bacteria (Staphylococcus aureus and Streptococcus mutans).

[0056] (1) Strains and recovery

[0057] Escherichia coli (CICC10899), Staphylococcus aureus (CICC10384), Porphyromonas gingivalis (ATCC33277) and Streptococcus mutans (ATCC25175) were stored at -80°C using the glycerol preservation method. Before the experiment, the bacteria were inoculated into liquid culture medium and cultured at 37°C for 12 hours to revive the strains and restore their vitality.

[0058] (2) Culture medium

[0059] The antibacterial experiment used a 90 mm inner diameter microbial culture dish. E. coli was cultured on 1.5% agar (g / L) with a bacterial concentration of 1×10 6 CFU / mL of LB broth medium, Staphylococcus aureus S. aureus containing 1.5% agar (m / v, g / L) bacterial concentration of 1×10 6 CFU / mL TSB broth medium, Streptococcus mutans S. mutans was 1×10 6CFU / mL BHI broth culture medium was purchased from Sangon Biotech (Shanghai) Co., Ltd. After high-temperature sterilization, the culture medium was naturally cooled to 55-65°C in a biosafety cabinet and poured into sterile microbial culture dishes, 20-25 mL per dish. The culture medium was then naturally cooled to room temperature in a biosafety cabinet and set aside.

[0060] Porphyromonas gingivalis P.gingivalis bacterial concentration is 1×10 6 Columbia blood agar culture plates with a CFU / mL were purchased from Nanjing Quanlong Biotechnology Co., Ltd. The culture medium was sealed and stored at 4°C until use.

[0061] (3) Antibacterial test

[0062] The antibacterial ability of lactoferrin was determined by the punching method. 8 holes were punched on the plate using a hole puncher, with the gap between two adjacent holes greater than 3 cm and the pore diameter 3.8±0.2 mm. Lactoferrin was dissolved in sterile water to 50 mg / mL and 25 mg / mL solutions and vortexed until completely dissolved. Samples were added at a volume of 20 μL / well, with 1 mg / well (50 mg / mL) and 0.5 mg / well (25 mg / mL) of lactoferrin added to each well. The positive control chlorhexidine (2MIC, 9.435 μg / mL) and the negative control sterile water were added in equal volumes. After adding the samples, the culture dishes were placed in a 37°C incubator for 18 hours to observe whether there was an inhibition zone, and the size of the inhibition zone was measured and recorded (n=3).

[0063] (4) Experimental results

[0064] Lactoferrin antibacterial results and inhibition zone diameters are shown in Figure 1 and Table 2. The results showed that lactoferrin had good inhibitory activity against a variety of tested strains and had broad-spectrum, stable and efficient antibacterial properties.

[0065] Figure 1 Lactoferrin antibacterial results Note: + represents chlorhexidine, + represents chlorhexidine, - represents sterile water, 25 represents 25 mg / mL lactoferrin, 50 represents 25 mg / mL lactoferrin

[0066] Table 2 Antibacterial activity of lactoferrin

[0067]

[0068] Note: The number of inhibition zone is the diameter of the inhibition zone

[0069] Example 3

[0070] Preparation of biofilm containing antimicrobial peptide lactoferrin

[0071] (1) Dissolve 0.2 g of lactoferrin powder (Mw = 78 kDa, purity 98%, iron saturation 12.2%) and 0.15 g of alginic acid (G molar content 42%) in 10 ml of deionized water, allow to swell fully, and stir until completely dissolved. Sterile filter the two solutions through a 0.22 μm filter membrane and set aside.

[0072] (2) Weigh 0.2 mg of KHPIKHQGLP polypeptide and dissolve it in alginate solution. Stir until completely dissolved.

[0073] (3) The lactoferrin solution and the alginate solution containing antimicrobial peptides were mixed thoroughly in a 1:1 volume ratio until homogeneous.

[0074] (4) Add 2% glycerol (final mass concentration) to the above mixture and stir with a magnetic stirrer at 100 rpm until thoroughly mixed. After standing for 10 min to degas, spread 2 mL of the solution onto a glass plate and allow it to flow naturally until it spreads horizontally. Let stand for 20 min.

[0075] (5) Place in a fume hood and dry for 60 minutes at room temperature, then remove the film.

[0076] Antibacterial performance investigation

[0077] The antibacterial activity of bovine lactoferrin was investigated using Gram-negative bacteria: Escherichia coli and Gram-positive bacteria: Staphylococcus epidermidis as target bacteria.

[0078] (1) Strains and recovery

[0079] Escherichia coli (CICC10899) and Staphylococcus epidermidis (CICC10398) were stored at -80°C using the glycerol preservation method. Before the experiment, the bacteria were inoculated into liquid culture medium and cultured at 37°C for 12 hours to revive the strains and restore their vitality.

[0080] (2) Culture medium

[0081] The antibacterial experiment used a 90 mm inner diameter microbial culture dish. E. coli was cultured on 1.5% agar (g / L) with a bacterial concentration of 1×10 6 CFU / mL of LB broth medium, Staphylococcus epidermidis S.epidermidis containing 1.5% agar (m / v, g / L) bacterial concentration of 1×10 6 CFU / mL TSB broth culture medium was purchased from Sangon Biotech (Shanghai) Co., Ltd. After high-temperature sterilization, the culture medium was naturally cooled to 55-65°C in a biosafety cabinet and poured into sterile microbial culture dishes, 20-25 mL per dish. The culture medium was then naturally cooled to room temperature in a biosafety cabinet and set aside.

[0082] (3) Antibacterial test

[0083] The antibacterial ability of lactoferrin was determined by the punching method. 8 holes were punched on the plate using a hole puncher, with the gap between two adjacent holes greater than 3 cm and the pore diameter 3.8±0.2 mm. Lactoferrin was dissolved in sterile water to 50 mg / mL and 25 mg / mL solutions and vortexed until completely dissolved. Samples were added at a volume of 20 μL / well, with 1 mg / well (50 mg / mL) and 0.5 mg / well (25 mg / mL) of lactoferrin added to each well. The positive control chlorhexidine (2MIC, 9.435 μg / mL) and the negative control sterile water were added in equal volumes. After adding the samples, the culture dishes were placed in a 37°C incubator for 18 hours to observe whether there was an inhibition zone, and the size of the inhibition zone was measured and recorded (n=3).

[0084] The antibacterial activity of the biofilms was determined using the disc method. After UV irradiation, the biofilms and PE films (0.05 mm) were cut into 4 mm diameter circular films using a hole punch. Sterile filter paper discs sterilized with high-pressure steam (121°C, 20 min) were cut into 4 mm diameter circular films. Using a pipette, 20 μL of sterile water, 10 μg / μL lactoferrin solution (final concentration 0.2 mg / filter paper disc), and 1 μg / μL antimicrobial peptide KHPIKHQGLP solution (final concentration 0.02 mg / filter paper disc) were each pipetted onto the sterile filter paper until completely absorbed. Sterile water served as a blank control, PE films as a negative control, and lactoferrin solution and antimicrobial peptide KHPIKHQGLP as positive controls. Using sterile tweezers, the filter paper discs for the experimental, blank, and control groups were evenly placed on the microbial culture plate and gently pressed until they were completely in contact. The filter paper discs were spaced approximately 20 mm apart to avoid interference caused by overlapping inhibition zones. After adding the sample, the culture dish was placed in a 37°C incubator for 18 h to observe whether there was an inhibition zone, and the size of the inhibition zone was measured and recorded. Each group of experiments was repeated three times.

[0085] (4) Experimental results

[0086] After 18 hours of incubation, the composite hydrogel biofilm exhibited inhibitory effects against both Escherichia coli and Staphylococcus epidermidis, with the diameters of the E. coli inhibition zone being 5.89±0.20 mm and the S. epidermidis inhibition zone being 5.45±0.34 mm. No inhibition was observed in the sterile water blank control group or the PE film negative control group. In the positive control group, the diameters of the lactoferrin inhibition zone against E. coli were 4.21±0.18 mm and 4.47±0.25 mm, respectively. The diameters of the antimicrobial peptide inhibition zone against E. coli and S. epidermidis were 4.37±0.21 mm and 4.62±0.19 mm, respectively. This demonstrates that the biofilm exhibits excellent antibacterial activity and is suitable for use as a repair dressing for superficial wounds such as epidermal abrasions and burns.

[0087] Example 4

[0088] Preparation of biofilm containing antimicrobial peptide lactoferrin

[0089] (1) Dissolve 0.45 g of lactoferrin powder (Mw=80 kDa, purity 96%, iron saturation 8.9%) and 0.2 g of alginic acid (G content 35%) in 10 ml of deionized water, allow to swell fully, and stir until completely dissolved. Sterile filter the two solutions through a 0.22 μm filter membrane and set aside.

[0090] (2) Weigh 0.05 mg of KHPIKHQGLP polypeptide and dissolve it in the alginate solution. Stir until completely dissolved.

[0091] (3) The lactoferrin solution and the alginate solution containing antimicrobial peptides were mixed thoroughly at a volume ratio of 2:1 until homogeneous.

[0092] (4) Add 1% 1,2-propylene glycol to the above mixture and stir with a magnetic stirrer at 100 rpm until thoroughly mixed. After standing for 15 min to degas, 3 mL of the solution was spread evenly on a glass plate (10 cm x 10 cm) and allowed to flow naturally until it spread horizontally.

[0093] (5) Place in a fume hood and dry for 120 minutes at room temperature, then remove the film.

[0094] Promotes osteoblast growth performance

[0095] 1) Culture of mouse embryonic osteoblast precursor cells MC3T3-E1

[0096] Remove the frozen MC3T3-E1 cells from the liquid nitrogen tank, quickly place them in a 37°C water bath, and gently shake them to completely thaw. Transfer the revived cell suspension to a 15mL centrifuge tube containing 5mL of high-glucose DMEM medium (Gibco) containing 10% FBS (FBS, Gibco). Centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 3mL of fresh medium to resuspend the cells, inoculate them into cell culture flasks, and culture them in an incubator at 37°C with 5% CO2 in air. Change the medium every other day, and disperse the cells with phenol red-free, EDTA-free digestion solution (Solarbio, China) containing 0.25% trypsin by volume. When the cell density grows to 80-85%, subculture or inoculate the cells.

[0097] 2) Biofilm treatment

[0098] The biofilm was placed under ultraviolet irradiation for 30 minutes. The sterilized biofilm was cut into small circular pieces with a diameter of 2 mm using a hole punch and placed in a 96-well plate.

[0099] 3) Group settings

[0100] MC3T3-E1 cells in the logarithmic growth phase were selected and diluted to 5×10 cells using high-glucose DMEM medium containing 10% FBS. 4 cells / mL, add 200 μL of cell suspension to each well to reach a seeding cell density of 1×10 4 cells / well.

[0101] Cells without biofilm served as the blank control group, cells treated with culture medium containing lactoferrin (50 μg / mL, final lactoferrin concentration of 10 μg / well) served as the experimental control group, and cells treated with biofilm served as the experimental group.

[0102] 4) Evaluation of osteoblast growth performance

[0103] The CCK8 assay was used to determine the effect of biofilms on the proliferation of osteoblast precursor cells MC3T3·E1 in vitro. Cells were precultured in culture medium (high-glucose DMEM supplemented with 10% fetal bovine serum (FBS, Gibco)) for 24 hours. The medium was then replaced. After 48 hours of continuous culture, the medium was removed and 100 μL of serum-free medium (high-glucose DMEM supplemented with 10% CCK8) was added to each well. The cells were incubated in a 37°C incubator with 5% CO₂ in air for 2 hours. The supernatant was then transferred to a fresh 96-well plate and absorbance was measured at 450 nm, with a reference wavelength of 630 nm.

[0104] Cell proliferation rate (%) = [A(experimental group) - A(blank)] / [A(blank control) - A(blank)] × 100

[0105] Note: Blank refers to culture medium, and blank control refers to the experimental group without biofilm cells.

[0106] Three replicates were set for each sample group. The results are shown in Table 3-1.

[0107] Antibacterial performance investigation

[0108] The antibacterial activity of the biofilm containing the antimicrobial peptide lactoferrin was investigated using Gram-negative bacteria: Porphyromonas gingivalis (ATCC33277) and Gram-positive bacteria: Streptococcus mutans (ATCC25175) as target bacteria.

[0109] (1) Strains and recovery

[0110] Porphyromonas gingivalis and Streptococcus mutans (ATCC25175) were stored at -80°C using glycerol preservation. Before the experiment, the bacteria were inoculated into liquid culture medium and cultured at 37°C for 12 hours to resuscitate the strains and restore their vitality.

[0111] (2) Culture medium

[0112] The antibacterial experiment used a 90 mm inner diameter microbial culture dish. Porphyromonas gingivalis was cultured in a 1.5% agar (g / L) agar solution with a bacterial concentration of 1×10 6 Columbia blood agar plates with a bacterial concentration of 1×10 CFU / mL and Streptococcus mutans (S. mutans, containing 1.5% agar (m / v, g / L)) 6 CFU / mL BHI broth culture medium was purchased from Sangon Biotech (Shanghai) Co., Ltd. After high-temperature sterilization, the culture medium was naturally cooled to 55-65°C in a biosafety cabinet and poured into sterile microbial culture dishes, 20-25 mL per dish. The culture medium was then naturally cooled to room temperature in a biosafety cabinet and set aside.

[0113] (3) Antibacterial test

[0114] The antibacterial ability of the biofilm was determined by the paper disc method. The biofilm and PE film (0.05 mm) sterilized by ultraviolet irradiation were cut into circular films with a diameter of 4 mm using a hole punch, and the sterile filter paper pieces sterilized by high-pressure steam (121°C, 20 min) were cut into circular films with a diameter of 4 mm. Use a pipette to draw 20 μL of sterile water and add it to the sterile filter paper until it is completely absorbed, which serves as a blank control. The PE film is the experimental control group. Use sterile tweezers to evenly place the filter paper pieces of the experimental group, blank control group and experimental control group on the microbial culture plate and press them gently until they are in full contact. The filter paper pieces are spaced about 20 mm apart to avoid interference caused by overlapping inhibition zones. After adding the sample, the culture dish is placed in a 37°C incubator for 18 hours to observe whether an inhibition zone appears, and the size of the inhibition zone is measured and recorded. Each group of experiments is repeated three times.

[0115] (4) Experimental results

[0116] The results showed that after 18 hours of incubation, the composite hydrogel biofilm exhibited inhibitory effects against both Porphyromonas gingivalis and Streptococcus mutans. The inhibition zones are shown in Table 3-2. No inhibition zones were observed in the negative control groups of sterile water and PE film. This suggests that the biofilm has dual efficacy in promoting osteoblast progenitor cell proliferation and inhibiting bacteria, and could be used for oral antibacterial and targeted interventions for orthodontic relapse.

[0117] Table 3-1 Biofilm promotes the proliferation of osteoblast precursor cells

[0118]

[0119] Table 3-2 Antibacterial activity of biofilm

[0120]

[0121]

Claims

1. A method for preparing a lactoferrin biofilm containing milk-derived antimicrobial peptides, characterized in that: The steps are as follows: 1) Lactoferrin and alginic acid powders are weighed separately and dissolved in water, and stirred until completely dissolved after swelling; the lactoferrin concentration is 0.5-8 wt% (preferably 2-6%, more preferably 3-4%), and the alginic acid concentration is 0.5-2% (preferably 0.8-1.6%, more preferably 1.2-1.5%); the resulting lactoferrin solution and alginic acid solution; 2) adding the antimicrobial peptide to the lactoferrin solution and / or the alginic acid solution; and uniformly mixing the lactoferrin solution and the alginic acid solution at a volume ratio of 10:1 to 1:10 (v / v) (preferably 1-4:1, more preferably 1-2:1) to obtain a mixed solution; 3) adding a plasticizer to the mixed solution obtained in step (2) to obtain a film-forming solution, allowing the solution to stand for degassing, coating the solution on a flat plate, allowing it to flow naturally until it spreads flat, allowing it to stand for 5 to 20 minutes to crosslink and form a film, and drying; 4) peeling off the dried gel film prepared in step (3).

2. The preparation method according to claim 1, wherein: described The molecular weight (Mw) of lactoferrin is 78-80 kD, the Fe saturation is 5%-16%, and it is derived from natural bovine lactoferrin.

3. The preparation method according to claim 1, wherein: The antimicrobial peptide sequence in step (3) is KHPIKHQGLP, and its amino acid sequence is shown in SEQ ID NO: 1, specifically Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro; its concentration in the film-forming solution is 0.01-1 wt% (preferably 0.5-1%, more preferably 0.8%-1%).

4. The preparation method according to claim 1, wherein: The antimicrobial peptide segment in step (3) has an isoelectric point of 10.6, a molecular weight of 1154.36 Da, and 10 amino acids; biological information of the amino acid sequence shown in SEQ ID NO: 1 obtained through an online tool indicates that the average hydrophobicity GRAVY of the peptide segment is -1.3, the instability coefficient is 16.89, and the Aliphatic index is 78; Information on SEQ ID No. 1 (a) Sequence characteristics *Length: 10 amino acids *Type: Amino Acid (b) Molecule type: protein Sequence description: KHPIKHQGLP.

5. The preparation method according to claim 1, wherein: The plasticizer in step (3) is one of sorbitol, mannitol, glycerol, ethylene glycol, and polyethylene glycol.

6. The preparation method according to claim 1, wherein: The concentration of the plasticizer in the film-forming solution in step (3) is 0.05 wt% to 1 wt% (preferably 0.4-0.9%, more preferably 0.5% to 0.8%).

7. The preparation method according to claim 1, wherein the crosslinking film forming temperature in step (4) is 20-37°C, the film is allowed to stand for degassing for 5-20 minutes, 1-5 mL is evenly coated on a horizontally placed flat plate (e.g., 50-300 cm 2 ).

8. The preparation method according to claim 1, wherein: The mass ratios of the components in the cross-linked film in step (4) are as follows: lactoferrin: sodium alginate: antimicrobial peptide: plasticizer = 0.5-80: 0.5-20: 0.01-1: 0.05-1 (preferably 2-24: 0.8-1.6:0.5-1:0.4-0.9 (w / w / w / w), more preferably 3-8:1.2-11.5:0.8-1:0.5-0.8).

9. A lactoferrin biofilm containing milk-derived antimicrobial peptides prepared by the preparation method according to any one of claims 1 to 8.

10. A use of the biofilm according to claim 9, characterized in that: This lactoferrin biofilm can be used as a dressing for wound repair such as cuts, burns / scalds, diabetic foot or pressure sores, or for targeted intervention in orthodontic relapse and other treatment processes.

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