A lactobacillus biofilm derivative for promoting wound healing, and preparation method and application thereof

Abstract

This scheme provides a method for preparing a lactobacillus biofilm derivative that promotes wound healing, including the following steps: S1: lactobacillus resuscitation and culture; S2: model preparation; obtaining a model with a porous rough surface having a uniform pore structure on the surface; S3: lactobacillus inoculation and biofilm preparation; S4: removing the live lactobacillus bacteria to obtain a sterile biofilm derivative filtrate, i.e., a lactobacillus biofilm derivative. The present invention uses a porous rough micro-surface mold with a uniform pore structure to culture lactobacillus biofilm, helping to expand its production, which is conducive to the large-scale production of biofilm. The extracted lactobacillus biofilm derivative retains the active ingredients in the lactobacillus biofilm, introduces and adopts the new probiotic drug delivery system concept of "bacteria-free biofilm derivatives", and has a very favorable promoting effect in the field of diabetic wound regeneration.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical wound surface materials, and in particular to a scalable extraction and preparation method for lactobacillus biofilm derivatives and applications thereof. Background Art

[0002] Diabetes has become a chronic disease that seriously threatens human health after tumors and cardiovascular diseases. The biggest threat of diabetes to patients comes from its complications. Among them, diabetic wounds have become a serious complication that seriously threatens the survival and quality of life of diabetic patients. They are difficult to cure, easy to infect, and have a high amputation rate. Their pathological mechanisms are complex. At present, diabetic wounds are mainly limited to traditional treatments, and the clinical treatment effect is not ideal. The effects of some newly developed single growth factor drugs are still relatively limited. There is a lack of simple, economical, safe and efficient new drugs and biological treatment methods. Therefore, how to effectively promote the healing of diabetic wounds has become a topic of common concern among multiple disciplines. New strategies and breakthroughs are needed to overcome the treatment of diabetic wounds.

[0003] Compared with healthy skin, diabetic wounds have more opportunistic microorganisms, lower bacterial diversity, and secretion of virulence factors and proteases, which is one of the reasons why diabetic wounds are chronically inflamed and difficult to heal. On the contrary, introducing certain multifunctional probiotics on the wound surface is very beneficial for regulating cell function and further promoting wound healing.

[0004] Under today's medical conditions, probiotic drug delivery systems still have great limitations. The weakly acidic wound environment, interference with the host immune system, and competition from other pathogens may reduce the viability and activity of probiotics. More importantly, when probiotics come into contact with the blood circulation system, they may induce bacteremia or sepsis. In the natural environment, probiotics mainly exist in the form of biofilms. Compared with free-living planktonic bacteria, biofilms are essentially different in biological functions and other aspects. Bacteria have a variety of secretory functions. When the secreted DNA, proteins, lipids, and peptidoglycans reach a certain level, these substances polymerize and form a membrane-like protective matrix to wrap the bacteria, helping the bacteria resist the harsh external environment. Successful separation of bacteria and biological components will effectively reduce the possibility of bacteria causing disease.

[0005] Lactobacillus has multifunctional pro-repair effects including immune regulation, and can regulate the secretion of immune cytokines by macrophages. Lactobacillus biofilms also have similar rich pharmacological effects. In the natural environment, bacterial biofilms attach to different matrix surfaces and secrete a large amount of bacterial extracellular matrix to wrap their own bacteria. However, not all matrix surfaces are suitable for the growth and reproduction of biofilms. In addition, Lactobacillus belongs to Gram-positive bacteria. Gram-positive bacterial biofilms have their own specific quorum sensing system, which will affect their own biofilm production through direct or indirect effects, and cannot be extracted and preserved on a large scale. Summary of the invention

[0006] The invention provides a bacteria-free lactobacillus biofilm derivative capable of promoting wound healing, especially diabetic wound healing, and a scalable extraction preparation method and application thereof.

[0007] In order to solve the above technical problems, the present invention adopts the following technical scheme: A method for preparing a lactobacillus biofilm derivative that promotes wound healing, comprising the following steps: S1: lactobacillus recovery and culture: lactobacillus is cultured using a sterile MRS medium, and the cells placed in an anaerobic culture bag are anaerobically cultured in a bacterial culture box at 37°C for 24 hours. h, and cultured for multiple generations until the bacterial properties were stable; S2: Model preparation: 20% silica-ethanol suspension that had been fully ultrasonically dispersed was evenly spread on a glass slide and air-dried to obtain a SiO2 particle template; a polystyrene toluene solution was used as a filler to fill the gaps in the SiO2 particle template and air-dried; the SiO2 particle template was fully etched with a 10% hydrofluoric acid solution, and the residual acid on the surface was washed off with pure water to obtain a model with a porous rough surface with a uniform pore structure on the surface; S3: Lactobacillus inoculation and biofilm preparation: Lactobacillus with stable properties was inoculated at a 1% inoculation rate on the sterilized porous rough model surface and cultured anaerobically for 24 hours; after removing the excess culture medium, a layer of white film was left on the film surface, which was the lactobacillus biofilm; S4: Removal of live lactobacillus cells: The lactobacillus biofilm formed on the surface of the porous rough model was collected and 1× The mixture was washed with PBS buffer until the washing solution was colorless and clear, and the lactobacillus and its active secretions were separated by ultrasound. The mixed solution after ultrasound was filtered through a filter with a pore size of 0.45 μm to obtain a sterile biofilm derivative filtrate, namely, the lactobacillus biofilm derivative.

[0008] As an improvement: in step S4, 130 kW, 20 kHz ultrasonic power and 50% amplitude are used to separate lactobacilli from their active secretions for 10 minutes.

[0009] As an improvement: the lactobacillus biofilm derivative filtrate obtained in step S4 is freeze-dried to obtain milky white lactobacillus biofilm derivative LBD freeze-dried powder.

[0010] As an improvement: it also includes a Lactobacillus biofilm derivative prepared by the above method for preparing a Lactobacillus biofilm derivative that promotes wound healing.

[0011] As an improvement: the metabolites of the lactobacillus biofilm derivative prepared by the above method for preparing the lactobacillus biofilm derivative for promoting wound healing include dibutyl phthalate which has a promoting effect on M2 macrophages.

[0012] As an improvement: it also includes applying the lactobacillus biofilm derivative prepared by the above method for preparing the lactobacillus biofilm derivative that promotes wound healing to the treatment of diabetic wounds, and applying the derivative to the diabetic wound by smearing.

[0013] As an improvement: the concentration of the Lactobacillus biofilm derivative applied to the diabetic wound surface is 1 mg / mL.

[0014] The beneficial effects provided by the present invention are: the present invention can target the defect of low yield of existing lactobacillus biofilms, use a porous and rough micro-surface mold with a uniform pore structure to cultivate lactobacillus biofilms, help expand their yield, and facilitate large-scale production of biofilms. Ultrasonic technology and filtration technology are used to remove harmful bacterial components in the biofilms, increase the safety of their application, and the extracted lactobacillus biofilm derivatives retain the active ingredients in the lactobacillus biofilms, and introduce and adopt the new probiotic drug delivery system concept of "bacteria-free biofilm derivatives", which solves the problem of safety hazards in existing probiotic administration, and has a very favorable promoting effect in the field of diabetic wound regeneration.

[0015] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a porous and rough micro surface with a uniform pore structure.

[0017] Figure 2 Comparison of biofilm yields grown on a smooth surface and on a porous rough surface structure.

[0018] Figure 3 Characterization diagram of Lactobacillus biofilm derivatives.

[0019] Figure 4 This is a diagram showing the biocompatibility experiment of Lactobacillus biofilm derivatives.

[0020] Figure 5 This is a diagram of wound changes and healing trajectories in the treatment of diabetic wounds using Lactobacillus biofilm derivatives.

[0021] Figure 6 This is a diagram of the experiment in which LBD promotes the polarization of macrophages RAW264.7 to the M2 phenotype.

[0022] Figure 7 This is a diagram of the experiment in which LBD promotes LPS-induced macrophage polarization toward the M2 phenotype.

[0023] Figure 8 This is an experimental diagram showing that LBD promotes the polarization of M1 macrophages to M2 macrophages in a concentration-dependent manner.

[0024] Fig. 9 Metabolomics analysis of LBD included the bioactive anti-inflammatory component dibutyl phthalate (DBP). DETAILED DESCRIPTION

[0025] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention. These all belong to the scope of protection of the present invention.

[0026] Example 1

[0027] A method for preparing a lactobacillus biofilm derivative that promotes wound healing. Since the adhesion ability of bacteria on a smooth surface is significantly weaker than that on a rough surface, the biofilm yield on a rough surface is significantly higher than that on a smooth surface. Therefore, the present invention uses a rough micro-surface mold with a hole structure to increase the yield, and combines ultrasonic technology and filtration technology to improve the safety of biofilm application.

[0028] The preparation of the lactobacillus biofilm derivative comprises the following steps:

[0029] S1: Recovery and culture of lactobacilli: Lactobacilli were cultured using sterile MRS medium. The cells were placed in anaerobic culture bags and cultured anaerobically in a 37°C bacterial incubator for 24 h. The cells were subcultured multiple times until the bacterial characteristics were stable.

[0030] S2: Preparation of porous rough surface structure model: 20% silica-ethanol suspension that has been fully ultrasonically dispersed is evenly spread on a glass slide, placed in a ventilated environment to dry naturally, and the SiO2 particle template is obtained by air drying; a polystyrene toluene solution is used as a filler to fill the gaps in the SiO2 particle template, and air-dried; the SiO2 particle template is fully etched with a 10% hydrofluoric acid solution, and the residual acid on the surface is washed off with pure water to obtain a model of a porous rough surface with a uniform pore structure on the surface. The micro-surface is as follows Figure 1 shown.

[0031] S3: Lactobacillus inoculation and biofilm preparation: Stable lactobacillus was inoculated at 1% on the sterilized porous rough model surface and cultured anaerobically for 24 h. After removing the excess culture medium, a layer of white film was left on the film surface, which was the lactobacillus biofilm.

[0032] S4: Removal of live Lactobacillus cells: The Lactobacillus biofilm formed on the surface of the porous rough model was collected, washed with 1× PBS buffer until the washing liquid was colorless and clear, and ultrasonicated for 10 min at an ultrasonic power of 130 kW, 20 kHz, and an amplitude of 50% to separate the Lactobacillus and its active secretions. The mixed solution after ultrasonication was filtered through a filter with a pore size of 0.45 μm to obtain a sterile biofilm derivative filtrate, i.e., the Lactobacillus biofilm derivative.

[0033] In order to facilitate storage and application, after step S4, the obtained lactobacillus biofilm derivative filtrate is freeze-dried to obtain a milky white lactobacillus biofilm derivative (hereinafter referred to as LBD) freeze-dried powder.

[0034] The porous rough surface structure model prepared in step 2 of the present invention can greatly increase the yield of the biofilm, as shown in the comparison chart between the yield of the biofilm grown on a smooth surface and the yield of the biofilm grown on a smooth surface. Figure 2 As shown. Figure 2 In this study, the method of extracting Lactobacillus biofilm from a porous rough surface structure model was compared with the control group extracting Lactobacillus biofilm from a smooth plate in a variety of ways from various angles to verify the yield improvement effect.

[0035] in Figure 2 ab Concanavalin A tetramethylrhodamine conjugate (TMR-Con A) was used to stain the Lactobacillus biofilm on the smooth plate and the Lactobacillus biofilm formed on the surface of the porous inverse opal film. Figure 2 As shown in a, lactobacilli and their secreted extracellular matrix are mixed with each other and difficult to separate. They are sparsely distributed and thin in thickness, and are very easy to detach from the biofilm and release planktonic bacteria, resulting in extremely low biofilm yield. Compared with the control plate culture group, the lactobacillus biofilm formed on the surface of the porous inverse opal film is Figure 2 As shown in b, the distribution is denser and the thickness is thicker.

[0036] exist Figure 2 In c, (i) is the biofilm grown on a plate, and (ii) is the biofilm grown on an inverse opal film, which are the results of concanavalin A tetramethylrhodamine conjugate staining and Hoechst staining, respectively.

[0037] like Figure 2As shown in de: After collecting the biofilm products per unit area and resuspending them with 1 ml PBS, it can be seen that the biofilm resuspension formed on the rough surface of the inverse opal film is significantly more turbid.

[0038] like Figure 2 As shown in f, the biofilm products per unit area were collected, freeze-dried and weighed, which once again proved that the biofilm grown on the inverse opal film had a higher yield.

[0039] Figure 2 The various yield comparison methods all show that the porous rough surface structure model prepared in step 2 can greatly increase the yield of lactobacillus biofilm.

[0040] Example 2

[0041] The bacteria-free lactobacillus biofilm derivatives prepared by the above method were subjected to experimental analysis, and their specific characterization diagrams and biocompatibility were as follows: Figure 3 , Figure 4 shown.

[0042] The milky white Lactobacillus biofilm derivative LBD freeze-dried powder is obtained by freeze drying, and after dissolving in water, it presents a colorless transparent solution, such as Figure 3 As shown in a, the Lactobacillus biofilm derivative LBD freeze-dried powder solid is very soluble in water.

[0043] exist Figure 3 In b, the protein of the obtained lactobacillus biofilm derivative (LBD) was analyzed by SDS-PAGE polyacrylamide gel electrophoresis, and it was observed that most of the active protein components were retained while the LBD obtained by this method was removed from the bacteria.

[0044] In order to improve the safety of Lactobacillus biofilm application, we exposed the Lactobacillus biofilm to an ultrasonic power of 130 kw, 20 Hz, and 50% amplitude, and subjected it to low-temperature ultrasound for 5 minutes to try to see whether the bacteria in the biofilm could be separated. The results of the scanning electron microscope are shown in Figure 2. Figure 3 As shown in Figure c, no obvious bacterial cell structure was found on the surface of the extracted LBD, that is, the extraction method of the present invention can effectively remove the bacteria in the biofilm, providing a guarantee for the safety of the application of lactobacillus biofilm.

[0045] To ensure the safety of application, we also conducted cytotoxicity tests on NIH3T3 cells at different concentrations of LBD for different periods of time. Figure 4 As shown in a; The cytotoxicity test of different concentrations of LBD on RAW264.7 cells at different times, as shown in Figure 4 As shown in b; the NIH3T3 cells treated with LBD were subjected to live / dead staining and compared with the blank control group. Figure 4 As shown in c.

[0046] like Figure 4 a shows the cytotoxicity test of different concentrations of LBD on NIH3T3 cells at different times, where the red dotted line indicates the survival rate of the blank control group. The results show that when the concentration of LBD is lower than 1 mg / ml, it has good biocompatibility and does not cause any significant inhibition on cell growth and proliferation. However, when the concentration of LBD is higher than 10 mg / ml, LBD will inhibit cell growth.

[0047] like Figure 4 b shows the cytotoxicity test of different concentrations of LBD on RAW264.7 cells at different times. The results show that when the concentration of LBD is lower than 1 mg / ml, it has good biocompatibility and does not cause any significant inhibition on cell growth and proliferation. However, when the concentration of LBD is higher than 10 mg / ml, LBD will inhibit cell growth.

[0048] like Figure 4 As shown in c, by comparing with the blank control group, after treatment with low concentration of LBD, most of the living cells appeared green, with only a few dead cells, which was no different from the blank control group.

[0049] Depend on Figure 4 The results verified that the best approach was to apply 1 mg / mL of Lactobacillus biofilm derivatives to the diabetic wound surface, which was both effective and safe.

[0050] Example 3

[0051] The lactobacillus biofilm derivatives prepared above and sterilized by 0.45 μm filtration were used in a diabetic wound application experiment in mice. Figure 5 As shown in the figure, the healing process of the full-thickness excision wound of diabetic mice was recorded at 1, 4, 7, 10 and 14 days. The experiment was divided into a PBS control group and a Lactobacillus biofilm derivative application LBD group. The PBS control group was a natural wound healing group of mice without any treatment. The Lactobacillus biofilm derivative application LBD group: the administration method was smear administration; the final concentration of the Lactobacillus biofilm derivative was 1 mg / mL; the LBD solution was applied to the wound every other day.

[0052] Diabetic wounds were constructed in high-fat diet-induced type 2 diabetes model mice, and LBD solution was applied to the diabetic wounds to observe the effect of LBD on wound closure. Figure 5 As shown in a.

[0053] In order to compare the wound healing effects of different treatment groups, the wound bed contours at different time points during the wound healing process were depicted, and representative images of the wound bed at different time points after different treatments were used. + PBS or + lactic acid bacteria biofilm derivatives. The unit scale bar in the image is 1 mm. The results are shown in Figure 5 As shown in b.

[0054] At the same time, digital images were used to observe the wound closure at different time points, such as Figure 5 c also shows that the wound healing rate in diabetic mice treated with LBD was significantly faster than that in mice treated with PBS.

[0055] The wound healing area was counted and quantified. Figure 5 As shown in Figure d, there was a difference in the wound healing area between the experimental group and the control group starting from the 4th day after injury. Figure 5 As shown in Figure e, the wound healing areas were counted and quantified, and it was found that there was a difference in the wound healing areas between the experimental group and the control group starting from the 4th day after injury.

[0056] Hematoxylin and eosin (H&E) staining images of wound tissues harvested on the 7th and 14th days after different treatments of the PBS control group and the Lactobacillus biofilm derivative application LBD group, as shown in Figure 5 f, scale bar: 1000um. After the diabetic wound was treated with Lactobacillus biofilm derivatives, the granulation tissue gaps in the wound were narrower, the granulation layer was thicker, and the number of layers was more, indicating that the experimental group had more abundant granulation tissue formation than the PBS control group, which was consistent with the trend of wound closure area. In addition, in the tissue sections of the wound on the 14th day, it was found that the number of skin appendages in the group treated with LBD increased significantly, almost the same as normal skin.

[0057] The above data all indicate that the lactobacillus biofilm derivatives extracted by the present invention play a positive role in the healing process of diabetic wounds.

[0058] The above is the in vivo research experiment that proves that LBD can significantly promote the healing of diabetic wounds. The following is an in vitro research experiment to illustrate.

[0059] During the inflammatory phase of wound healing, macrophages play a key anti-inflammatory role. When wound healing enters the late inflammatory phase, around the seventh day after injury, macrophages change their phenotype, promoting tissue repair and transitioning it to the repair phase. However, in diabetic wounds, the ability of macrophages to undergo this phenotypic change is impaired, causing diabetic wounds to remain in the inflammatory phase, which is a very important reason why diabetic wounds do not heal for a long time.

[0060] Different phenotypes of macrophages can show different morphological characteristics. For example, M1 macrophages are mostly round in outline, while M2 macrophages are elongated. Therefore, the phenotypic changes of macrophages can be inferred by observing the changes in cell characteristics.

[0061] LBD promotes the polarization of macrophages RAW264.7 toward the M2 phenotype, such as Figure 6 As shown. Among them, Figure 6 a: Microscope image of cell morphology; Figure 6 bd: Representative fluorescence images and quantified mean fluorescence intensity of macrophage cytokines; Figure 6 e: Western blot analysis of macrophage-related protein expression; Figure 6 f: Qualitative and quantitative analysis of CD206+ in LBD-treated macrophages RAW264.5; Figure 6 g: Qualitative and quantitative analysis of iNOS+ in RAW264.5 macrophages treated with LBD.

[0062] like Figure 6 As shown in a, mouse macrophages RAW 264.7 were cultured with LBD-containing culture medium for 24 hours, and the morphology of some cells changed from the original spherical shape to an elongated shape. This shows that LBD promotes the polarization of macrophages. Next, immunofluorescence staining was performed on mannose receptor (CD206 wound tissue anti-inflammatory factor), arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS pro-inflammatory factor) in macrophages to confirm the phenotype of macrophages. Mannose receptor (CD206) and arginine kinase-1 (Arg-1) are markers of M2 macrophage activation, while nitric oxide synthase (iNOS) is a marker molecule for M1 macrophage activation. As shown in Figure 6 As shown in bd, macrophages cultured with LBD-containing medium showed elongated cell morphology, and the expression levels of CD206 and Arg-1 were significantly higher than those of the control group. In contrast, the expression level of iNOS was significantly lower than that of the control group.

[0063] Western blotting was used to detect the expression of related proteins in macrophages. Figure 6 As shown in e. LBD treatment can significantly downregulate the expression of pro-inflammatory protein IL-1β in macrophages and upregulate the expression of pro-healing proteins such as CD206 and IL-10. In addition, flow cytometry analysis showed that LBD treatment can induce macrophages to downregulate the inflammatory gene iNOS and upregulate the pro-healing gene CD206 associated with M2 macrophages. Figure 6 e. Figure 6 f. These experimental results all indicate that LBD can promote the polarization of mouse macrophages RAW264.5 to M2 phenotype.

[0064] LBD promotes LPS-induced macrophage polarization toward the M2 phenotype, such as Figure 7 As shown. Among them, Figure 7 a: Microscope image of cell morphology; Figure 7 b: Representative fluorescence images; Figure 7 c: Quantitative mean fluorescence intensity of macrophage cytokines; Figure 7 d: Western blot analysis of macrophage-related protein expression; Figure 7 e: Qualitative and quantitative analysis of CD206+ in LBD-treated macrophages RAW264.5; Figure 7 f: Qualitative and quantitative analysis of iNOS+ in LBD-treated RAW264.5 macrophages.

[0065] The local high-sugar and high-fat environment in diabetic wounds causes the wound to remain in the inflammatory phase, and macrophages in the wound express high levels of pro-inflammatory factors, thereby delaying wound healing. Macrophages mature into the classic M1 phenotype after exposure to lipopolysaccharide. Macrophages RAW264.7 were pretreated with LPS to polarize them to the M1 phenotype. It was found that after LPS pretreatment, most macrophages showed a classic spherical morphology. After LBD treatment, it was found that most macrophages turned into elongated cell morphology, such as Figure 7 As shown in a. The fluorescence expression of proteins in the cells was detected at this time. The expression of CD206, a marker of M2 macrophages, increased, while the expression of iNOS, a marker of M1 macrophages, decreased. Figure 7 b and Figure 7 As shown in c. Figure 7 The same results were also shown by Western blotting in d. The cell phenotypes were further analyzed by flow cytometry. Figure 7 e and Figure 7 As shown in Figure f, there are significantly more M2 macrophages in the group treated with LPS and LBD successively, which is manifested by high expression of CD206 and low expression of iNOS. These experimental results all indicate that LBD can promote the polarization of macrophages from M1 phenotype to M2 phenotype.

[0066] The concentration experiment of lactobacillus biofilm derivatives was conducted by treating them with different concentrations of LBD solution, such as Figure 8 As shown. Among them, Figure 8 ab, Representative fluorescence images of macrophage cytokines; Figure 8 c: Quantitative Figure 8 a Mean fluorescence intensity; Figure 8 d: Quantitative Figure 8 b Mean fluorescence intensity; Figure 8 e: Qualitative characterization of CD206+ in RAW264.5 macrophages after LBD treatment; Figure 8 f: Quantification of CD206+ in RAW264.5 macrophages after LBD treatment; Figure 8 g: Characterization of iNOS+ in LBD-treated macrophages RAW264.5; Figure 8 h: Quantification of iNOS+ in LBD-treated RAW264.5 macrophages.

[0067] LBD increases Arg-1 and the anti-inflammatory factor CD206 in wound tissue in a concentration-dependent manner and reduces the expression of iNOS in LPS-pretreated macrophages. Low concentrations of LBD have little effect on M1 macrophages, while higher concentrations of LBD within the safety range can significantly promote the polarization of macrophages from M1 to M2 phenotype. LBD promotes the polarization of M1 macrophages to M2 macrophages in a concentration-dependent manner. The final concentration of the preferred lactobacillus biofilm derivative used is 1 mg / mL; applying LBD solution to the wound once every other day is a safe and effective solution.

[0068] In summary, Lactobacillus biofilm derivative LBD can promote the polarization of macrophages from M1 to M2, and the structure of Lactobacillus biofilm derivative without live bacteria promotes the polarization of macrophages to M2 pro-healing phenotype in a concentration-dependent manner. The immune microenvironment at the wound surface is improved by regulating the polarization of M2 macrophages at the diabetic wound surface. The improvement of the polarization-promoting function of M2 macrophages will mediate tissue repair and regeneration by secreting anti-inflammatory factors, thereby transforming the wound surface from the inflammatory phase to the proliferative phase and accelerating the healing process of the diabetic wound surface.

[0069] The LBD extracted by the present invention is subjected to metabolomics analysis as a complex compound. Fig. 9 As shown, its metabolites include dibutyl phthalate DBP, which is a compound that promotes M2 macrophages, and the content of DBP is relatively high. Ultra-high performance liquid chromatography was used to detect the content of DBP in LBD, such as Fig. 9 As shown in a. Since the concentration of each metabolite is proportional to the peak area, it is calculated that each 1 mg of LBD contains approximately 41.3 μg of DBP. In other words, DBP accounts for approximately 4.13% of all metabolites of LBD. Fig. 9 b. When the M1 macrophages were treated with DBP standard and LBD with the same DBP content, it was found that the proportion of M2 macrophages in the LBD treatment group was higher than that in the other two groups. Fig. 9 As shown in c-9h.

[0070] In summary, the method provided by the present invention can extract lactobacillus biofilm derivatives simply and quickly, achieve high-yield and high-efficiency preparation thereof, and greatly ensure the safety of application in the field of wound regeneration, so that the active ingredients such as proteins carried by them are not destroyed, and subsequent Western Blot, IF, IHC and other tests can be continued, thereby providing a simple and effective extraction method and application idea for research in the field of biofilms.

[0071] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a lactobacillus biofilm derivative that promotes wound healing, characterized in that: The following steps are involved: S1: Lactobacillus recovery and culture: Lactobacillus was cultured using sterile MRS medium. The cells placed in anaerobic culture bags were cultured anaerobically in a 37°C bacterial incubator for 24 h and subcultured multiple times until the bacterial characteristics were stable. S2: Model preparation: 20% silica-ethanol suspension that has been fully ultrasonically dispersed is evenly spread on a glass slide and air-dried to obtain a SiO2 particle template; a polystyrene toluene solution is used as a filler to fill the gaps in the SiO2 particle template and air-dried; the SiO2 particle template is fully etched with a 10% hydrofluoric acid solution, and the residual acid on the surface is washed off with pure water to obtain a model with a porous rough surface having a uniform pore structure; S3: Lactobacillus inoculation and biofilm preparation: Stable lactobacillus was inoculated at 1% on the sterilized porous rough model surface and cultured anaerobically for 24 h. After removing the excess culture medium, a layer of white film was left on the film surface, which was the lactobacillus biofilm. S4: Removal of live Lactobacillus cells: Collect the Lactobacillus biofilm formed on the surface of the porous rough model, wash it with 1×PBS buffer until the washing liquid becomes colorless and clear, ultrasonically separate the Lactobacillus and its active secretions, and filter the mixed solution after ultrasonication through a filter with a pore size of 0.45 μm to obtain a sterile biofilm derivative filtrate, i.e., the Lactobacillus biofilm derivative.

2. The method for preparing a lactobacillus biofilm derivative for promoting wound healing according to claim 1, characterized in that: In step S4, 130 kW, 20 kHz ultrasonic power and 50% amplitude were used for 10 min to separate lactobacilli from their active secretions.

3. The method for preparing a lactobacillus biofilm derivative that promotes wound healing according to claim 1 or 2, characterized in that: The lactobacillus biofilm derivative filtrate obtained in step S4 is freeze-dried to obtain milky white lactobacillus biofilm derivative LBD freeze-dried powder.

4. A lactobacillus biofilm derivative for promoting wound healing, characterized in that: The biofilm derivative of Lactobacillus that promotes wound healing is prepared according to the method for preparing the biofilm derivative of Lactobacillus that promotes wound healing according to any one of claims 1 to 3.

5. The lactobacillus biofilm derivative according to claim 4, characterized in that: Its metabolites include dibutyl phthalate, which has a promoting effect on M2 macrophages.

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